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GAN STATE UNlVE

IHIIIJIIIHIEIIHII llll i‘lllltl'llllfllllllllllll

1293 00877 3461

 

 

 

 

 

 

 

 

 

 

This is to certify that the

dissertation entitled

Molecular Physiology of Xylan Degradation
by Thermoanaerobes
presented by

Yong-Eok Lee

has been accepted towards fulfillment
of the requirements for

Microbiology

 

Ph.D. degmin

4 WW
fl /"°‘“‘°'

7/1/92

 

MS U is an Affirmative Action/Equal Opportunity Institution

 

 

 

 

LIBRARY
Mlchlgan State
Unlverelty

 

 

 

PLACE IN RETURN BOX to remove thie checkout from your record.
TO AVOID FINES return on or before date due.

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

cmeI

 

MOLECULAR PHYSIOLOGY OF XYLAN DEGRADATION

BY THERMOANAEROBES

By

Yong -Eok Lee

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health
1992

 

(ff, 055’ 7

ABSTRACT

MOLECULAR PHYSIOLOGY OF XYLAN DEGRADATION
BY THERMOANAEROBES

By

Yong -Eok Lee

Physiological, biochemical and molecular mechanisms of xylan
degradation were investigated in Thermoanaerobacter strain B6A-RI, which
was described as a new thermoanerobe species. This organism produced
endoxylanase, B-xylosidase, arabinofuranosidase, acetyl esterase and xylose
isomerase, and the first three activities were produced coordinately. When
grown on xylan, the xylanolytic enzyme activities were cell associated rather
than secreted. The organism produced multiple endoxylanases which were
glycosylated and thermostable. Endoxylanase (xynA), B-xylosidase (xynB) and
xylose isomerase (xylA) genes were cloned into Escherichia coli and
sequenced, and the expressed enzymes were purified and characterized.
XynB and xynA but not xylA were oriented in the same direction on the 28
kb DNA fragment. The thermostable B-xylosidase hydrolyzed xylopentose,
xylotriose, xylobiose and PNPX, but had no activity on xylan. The deduced
amino acid sequence of xylose isomerase showed very high homology to
those from thermoanaerobes but not mesophilic aerobes. xynA gene was
similar to genes from family F B-glycanases grouped by hydrophobic cluster
analysis. Endoxylanase A cleaved xylan and xylooligosaccharides, but not

xylobiose. Deletion of part of the N-terminal region of xynA resulted in a

i‘

mutant enz.“
part Of the C
sub-stir tion,
asparagine a
activity, and
predicted for
surface strut“
transmissior
xylanosome
endoxylanasi
xylanosome i

not ceilulose

mutant enzyme that retained activity but lost thermostability. Deletion of
part of the C-terminus did not alter thermostability or activity. Individual
substitution, by site directed mutagenesis, of Asp-504 and Asp-569 by
asparagine and Glu—567 by glutamine completely destroyed endoxylanase
activity, and implicated their role in the general catalysis mechanism
predicted for hydrolytic enzymes such as lysozyme. Negatively charged cell
surface structures were visualized on cells grown on xylan by scanning and
transmission electron microscopy. The presence of these putative
xylanosome structures coincided with the production of cell bound
endoxylanases and the ability of the cells to bind tightly to xylan. The
xylanosome was specific for cellular adhesion to and degradation of xylan but

not cellulose.

T01

To my late father, Jung-Woo Lee, my mother, Young—Sock Kim,
my wife, Pyung-Ok Lim, and my son, John Lee

with love.

 

l woul.
have made th
First ar
intellectual 51
stidy and allt
Ishoul
correcting m.
works. Withc
I won‘.
it Bagdasari
for their yak
1 W011
MOOSCOP}! I
1 also

L ' c

ACKNOWLEDGMENTS

I would like to acknowledge my gratitude to the many people who
have made the completion of this dissertation possible.

First and foremost I want to thank Dr. J. G. Zeikus for his guidance and
intellectual support throughout this project. He gave me a opportunity to
study and allows me to develop and mature as a scientist.

I should particularly like to thank Dr. Sue Lowe for her painstakingly
correcting many versions of this dissertation and for collaboration of many
works. Without her assistance, this thesis would not have been possible.

I would like to thank all of the members of my guidance committee Dr.
M. Bagdasarian, Dr. J. A. Breznak, Dr. R. Hollingsworth, and Dr. L. R. Snyder
for their valuable advices and comments.

I would like to thank Mr. Pankratz H. Stuart for his excellent electron
microscopy works.

I also would lke to acknowledge the assistances and encouragements I
have received from the past and present members of the Zeikus laboratories:
Dr. M. Iain, Dr. Bhatnagar Lakshumi, Dr. Badal Saha, Dr. M. V. Ramesh, Saroj
Mathupala, John Kemner, Maris Laivenicks, Doug Burdette, and Keith
Strevett.

I am very grateful to my parents for their love and support.

Words cannot express my thanks to my wife, Pyung-Ok, for all of her
understanding, patience, friendship and love. Without her emotional
encouragement the completion of this degree would not have been possible.

And most importantly, thanks and praise to the LORD, who truly

" causes all thing to work for good for those who love Him".

iv

 

LIST OF TABL
LIST Of FIGL']
ABBRE‘I'LAIIC

CRAFT ER 1.
In:
Lit

CRAFT ER 11.
COMP.
THER).
XEW I

A!
In
M
RI
D

TABLE OF CONTENTS

 

 

 

 

page
LIST OF TABLES viii
LIST OF FIGURES x
ABBREVIATIONS xiv
CHAPTER I. INTRODUCTION AND OBJECTIVES ................................. 1
Introduction 2
Literature cited 22

 

CHAPTER II. DEOXYRIBONUCLEIC ACID HOMOLOGY AND OTHER
COMPARISONS AMONG XYLANASE PRODUCING
THERMOANAEROBIC BACTERIA, WITH A PROPOSAL FOR

 

 

 

 

 

 

NEW TAXONOMIC DESIGNATIONS 33
Abstract 34
Introduction 36
Materials and Methods 39
Results 45
Discussion 57
Literature cited 70

 

CHAPTER III. REGULATION AND CHARACTERIZATION OF
XYLANOLYTIC ENZYMES OF Thermoanaerobacter STRAIN

 

 

B6A-RI 74
Abstract 75
Introduction 76

 

Materials and Methods 79

 

Clikl’f ER .
CHA
TIE?!

 

’ Results

Discussion

 

Literature cited

 

CHAPTER IV. CLONING, SEQUENCING AND BIOCHEMICAL
CHARACTERIZATION OF ENDOXYLANASE FROM
Thermoanaerobacter STRAIN B6A-RI

 

 

Abstract

Introduction

 

Materials and Methods

 

Results

 

Discussion

 

Literature cited

 

CHAPTER V. GENETIC ORGANIZATION, SEQUENCE AND
BIOCHEMICAL CHARACTERIZATION OF RECOMBINANT
B-XYLOSIDASE FROM Thermoanaerobacter
STRAIN B6A-RI

 

 

Abstract

Introduction

 

Materials and Methods

 

Results

 

Discussion

 

Literature cited

 

CHAPTER VI. CLONING, SEQUENCING AND BIOCHEMICAL
CHARACTERIZATION OF XYLOSE ISOMERASE FROM
Thermoanaerobacter STRAIN B6A-RI

 

97
101

106
107
108
110
117
I41
144

149
150
151
153
159
180
182

185

Abs:

Intrc

CiikI’I ER VII.
THERV.

Therm;

Abstract 186

 

 

 

 

 

Introduction 187
Materials and Methods 188
Results 192
Discussion 205
Literature cited 208

 

CHAPTER VII. CHARACTERIZATION OF THE ACTIVE SITE AND
'THERMOSTABILTTY REGIONS OF ENDOXYLANASE FROM

 

 

 

 

 

 

Thermoanaerobacter 212
Abstract 213
Introduction 214
Materials and Methods 216
Results 220
Discussion 230
Literature cited 233

 

CHAPTER VIII. EVIDENCE FOR XYLANOSOMES ON THE CELL

 

 

 

 

 

 

SURFACE OF Thermoanaerobacter B6A-RI 237
Abstract 233
Introduction 239
Materials and Methods 241
Results 244
Discussion 251
Literature cited 255

 

CHAPTER IX. CONCLUSIONS AND RECOMMENDATIONS FOR
FUTURE RESEARCH 258

 

vii

1. Gem

Z Gent

Iii]
dint

Chapter II

to
n
O
:1

tner

4. Cor

stra

5- Pro

has

sac
Clapterrn

 

 

 

 

 

 

 

LIST OF TABLES
page
Chapter I
1. General properties of xylanases from bacteria and fungi ---------- 9
2. General properties of the purified B—xylosidases from
different microorganisms 11
Chapter II
1. Strains used in this study 40
2. Comparison of substrate utilization by thermoanaerobes --------- 51
3. Comparison of xylanase activity from various
thermoanaerobes 52
4. Comparison of DNA homology between thermoanaerobic
strains that display xylanase activity 54
5. Proposed nomenclature for xylanolytic thermoanaerobic
bacterial species of uncertain phylogenetic affiliation -------------- 59
6. Taxonomic key for preliminary identification of
saccharolytic, ethanologenic thermoanaerobes ----------------------- 61
Chapter III
1. Effect of growth substrate on xylanolytic enzyme levels of
Thermoanaerobacter strain B6A—RI 85
2. Effect of xylan concentration on cell bound versus
excreted endoxylanase activity in Thermoanaerobacter
strain BéA—RI 91
Chapter IV
1. Comparison of the translated amino acid sequence of
endoxylanase (xynA) from Thermoanaerobacter B6A-RI
with other endoxylanases 133
2. Summary of purification of endoxylanase from
E. coli (pZEP12) 134

 

Chapter V

l. Rela
Tilt‘.’

2 Sun
E. c

3. H}'(

pur
Chapter VI

1. Re]
The

Z A I
ge:
im

Chapter VII

1. 5}
ET

Chapter V

1. Relative homology between B-xylosidase from
Thermoanaerobacter B6A-RI and other B—xylosidases -------------- 170

2. Summary of purification of B-xylosidase from

 

 

E. coli (pXPH3) 172
3. Hydrolysis of various nitrophenyl-glycosides by the
purified B-xylosidase 176
Chapter VI

1. Relative homology between xylose isomerase from
Thermoanaerobacter B6A-RI and other xylose isomerases -------- 198

2. A comparison of the codon usage frequency in the three
genes from Thermoanaerobacter B6A-RI which are
involved in xylan degradation 202

 

Chapter VII

1. Synthetic oligonucleotides used to isolate mutant
endoxylanases 219

 

2. Comparison of temperature optimum and thermostability

 

of endoxylanase with its deletion derivatives ---------------------- 222

3. Effect of EDAC on recombinant endoxylanase A activity -------- - 227

4. Specific activity of mutated endoxylanases 229
ix

Chapter I
l. Poss

2. A.h}
enzy

Chapterll

3. E19:
ther

ChapterIII

1- Fern
cou:
grow

2— Fern
C0ur
grov

3'SHDS
IDuri
gTOr.

Titer;

U1

The:

fl'Om

Chapter [\7

I. Linea
Then

LIST OF FIGURES

 

 

 

 

 

 

 

 

page
Chapter I
1. Possible configurations for xylans 5
2. A hypothetical xylan and the sitesd of its attack by xylanolytic
enzymes 6
Chapter II
1. Phase-contrast photomicrographs of sulfur-depositing
cultures of Thermoanaerobacter strain LX-11 (A) and
B6A-RI (B) 46
2. Electron micrograph of an endospore of Thermoanaerobacter
strain LX-11 in thin section 48
3. Electrophoretic comparison of cellular proteins from
thermoanaerobic strains that display xylanase activity -------------- 56
Chapter III
1. Fermentation and xylanolytic enzyme synthesis time
course ofThermoanaerobacter straibn BéA-RI during
growth on xylose 86
2. Fermentation and xylanolytic enzyme synthesis time
course of Thermoanaerobacter strain BéA-RI during
growth on xylan 88
3. SDS—PAGE (A) and a zymogram (B) of endoxylanase
purification from Thermoanaerobacter strain B6A-RI
grown on xylose 93
4. The pH profile of partially purified endoxylanase from
Thermoanaerobacter B6A-RI 95
5. The temperature profile of partially purified endoxylanase
from Thermoanaerobacter B6A-RI 96

 

Chapter IV

1. Linear restriction map of plasmid pZXAl which carries the
Thermoanaerobacter B6A-RI DNA insert expressing

X

Z Phy:
the 1

3. Sou

prni

frao

4. Ph}'
con

5. XUI
for
Be;

6. SEX

fro!

7.'Ihé
fro:

8. 1}“

enc

9- Iin
xyL

E‘IIC

Chapter V

1. Pin
28
Bér
a-..

2. Pig
con

86;

3. 111%
offi
ancj

 

thermostable endoxylanase 1 18

2. Physical map of plasmid pXDM1 and functional mapping of
the endoxylanase and B-xylosidase gene domain ----------------- -— 120

3. Southern hybridization analysis of Thermoanaerobacter
B6A-RI and E. coli genomic DNA digests with random-
primed DNA synthesized from the 1.2 kb HincII-PstI
fragment from pZEPlZ 122

 

4. Physical and genetic map of the deletion plasmid pZEP12
containing xynA constructed from pXDM1 — ------------------------- 125

5. Nucleotide sequence and translated amino acid sequence
for the endoxylanase A gene from Thermoanaerobacter
B6A-RI 126

 

6. SDS-PAGE analysis of recombinant endoxylanase purification
from E. coli (pZEP12) 136

 

7. The effect of pH on the activity of recombinant endoxylanase
from E. coli (pZEP12) 137

 

8. The effect of temperature on the activity of recombinant
endoxylanase from E. coli (pZEP12) 138

 

9. Time course of hydrolysis of the soluble fraction of oat spelt
xylan and xylooligosaccharides by the recombinant
endoxylanase 139

 

Chapter V

1. Physical map of cosmid clone pXDM1 which contains the
28 kb chromosomal DNA of Thermoanaerobacter strain
BéA-RI expressing the endoxylanase (xynA) and

B—xylosidase (xynB) genes 160

 

2. Physical and genetic map of the deletion plasmid pXPH3

containing the B-xylosidase (xynB) from Thermoanaerobacter
B6A-RI 161

 

3. The nucleotide sequence anddeduced amino acid sequence

of B-xylosidase (xynB) from Thermoanaerobacter B6A-RI
and its flanking region 162

 

4. South
Bea-i
prime
from 1

 

5. EDS-I

B-xy‘u
6. The r

7. The

8. Tim

xvia

B-x
Chapter VI

1. Th.
ins

2. T‘r
of

CL.
“apier I

. Southern hybridization analysis of Thermoanaerobacter

B6A-RI and E. coli genomic DNA digests with random-
primed DNA synthesized from the 1.2 kb HincII-Pstl fragment
from pZEP12 and 0.9 kb SspI-HindIII fragment from pXPH3 ---—— 168

. SDSoPAGE analysis of the purification of recombinant

 

B—xylosidase from E. coli (pXPH3) cells 173

. The pH profile of recombinant B-xylosidase --—-—---— ------------ - 174

. The temperature profile of recombinant B—xylosidase ------------- 175

. Time course of hydrolysis of the soluble fraction of oat spelt

xylan and xylooligosaccharides by the recombinant
B—xylosidase 178

 

Chapter VI

1.

The physical map of pZXI6 containig vector pUC18 with the
insert fragment containing the xylose isomerase gene -------------- 193

. The nucleotide sequence and deduced amino acid sequences

 

of the cloned xylose isomerase 194

. Comparison of the xylose isomerase amino acid sequences ------- 199

. SDS—PAGE analysis of the thermostable Thermoanaerobacter

B6A-RI xylose isomerase expressed in E. coli (pZXIé) ------------- - 204

Chapter VII

1.

The physical map of the DNA fragment containing the
Thermoanaerobacter B6A-RI xynA gene and the deletion
mutants 21

 

. Alignment of the deduced amino acid sequences of

endoxylanase from Thermoanaerobacter B6A-RI with those
of family F B-glycanases 224

 

. Amino acid sequence alignment of the endoxylanase (xynA)

from the Thermoanaerobacter B6A-RI with those of the other
five enzymes in family F B—glycanases 225

 

Chapter VIII

 

A

1. Light
86A-

2. Scan:
Tiit‘fl

xylo:

3. Irar
strai
catic

1. Light microscopy of cells of Thermoanaerobacter strain
B6A-RI grown on (A) xylan, (B) xylose and (C) glucose -—----—---- 245

2. Scanning electron microscopy of gold l;abelled cells of
Thermoanaerobacter strain B6A-RI grown on glucose (a, b),
xylose (c, d), and xylan (e, f) 248

 

3. Transmission electron microscopy of cells Thermoanaerobacter
strain B6A-RI grown on xylan and treated with gold and
cationized ferritin 250

 

xiii

 

RBB-xylan 4

Im
HEPES
SDS
PAGE
G+C
kDa
MOPS
MES
PAS
TLC
CMC
ORP
kb

Mr

5. D.
PNPx
FPLC

I

b

RBB-xylan
T m
HEPES
SDS
PAGE
G+C
kDa
MOPS
MES
PAS
TLC
CMC
ORF
kb

Mr

S. D.

PNPX

EDAC

ABBREVIATIONS

4-O-methyl-O-glucurono-D-xylan-Remazole Brilliant Blue R
midpoint thermal denaturation temperature
N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid
sodium dodecyl sulfate

polyacrylamide gel electrophoresis
guanine-plus-cytosine

kilo-dalton

3-(N-morpholino)propanesulfonic acid
2-(N—morpholino)ethanesulfonic acid

periodic acid, Schiff's reagent

thin layer chromatography

carboxymethyl cellulose

open reading frame

kilo-base

molecular weight

Shine-Dalgarno

p-nitrophenyl-b-D-xylopyranoside

fast performance liquid chromatography

1-(3-dimethylaminopropenyl)-3-ethylcarbodiimide
hydrochloride

 

INTRODUCTION

 

‘ ’ w and composition of lignocellulmic materials

mm years there has been considaabkc interest in the utilization of
h-“ as a 'IP‘U'T': at fuels and chemicals Plant material is composed
“I, of cellulose. hemittlwi'J-a‘ and ilglllz't. For example, when contains
Wiy 2540‘!) cell-41mg, 354.2'2-3 lxemtceliulow and 10-309'0 lrgnin (16).
W19 e linear polymer of glucose molecules linked in the 8-1.4
mama. (”enclave :s ur-‘zam‘fiflo iamma: crystallites which on
W into rm'crolequ'xts .31 the pianr cell wall Each microfibn'l contain
mm of amorphouhm ded intertwined with
Maine. Hernicellulose .:r xylan IS a linen heteropolymcr of xylose.
Mg side chains of 4-( I-nmihvt IJ-giurunm'li‘ Add and tantalum
“Italic linked in the ll-i -l-un".7;gurr19iuh The hernncelltfiuae it in turn
wee-linked t0 lignm which 15 a ‘tmlvzncr m Vinita»“urn-1:70am up“.
“WC-C and C434: linkages.

WW

“Wot hemmlluhm .

M :mlutese is the term gamut to W «out
Widen that occur together with “1C! 9” m.

   
 

l

 

A Structure;

In recer
plant material
mainly of ce‘;
approximatek
Cellulose is
configur ado
bundled int
“3.10% of
hemicenulc
Possessing
“'hiCh is a]
UOSSlinka

C‘mne‘cted

8' Herni
[11 Abu
He
POKySaCC
“Emicel
biomaSS
FOIySaCC
Can be

Xv} ‘
.. an-c

INTRODUCTION

A. Structure and composition of lignocellulosic materials

In recent years there has been considerable interest in the utilization of
plant material as a source of fuels and chemicals. Plant material is composed
mainly of cellulose, hemicellulose and lignin. For example, wheat contains
approximately 25-40% cellulose, 25-50% hemicellulose and 10-30% lignin (16).
Cellulose is a linear polymer of glucose molecules linked in the B—1,4
configurations. Cellulose is organized into laminar crystallites which are
bundled into microfibrils in the plant cell wall. Each microfibril contains
regions of amorphous cellulose interspread and intertwined with
hemicellulose. Hemicellulose or xylan is a linear heteropolymer of xylose,
possessing side chains of 4-O-methyl-D-glucuronic acid and L-arabinose,
which is also linked in the B-1,4-configuration. The hemicellulose is in turn
crosslinked to lignin which is a polymer of p-hydroxyphenylpropane units

connected by C-C and C-O-C linkages.

B. Hemicellulose
[1] Abundance of hemicellulose

Hemicellulose is the term given to low-molecular weight
polysaccharides that occur together with cellulose in plant tissues.
Hemicellulose comprises up to one third of the content of dry cellulosic
biomass (97). After cellulose, it is the next most abundant renewable
polysaccharide in nature. Xylan, a major component of plant hemicellulose,
can be converted to monosaccharides by enzymatic hydrolysis. However,

xylan-degrading enzyme systems have been studied less than the cellulose-

‘

degrading en;
heterogeneous
does. not lead
which result
hemicellulos

from the eff

l2] Struct‘
Cnl‘.

composing

3
degrading enzyme systems. One of the reasons for this might be the

heterogeneous composition of hemicellulose. The hydrolysis of this polymer
does not lead to a single product, differing from the degradation of cellulose,
which results in the formation of glucose. Presently, large amounts of
hemicellulosic materials are wasted or dumped, contributing to pollution

from the effluent of mills in the pulp and paper industry.

[2] Structure and composition of hemicellulose
Unlike cellulose, hemicelluloses show variability in both structure and
composition and are heteropolymers of various hexoses, pentoses and uronic
acids. Most hemicelluloses contain 2 to 6 different sugar residues and are
often classified according to the major carbohydrate backbone, for example, as
glucans, xylans, mannans, galactans and galacturonans (96). Mannans are the
predominant hemicellulose in gymnosperms, whereas xylans predominate

in angiosperms (97).

C. Occurrence, structure and composition of xylans

Xylans are the most abundant noncellulosic polysaccharides in
angiosperms, where they account for 20 to 30% of the dry weight of woody
tissue. In gymnosperms, where galactoglucomannans and glucomannans
form the major hemicelluloses present, xylans are less abundant, accounting
for about 8% of tissue dry weight (2). A substantial proportion of all known
xylans are acidic due to the attachment of 4-O-methyl-D-glucuronic acid
groups. Typical xylans from gymnosperms, for example, contain 14 to 18% 4-
O-methyl-D-glucuronic acid, with low variable levels of L-arabinofuranose

side chains (101).

    
 

B-lpl-Xj“
component 02'
represent the
generally cons
Xylans from
containing n.
branched ar.
occurring in

The ‘
XYlOpyr ano
Xi'bpyrano

(9’5). Figu

e. . .
“81 th

4

[3-1,4-Xylans are mainly found in secondary walls, the major
component of mature cell walls in woody tissue (97). Although they also
represent the major hemicellulose in the primary walls of monocots, xylan
generally constitutes a minor component of the primary wall in dicots (2, 64).
Xylans from monocots are of two types; highly branched arabinoxylans,
containing no glucuronic acid, as found in cereal endosperms, and less-
branched arabinoxylans with uronic acid and galactose side chain types
occurring in more highly lignified tissue (104).

The backbone of xylan consists of a polymer of B-1,4-linked
xylopyranose units. Side chains are linked to the main chain via B-1,6-
xylopyranose, a-1,3—arabinofuranose, or or-1,2-(4-O-Me)-glucosyluronate bonds

(96). Figure 1 shows possible configurations for various xylans.

D. Enzymes involved in the degradation of xylan

Complete breakdown of a branched acetyl xylan requires the action of
several hydrolytic enzymes (Fig. 2). The best known are endo-1,4-B-xy1anase
(xylanase), which attack the polysaccharide backbone, and B-xylosidase, which
hydrolyze xylooligosaccharides to D-xylose. The resulting D-xylose is
isomerized to xylulose by xylose isomerase and further catabolized after
phosphorylation by xylulokinase. The xylanolytic enzymes have recently

been reviewed elsewhere (9, 19, 105).

[1] D-xylanases
D-xylanases have been reported to occur in bacteria, fungi and protozoa
(19). In general, D-xylanases are predominantly extracellular in bacteria and
fungi though some microorganisms, for example, rumen bacteria (15, 40) and

protozoa (3) also have intracellular D-xylanas

A

- X — X — X — X - X -
l Esparto xylan
X - X - X
- X - X ' X ' X ’ X ‘ Xl ' Glucuronoxylan
Ac GP
- X - X - X - )|( - X ' )i ' XI Glucuronoarabinoxylan
-x-x-x-x-x-X-X-X-
I I I l Xylan gums
x - x - x Ar H Gp
| I
H GP

X = [3-1,4-linked D—xylopyranosyl units

Ac = Acetyl
Ar = L—Arabinofuranosyl unit
Gp = D-Glucuronopyranosyl or 4—O-Methyl-D-glucuronopyranosyl unit

H = Hexose unit

Figure 1. Possible configurations for xylans (Adapted from Thompson, 1983).

A

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xvii

 

 

7
D-xylanases are enzymes capable of hydrolyzing the B-1,4-D-

xylopyranosyl linkages of the B-1,4-D-xylans, namely arabinoxylan,
arabinoglucuronoxylan, arabino-4-O-methyl-D-glucuronoxylan and glucuron-
oxylan. D-xylanases of this type are referred to as B—1,4-D-xylan
xylanohydrolase or endoxylanase (EC 3. 2. 1. 8) and B-1,4-D-xylan xylohydro-
lase or exoxylanase (EC 3. 2. 1. 37).

Fungal D-xylanases are potentially of either the exo or endo type,
although the production of exoxylanase has not been unequivocally proven
(19). This enzyme would release xylose monomers by attacking the ends of a
xylan molecule and confusion arises because the same results would be
obtained if the enzyme consisted of a mixture of both B-D-xylosidase and an
endoxylanase. The endoxylanases can be divided into two groups, those that
liberate L-arabinose from the enzymatic hydrolysis of arabinoxylans and
arabinoglucuronoxylans (i.e. arabinose-liberating xylanases) and those that do
not liberate L-arabinose from substrates (i.e. non-arabinose-liberating
xylanases). The cleavage of the arabinose side chains would involve the
enzyme a-L-arabinofuranosidase (EC 3. 2. 1. 55). Endoxylanases attack the
polysaccharide backbone and release xylose, xylobiose and
xylooligosaccharides.

The activity of endoxylanase is determined by incubating the enzyme
with the substrate at a particular pH and temperature and measuring the
release of reducing sugars (xylose). Substrates that may be used for this
purpose include oat spelt xylan, birchwood xylan, larchwood xylan, and
arabinoxylan from rice straw. The definition of the unit of xylanase activity
usually given is the amount of enzyme required to produce 1 umol of

reducing sugar in a specified time under the conditions of the assay.

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Endo-1,4-B-D-xylanases usually have pH optima in the region pH 3.5 to

6.5. The enzymes produced by fungi have pH optima in the range 3.5 to 5.5.
pH optima of bacterial xylanases tend to be around 6.0 (19, 101, 105).
Temperature optimum and temperature stability values may vary
significantly with source (Table 1). Thermostable xylanases have been
reported from a number of microorganisms including Bacillus sp. C—125 (39),
Clostridium acetobutylicum (54), Clostridium stercorarium (5), Caldocellum
saccharolyticum (57), Clostridium thermocellum (33), Streptomyces lividans
(66) and Trichoderma harzianum (93, 94).

The molecular weight of xylanases varies greatly depending on the
microbial source (105). Generally the molecular weights of the xylanases like
those of other hemicellulases, are relatively low, ranging from 15 kDa to 85

kDa (Table 1).

[2] B—D-Xylosidase (EC 3. 2. 1. 37)

B—D-Xylosidase hydrolyzes xylose units from the nonreducing
terminus of D-xylan chains with the products retaining configuration (20, 92).
B-xylosidase activity decreases with increasing xylooligosaccharide chain
length (40, 92). B-xylosidase also have substantial transferase activity (20, 92).
The enzyme is usually assayed colorimetrically since it hydrolyzes p-nitro-B-
D-xylopyranoside to D-xylose and p-nitrophenol. Activity can also be assayed
by detecting cellobiose hydrolysis leading to the formation of glucose. Some
of the microorganisms reported to produce B-D-xylosidase are listed in Table
2.

[3] Other enzymes liberating the side-chain sugars and their synergistic

effect on xylan degradation

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Relatively little is known about the enzymes which liberate the side-
chain sugars, which include a—L-arabinofuranosidase and a—glucuronidase, or
the enzymes involved in the degradation of acetyl xylan, such as acetyl
esterase.

a-L-Arabinofuranosidases have been reviewed by Kaji (44), who
subdivided them into two groups, an Aspergillus niger type of oz-L-
arabinofuranosidase, which can hydrolyze the terminal non-reducing a-L-
arabinofuranoside residues from arabinoxylans, and a Streptomyces
purpurascens type of a—L-arabinofuranosidase, which cannot hydrolyze
terminal non-reducing a—L-arabinofuranoide residues from arabinoxylan.
There are claims that a-L—arabinofuranosidase activity is an inherent property
of some xylanase (19). a-L-Arabinofuranosidases have been purified from
several microorganisms (34, 38, 52) and cooperative interactions between a-L-
arabinofuranosidase and xylanase have been demonstrated (34).

a—Glucuronidase was detected from Agaricus bisporus and Pleurotus
ostreutus. These enzymes act synergistically with xylanases and liberates 4-0
methylglucuronic acid from 4-O-methy1glucuronic acid-substituted
xylooligomers (80).

Esterases that deacetylate acetyl xylan have been reported to occur in
fungi (12, 79) and bacteria, including Fibrobacter succinogenes (63), and
Caldocellum saccharolyticum (56). The fungal esterases act synergistically
with xylanases in the degradation of acetyl xylan. In the absence of esterase,
xylanases break only a limited number of glycosidic bonds. Both the rate and
the extent of breakdown of xylan increases when esterase is present. The two

activities also act synergistially to liberate acetyl residues (10).

 

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[4] Xylose isomerase

Xylose isomerase (EC 5. 3. 1. 5) is an intracellular enzyme that catalyzes
the isomerization reaction between D-xylose and D-xylulose during xylose
metabolism in various microorganisms. Because this enzyme can also
convert D—glucose to D-fructose, the enzyme is often referred to as glucose
isomerase (91). Production of glucose isomerase has been reported from a
large number of bacteria and actinomycetes that can grow on xylose as an
energy source. Among these, Streptomyces species have been studied most
extensively and used as a source of industrial enzymes (90, 91). Most
microorganisms require xylose to induce glucose isomerase production and
enzyme synthesis is catabolite repressed by glucose. Most purified xylose
isomerases have molecular weights ranging from 80,000 to 190,000 and are
composed of 2 or 4 identical subunits (1, 14). The pH optima for different
xylose isomerases lie in the range of 6.5 to 9.5 (14). Temperature optima for
enzyme activity and stability vary greatly depending on the source of xylose
isomerase. Xylose isomerase is a metalloenzyme, and it requires a divalent
cation such as Mn“, Co”, or Mg++ for both catalytic activity and enzyme

stability.

E. Occurrence of multiple xylanases in microorganisms

Multiple xylanases have been reported in several microorganisms
(105). Five different xylanases have been purified and characterized from
Aspergillus niger species (24, 25, 42, 43, 86). The occurrence of multiple
xylanases have also been detected in Clostridium stercorarium (5).
Streptomyces species (51, 60, 67), Trichoderma harzianum (93, 94, 106),

Aeromonas sp. strain 212 (69) and Penicillium janthinellum (92.

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The extent of xylanase multiplicity in microorganisms remains to be

answered, particularly since a zymogram technique has detected five major
and ten minor xylanases in the culture filtrate of Aspergillus niger 14 (11).
The nature and relevance of minor xylanases may not be determined, since
often the major xylanolytic activities are the enzymes purified. Minor
xylanases may have functions that are not required in large quantities such as
hydrolysis of bonds which are not prevalent within the polymer. These
minor xylanases may be produced in larger amounts if the culture conditions
were altered, or they may be lost during the purification procedure either due
to adsorption on to the substrate, or degradation.

Xylan is a heteropolymer and its structure differs from plant to plant.
Therefore the production of multiple xylanases, with each enzyme with
having specialized functions, may be one strategy whereby a microorganism
achieves efficient xylan hydrolysis. However the regulation, the substrate
cross-specificity (87, 98), and the posttranslational modifications of these
enzymes must be considered, because each of these factors can result in the
occurrence of multiple enzymes with xylanolytic activity.

In fungi, some of the multiple xylanases may also be allozymes,
products of different alleles of the same gene. Multiple xylanases may be
proteolytic degradation products or differential glycosylation products (5, 6, 24,
25, 50, 110). In some cases, each of the multiple xylanases may be a distinct
gene product as found with T. harzianum which has three distinct xylanases
(106), Bacillus sp. C-125 with at least two types of xylanases (39), and C.
acetobutylicum with two different endoxylanases which have been
characterized (54).

Multiplicity in B—xylosidase also has been suggested by the detection of

multiple B-xylosidases in A. niger 14 (81), in Trichoderma harziunum E58

gm

15
with two B-xylosidases (106), and the presence of four B-xylosidases from
Penicillium wortmanni IFO 7237 (61).

The complexity of xylan must be considered in questions concerning
xylanase multiplicity. Multiple xylanases and B—xylosidases may be more
effective in the hydrolysis of xylan due to the heterologous nature of this
polymer, as the different forms may also have varying abilities to interact
with other xylanases in xylan hydrolysis. The significance of multiple
xylanases may lie in the hydrolysis of other xylose-containing polysaccharides,
or of secondary substrates. As xylan is important in fiber cohesion, the
primary functions of multiple xylanases may in fact relate to disruption of the
fibers and exposure of the other lignocellulosic components to other
hydrolases. Such functions might relate to debranching and transferase
activity in some xylanases rather than in hydrolysis of xylosidic linkages
alone. In addition the possession of a number of enzymes which catalyze
the same reaction, but which function under different conditions, such as
temperature or pH, may be of great advantage to the organism when
competing for substrates in a changing environment.

F. Regulation of xylanases

In non-cellulolytic bacteria and yeasts, xylanolytic enzymes appear to
be inducible: xylanase and B-xylosidase are produced in high amounts during
growth on xylan and synthesis of the enzyme is catabolite repressed by well
metabolized carbon sources such as glucose or xylose (9). Xylan cannot enter
the cells, so that the signal for accelerated synthesis of xylanolytic enzymes
must involve low molecular weight fragments, such as xylobiose and
xylotriose. The oligosaccharides are formed by hydrolysis of xylan in the

medium by small amounts of enzymes produced constitutively (9).

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16
G. Biotechnology of xylanases

[1] Bioconversion of hemicellulose to chemicals

Bioconversion of lignocellulose to fermentative products requires that
the highest yield of various polymeric sugars is obtained. Current thought
holds that the complete xylanolytic (and cellulolytic) systems are required to
achieve maximum hydrolysis of this complex substrate, to small
oligosaccharides and monomers. For the utilization of hemicellulose, such a
system would require xylanases, B-xylosidases and other hemicellulases. This
could be achieved in a number of ways, either by using the xylanase system of
a very effective xylanolytic microorganism, or alternatively using a
combination of specialized enzymes from different microorganisms, or from
microbes grown under different conditions for enzyme induction. With the
current research into alteration of catalytic activity through the use of site-
directed mutagenesis, protein engineering may provide new enzymes with
unique properties which would facilitate in the bioconversion of

lignocellulose.

[2] Biobleaching

In paper manufacturing, xylanase treatment has been suggested because
it can reduce energy cost of refining pulp (73, 78) and eliminate the formation
of toxic compounds. Multiple xylanases will probably be required to
maximize xylan removal from these pulps (105). The feasibility of using
xylanase for cellulose purification during rayon manufacture, would be
highly dependent on the intrinsic selectivity of the enzymes, the absence of
cellulolytic activities, and the availability of enzyme preparations in
inexpensive bulk quantities (105). One strategy for obtaining cellulase-free

xylanase is the isolation of xylanolytic microorganisms which are free of

{I

are

mic

17
cellulases. This could be achieved by one of two means, the isolation of

cellulase deficient mutant strains, or alternatively another strategy would be
the cloning of xylanase genes into noncellulolytic organisms such as
Escherichia coli which has been achieved from numerous organisms (7, 50,
65, 75, 83, 88, 111). This could result in the overexpression of the enzyme and
excretion from the cell, which would be advantageous due to the ease of

working with cell-free systems.

[3] Other applications of xylanases

Along with cellulases and pectinases, the use of xylanase has also been
suggested in applications such as the clarification of juices (9, 18), the
preparation of dextrans for use as food thickeners (96), and the production of
fluids and juices from plant materials (9, 107). Also there are some
applications for xylanases in the baking of high fiber breads to enhance rising
and water retention for the manufacture of low calorie bread (58), and for the
pretreatment of animal feed to enhance fiber utilization and reduce manure

production.

[4] Thermostable xylanases for industrial processes.

The efficient use of enzymes in many industrial processes requires that
the overall amount of substrate converted per unit quantity of enzyme is
maximized. Reaction temperature is an important physical factor that can be
optimized. For every 10°C increase in temperature, reaction rates
approximately double. Therefore, using thermostable enzymes, the amount
-of enzyme required can be reduced, or the conversion time shortened. There
are a number of advantages of running processes at elevated temperatures,

microbial contamination can be reduced at higher temperatures, and

an-

h-
in

in:

18
solubility of substrates can be increased. Thermophilic organisms are good

sources of thermostable enzymes and recombinant DNA technology provides
the opportunity for overproduction of these enzymes by cloning of the genes
into mesophilic hosts, with the production of enzymes which usually retain
thermostability and can be purified almost to homogeneity in a one step heat
treatment. Protein engineering techniques such as site-directed mutagenesis
can also produce catalytically more efficient enzymes or enzymes specific in

activity, for industrial applications.

H. Molecular biology of xylan degradation
[1] Cloning of the genes involved in xylan degradation

Recombinant DNA technology offers opportunities for the isolation
and molecular characterization of individual genes encoding for xylanolytic
enzymes. Cloning of hemicellulases could facilitate xylan bioconversion by
introducing a number of different enzymes into one organism to provide
numerous activities required to degrade this heterologous substrate, creating
a hemicellulolytic enzyme system. Such a system could be introduced into an
industrially important xylose-fermenting organism so that the direct
fermentation of xylan would be possible.

All the cloning work published so far has been restricted to bacterial
genes. Several xylanases from Bacillus sp. have been cloned and expressed in
E. coli (108, 109). Xylanases from Aeromonas sp. (50), Bacteroides spp. (88,
102, 103), Butyrivibrio fibrisolvens (55, 59), Fibrobacter succinogenes (41),

Ruminococcus ," f MN (22), (‘Pllu’c-“nas sp. (8) and Clostridium sp. (23,

 

 

111) were also cloned and characterized. Relatively few xylanases have been

cloned from thermophilic organisms (28, 57).

 

19
In comparison to the studies on xylanases there have been only a few

reports on the cloning of B—xylosidase genes. Two B-xylosidase genes were
isolated from Bacillus pumilus (76) and cloned and expressed in E. coli. B-
Xylosidases from Butyrivibrio fibrisolvens (85) and from Caldocellum

saccharolytin (57) were also cloned and expressed in E. coli.

[2] Domains in xylanases

Cellulases and xylanases hydrolyze B-1,4-glycosidic bonds between
pyranose units, but they show subtle differences in specificity. Structural
features common to such enzymes may be related to their general catalytic
activities as glycosidases. Those peculiar to each enzyme may be related to
their specificities, i.e., exoglycanase or endoglycanase, cellulase or xylanase.
Such features should be reflected in the amino acid sequences of the enzymes.
Cellulases contain two distinct functional domains, a catalytic domain (CD)
and a cellulose-binding domain (CBD) (4, 29, 71), but the existence of similar
domains in xylanases remains to be elucidated.

Cellulases and xylanases can be grouped into nine families of related
enzymes on the basis of amino acid sequence identities in their putative
catalytic domains (4, 37, 49) and hydrophobic cluster analysis (30). Most
xylanases belong to either family F or family G. Family F xylanases are
usually large molecular weight proteins and some of them have cellulase
activity. Family F 81,4-glycanases include xynA from Bacillus sp. strain C-125
(36), xynA from Butyrivibrio fibrisolvens (59), CelB, xynA and ORF4 from
Caldocellum saccharolyticum (57, 84), cex from Cellulomonas fimi (70), xynZ
from Clostridium thermocellum (33), xyn from Cryptococcus albidus (13),
xynA and xynB from Pseudomonas fluorescens subsp. cellulosa (35, 46) and

xyn from Thermoascus aurantiacus (89). Family G xylanases are small

nc
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an
Sig

XV

en.
the

alt

20
molecular weight enzymes and include xyn from Bacillus circulans (109),

xynA from B. pumilus (27), xyn from B. subtilis (72) and xynB from
Clostridium acetobutylicum (112).

It seems that various 8-1,4-glycanases arose from a few progenitor
sequences by mutation and domain shuffling. In this regard, it should be
noted that some enzymes show a mixed specificity in that enzymes that
hydrolyze B-1,4 bonds in cellulosic substrates may also hydrolyze xylan, chitin,
and related substrates. This could in part provide an explanation as to the
significant homology in the putative catalytic domains of cellulases and

xylanases.

1. Objectives

This research was undertaken because little is known about the
physiological, biochemical and molecular genetic features of xylanolytic
enzymes from thermophilic bacteria that are known to produce intrinsically
thermostable and thermoactive enzymes (e.g., amylase, cellulase, protease, or
alcohol dehydrogenase). '

In order to elucidate the molecular features of xylanases produced by
thermoanaerobes, their physiological function and compartmentalization,
their biochemical mode of action, and molecular characteristics of the genes
involved in xylan degradation, the following major research objectives were
undertaken:

(i) Isolation and general characterization of xylanolytic microorganisms for
selection of a model thermoanaerobe.
(ii) Physiological characterization and regulation of xylanase activity in a

model organism.

 

21
(iii) Purification and biochemical characterization of thermophilic

endoxylanase, B-xylosidase and xylose isomerase activities.

(iv) Cloning, organization, sequencing, and expression of endoxylanase, B-
xylosidase and xylose isomerase genes.

(v) Identification of the catalytic and thermostability domains of
endoxylanase.

(vi) ultrastructural studies to characterize any cell surface features involved

in xylan adhesion and hydrolysis.

10.

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.13

~10

3S

n
U

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all

1.1

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25

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Chapter II
Deoxyribonucleic acid homology and other comparisons

among xylanase producing thermoanaerobic bacteria,

with a proposal for new taxonomic designations

33

an

ch

Tl

ABSTRACT

Two thermophilic, anaerobic xylan degrading bacteria, BéA-RI and LX-
11 were isolated from Frying Pan Springs in Yellowstone National Park. The
organisms grew chemoorganotrophically utilizing xylan and starch, but not
cellulose, and a number of di- and monosaccharides including glucose and
xylose. Both organisms had the same optimum temperature and pH for
growth of 60°C and pH 6.0 respectively. Fermentation products included
acetate, ethanol, lactate, C02 and H2. Both organisms grew as rods, although
B6A-RI formed longer cells than LX—11, and both deposited sulfur on the cells.
The major difference between the two organisms was in spore formation, LX-
11 was found to sporulate whereas B6A-RI was not. Comparison of LX-11 and
B6A-RI was made with other thermophilic anaerobic xylan degrading bacteria
in respect to DNAzDNA hybridization and total protein analysis in order to
determine the relatedness of these organisms. From these findings three
different groups emerged and new taxonomic assignments were proposed.
Clostridium thermocellum LQRI was least related .to the other seven strains
and is placed in group I retaining its original taxonomic assignment without
change. Clostridium thermosulfurogenes strain 48 and the new isolates B6A-
RI and LX-ll are closely related and fall into group II for which a genus name
Thermoanaerobacterium gen. nov. is given. Isolate LX-11 is given a
taxonomic assignment as Thermoanaerobacterium xylanolyticum sp. nov.
Isolate B6A-RI is named and classified as Thermoanaerobacterium
saccharolyticum sp. nov., and Thermoanaerobacterium thermosulfurigenes
strain 4B (original C. thermosulfurogenes) is the type species for the genus.
Group III is termed Thermoanaerobacter and includes Thermoanaerobacter

ethanolicus strain JW200, Clostridium thermohydrosulfuricum strain 39B

34

3 5
and strain E100-69, and Thermoanaerobium brockii strain HTD4. T. brockii

strain HTD4 is renamed as Thermoanaerobacter brockii comb. nov. and C.
thermohydrosulfuricum E100-69 is renamed as Thermoanaerobacter
thermohydrosulfuricus comb. nov. C. thermohydrosulfuricum strain 39B is
nearly identical to T. ethanolicus strain JW200 and are considered to belong to
the same species. Therefore, strain 39B is renamed as Thermoanaerobacter

ethanolicus strain 39E, with T. ethanolicus strain JW200 as the type strain.

lniti
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Thai
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laxm

INTRODUCTION

In recent years, understanding of the biology of thermophilic
microorganisms has been greatly advanced. Thermophilic bacteria, which are
found in both the Archaebacteria and the Eubacteria kingdoms, have been
found in association with all types of thermal habitats including hot springs
(3). Intensive studies on the biology of anaerobic thermophiles have been
initiated in the last fifteen years in order to learn more about their function in
nature, and because of their potential applications in biotechnology (3, 35).
Most studies on eubacterial themioanaerobes are focused on saccharolytic
bacteria that form ethanol and lactate because of their importance in
carbohydrate turnover in thermal environments and because of the utility of
their thermostable enzymes in industrial processes (12). Saccharolytic
thermoanaerobic eubacteria include species in the genus
Thermoanaerobacter, Thermoanaerobium and Clostridium. Although
several strains in these groups have been isolated and characterized, the
taxonomy of these groups is uncertain and remains in a state of confusion.

Little is known on the microbial transformation of hemicellulose in
thermoanaerobic habitats. Wiegel et a1. (31, 34) studied a variety of
thermophilic, anaerobic, saccharolytic bacteria, including
Thermoanaerobacter ethanolicus, Thermobacteroides acetoethylicus,
Thermoanaerobium brockii, and Clostridium thermocellum, which all
ferment xylan albeit at a very slow rate. Studies were also conducted on
Thermoanaerobacter strain 86A, an organism isolated from an algal mat
present in Big Spring, Thermopolis, Wyoming (29), which was shown to
extensively degrade xylan (28). Xylan, a major polysaccharide in

hemicellulose, can be hydrolyzed by microbial enzymes to xylose (2).

36

an
re}

1.
Du;

Str-

D.\

res<

3 7
Thermostable xylanolytic enzymes have potential applications in the pulp

and paper industry (21, 22), and so thermophilic microorganisms have
attracted considerable attention as sources of thermostable xylanases. In the
present study we describe the general morphological, cellular and metabolic
characteristics of two new xylanolytic species that were isolated from Frying
Pan Springs in Yellowstone National Park, Wyoming by selective enrichment
with xylan as the energy source for growth. This site was chosen because of its
low pH (below pH 6.0) as we were interested in isolating xylan degrading
anaerobes which grow at low pH and may have industrial importance. We
also compare these strains with other described thermophilic saccharolytic
anaerobes which have xylanolytic activity to investigate the taxonomic
relationship between these organisms. These strains include Thermoanaero-
bacter ethanolicus (32), Thermoanaerobium brockii HTD4 (37), Clostridium
thermocellum LQRI (13), Clostridium thermohydrosulfuricum E100-69, 39E
(7, 36), Clostridium thermosulfurogenes 4B (25) and Thermoanaerobacter
strain 86A (29).

Although morphological and biochemical characteristics are useful,
DNA-DNA hybridization techniques have been especially successful in
resolving taxonomic relatedness between closely related organisms (species
level and below). In this study we performed DNA-DNA hybridization and
total protein analysis to investigate the taxonomic relationships of xylanolytic
thermoanaerobic species. The results of our protein profile and DNA-DNA
relatedness studies indicate that there are three independent groups in
saccharolytic thermoanaerobes and these results are supported by biochemical
and physiological data, as well as partial 16S-rRNA cataloging studies of
Bateson et a1. (1). On the basis of our results, we propose new taxonomic

assignments for these organisms. In addition, we propose that

38
Thermoanaerobacter and Thermoanaerobacterium, like Clostridium be

recognized as genera which can be comprised of sporeforming species.

Ba

lso

MATERIALS AND METHODS

Bacterial strains and culture conditions

The strains used in the present study are listed in Table 1. Stringent
anaerobic culture techniques described previously (36) were employed for
medium preparation and cultivation of organisms. Cultures were grown in
crimp-sealed anaerobic tubes (Bellco Glass, Vineland, NJ) that contained 10
ml of medium with 0.5% substrate and N2 gas headspace. Mass cultures to
obtain cells for DNA isolation were carried out in a 1 L round bottom flask
that contained 500 ml medium under a N2 gas phase. Clostridium
thermocellum LQRI was grown on G8 medium (20) with 0.5% cellobiose. All
other strains were grown on yeast extract (TYE) medium (36) with either
glucose or xylose (0.5%) as a substrate. CM5 medium (29) without yeast extract
but with vitamin solution and glucose was used to induce spore formation of
strain LX-ll. All cultures were incubated at 60°C without shaking. Xylanase
activity was detected by the addition of the chromogenic substrate 4-O-methyl-
O-glucurono-D-xylan-Remazol Brilliant Blue R (RBB) (Sigma Chemical Co.,

St. Louis, Mo.) to agar medium.

Isolation and enrichment

Sediments from the Frying Pan thermal acid spring in Yellowstone
National Park was used for enrichment of xylanolytic thermoanaerobes.
After several transfers in TYE medium with 0.5% oat spelt xylan, the
enrichment culture was plated onto homologous media containing agar at
2.5% and with RBB-xylan (0.2%) as substrate, in an anaerobic glove box (Coy
Lab Products, Ann Arbor, Mich). The plates were incubated anaerobically at

60°C using a modified paint tank (8). After 3 days, the colonies which gave a

39

150'

4“,
bit

40

TABLE 1. Strains used in this study.

Name as Received Strain Other Strain Source Reference(s)
Designationsa

Clostridium ATCC 35609 farm soil 13

thermocellu m LQRI DSM 2360 ‘ '

Isolate B6A-RI ATCC 49915 thermal spring This
DSM 7060 study

Isolate LX-11 ATCC 49914 thermal spring This

study

Thermoa naerobacter B6A None thermal spring 29

Clostridium ATCC 33743

thermosulfurogenes 4B DSM 2229 thermal spring 25

Thermoanaerobium ATCC 33075

brockii HTD4 DSM 1457 thermal spring 37

Thermoanaerobacter ATCC 31550T

ethanol icus JW200 DSM 2246T thermal spring 32

Clostridium
thermohydrosulfuricum 39E ATCC 33223 thermal spring 36

Clostridium
thermohydrosulfuricum E100-697 ATCC 35045T
DSM 56717 farm soil 7

a ATCC, American Type Culture Collection, Rockville, Md.; DSM, Deutsche
Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Federal
Republic of Germany; T = type strain.

4 1
clear halo were picked from plates and placed into liquid media tubes in the

anaerobic glove box.

Morphological and biochemical studies

Bacterial morphology and sulfur formation were investigated using a
phase-contrast microscope model BHS (Olympus). Agar-coated glass slides
(23) were used for photomicroscopy. To prepare specimens for electron
microscopy, cells were harvested by centrifugation at 5,000 X g for 10 min and
fixed overnight in 2.5% glutaraldehyde in 0.1 M NazHPO4-KH2PO4 (pH 7.2)
buffer solution at 4°C. The cells were embedded in agar and, after a buffer
wash, postfixed for 1 h at room temperature in 1% 0504 in the same buffer
described above. Cells were then dehydrated through an ethanol series,
treated with propylene oxide, and embedded in either Poly/ Bed 812
(Polysciences, Inc., Warrington, Pa.) or VCD/HXSA (Ladd Research
Industries, Inc., Burlington, Vt.) epoxy resin. Thin sections were cut with a
diamond knife mounted on an Ultratome III (LKB Instruments, Inc.,
Rockville, Md.), stained with uranyl acetate and lead citrate, and examined
with an electron microscope (CM-10; Philips Electronic Instruments, Co.,
Mahwah, N.J.). Growth was determined by measuring the increase in
turbidity at 660 nm with a spectronic 20 spectrophotometer (Bausch 6: Lomb,
Rochester, N.Y.). Fermentation products were measured directly in liquid or
gas samples removed from the culture tubes by gas chromatography as
described previously (6). Routine biochemical tests were performed by

standard methods of Smibert 8: Krieg (27).

Preparation of high molecular weight DNA

The cells were harvested at the early exponential phase of growth and

4 2
washed with saline-EDTA (0.15 M NaCl, 1 mM EDTA, pH 8.0). DNA was

isolated and purified by the method of Marmur (18). The purity of the DNA
was tested by spectrophotometry from the ratio of A260 to A280.

Determination of DNA base compositions

The midpoint thermal denaturation temperatures (Tm) were
determined as described by Mandel & Marmur (17) with a Gilford
ResponseTM spectrophotometer equipped with a thermoprogram (Gilford
Instrument Laboratories, Inc., Oberlin, Oh.) The temperature was raised
0.5°C/ min, starting at 70°C, and denaturation was monitored at 260 nm. The
guanine-plus-cytosine (G+C) contents were calculated by using the
thermoprogram and the equation described by Mandel & Marmur (17). DNA
of Escherichia coli B (Sigma Chemical Co.) (57.0 mol%G+C) was used as the

reference.

DNA-DNA hybridizations

DN As were labeled by nick translation with a commercial kit (Bethesda
Research Laboratories, Gaithersburg, Md.), using [1',2',5-3H]dCTP (NEN). The
average specific activity obtained with this procedure was 5 x 105 cpm/ug of
DNA. The labeled DNAs were denatured before hybridization by being
heated at 100°C for 5 min and then placed on ice. The S] nuclease procedure
described by Johnson (9) was used for the DNA homology experiments. Each
reassociation reaction mixture contained 10 ul of heat denatured labeled DNA
(0.03 to 0.05 pg), 50 ul of denatured unlabeled DNA (20 pg), 25 ul of 5.28 M
NaCl in 1 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic
acid) (pH 7.0), and 25 ul of deionized formamide. The mixtures were

reassociated at 53°C (25°C below the thermal melting point in this buffer

syfie:
Ines;
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nun:

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4 3
system) for 24 h. The Sl-resistant duplexes and 50 ug of yeast t-RNA were co-

precipitated by adding 10% trichloroacetic acid and collected on glass fiber
filters (Whatman GF / C). Radioactivity was measured with a Packard gamma
scintillation counter. Sheared salmon sperm DNA was hybridized against
radiolabeled DNA in each experiment to determine the background level of

reannealing of the test DNA.

Polyacrylamide gel electrophoresis of soluble proteins

For total cellular protein analysis, 10 ml of cultures of the late
exponential growth phase of cultures were harvested by centrifugation at
4,000 X g for 15 min. The cells were washed with saline-EDTA (pH 7.0) and
were suspended in 0.3 to 0.5 ml of deionized water and sonicated on ice for 5
min with an ultrasonicator (Kontes, Vineland, N.J.). The cell debris were
removed by centrifugation (15,000 X g) for 5 min and the protein
concentration of the supernatant determined by the method of Lowry et a1.
(16); 50 ug of protein from each strain was used as a sample for gel
electrophoresis. SDS-polyacrylamide gel electrophoresis (PAGE) by the
discontinuous buffer system of Laemmli (11) was performed with a model
Protean 11 vertical slab gel unit (Bio-Rad). The separating gel contained
acrylamide at a final concentration of 7.5% and the stacking gel contained 4%
acrylamide. The gel was stained with Coomassie blue (0.125% Commassie
blue R—250, 50% methanol, 10% acetic acid) and destained in methanol-acetic

acid-distilled water (4:1:5 vol / vol).

Enzyme assays

Endoxylanase activity was determined by incubating a 1 ml reaction

mixture containing 1% oat spelt xylan (Sigma Chemical Co.) and enzyme

4 4
solution in 50 mM acetate buffer (pH 5.5) for 1 hr at 65°C. The reducing sugar

formed was determined by the dinitrosalicylic acid method (19) by using D-

xylose as standard. One unit of xylanase was defined as the activity releasing 1

umol of xylose in 1 min.

RESULTS

Enrichment and isolation

Frying Pan Springs in Yellowstone National Park are located in the
Sylvan Springs thermal area, and are small, shallow hydrothermal areas.
Samples were taken during the summer and at this time the pH of these
springs ranged from 4.5 to 6.0 and the temperature was around 60°C.
Collection was made by passing a jar close along the surface of the bottom of
the spring and collecting the sediment. Enrichments for thermophilic
anaerobic xylan utilizing bacteria were made by subculturing in TYE medium
containing xylan and transferring onto agar plates, where single colonies were
picked to obtain isolates in pure culture. The two isolates studied in more

detail were LX-ll and B6A-RI.

Morphology and ultrastructure

From the light microscope, morphological differences between LX-11
and B6A-RI were very apparent. The former organism forms short rods,
either singly or in pairs (Fig. 1A), whereas B6A-RI is composed of much
longer rods which occur in chains of varying length (Fig. 18). Both strains
utilize thiosulfate as an electron acceptor and convert thiosulfate to elemental
sulfur which is deposited on the cell and in the medium (Fig. 1A and 18).
The organisms also differ in their growing patterns. In stationary phase, cells
of B6A-RI become more elongated and tend to aggregate and eventually settle
to the bottom of culture vessels; occurrences which are not observed with LX-
11. The most striking morphological difference is that LX-11 forms spores
(Fig. 2) whereas despite numerous attempts under various conditions, B6A-

RI has not been found to sporulate. Approximately 20% of LX-11 cells contain

45

46

Figure 1. Phase-contrast photomicrographs of sulphur-depositing
cultures of Thermoanaerobacter strain LX-11 (A) and strain B6A-RI
(B) grown on LPBB, 0.5% (w/v) glucose, 0.1% (w/v) yeast extract
and 20 mM Na28203.

Note that phase-bright sulfur accumulates in the medium

and on the cells. Bar = 3 um.

47

 

48

Figure 2. Electron micrograph of an endospore of Thermoanaerobacter
strain LX-11 in thin section.
The exosporium (EX) surrounds the spore coat, which encloses

the cortex (CX) and the cell wall (CW). Bar = 0.2 pm.

49

 

5 0
spores, which are located in the terminal part of the cell. The mature spores

always remained within the mother cell, rather than free in solution.

Metabolism

In order to assist in the taxonomic assignment of B6A-RI and LX-11,
their characteristics were compared with those of other thermoanaerobes. The
organisms selected are listed in Table 1. LX-11, B6A-RI, and 86A utilized the
same range of substrates, growing well on xylan and starch but unable to
degrade cellulose (Table 2). Growth occurred on a number of disaccharides
including maltose, lactose, sucrose and cellobiose, and of the
monosaccharides tested, glucose, xylose, galactose, mannose, fructose and the
sugar alcohol mannitol supported growth (Table 2). The fermentation
products formed by LX-ll and B6A-RI included acetate and ethanol in
approximately equal ratios and hydrogen and carbon dioxide (results not
shown). Substrate utilization by the other thermoanaerobic strains were
similar to B6A-RI and LX-11 with the exception of C. thermosulfurogenes 4B
which could not utilize lactose, and T. ethanolicus JW200 which could not
ferment mannitol. The metabolic properties of strain B6A-RI and
Thermoanaerobacter strain 86A were identical. C. thermocellum LQRI was
the only cellulolytic species among this group of bacteria.

Growth of LX-11 and B6A-RI occurred on xylan, and xylanase activities
were determined and compared with other xylan utilizing thermoanaerobic
bacteria (Table 3). B6A-RI and LX-11 produced high levels of xylanase during
growth on xylose, in comparison with the other organisms, with the
exception of C. thermocellum LQRI which has active xylanase along with an

active cellulase system.

51

TABLE 2. Comparison of substrate utilization by thermoanaerobes.

Substrate B6A-RIa LX-11b B6AC Iwzood 39138 Eioof HTD48 43h

Glucose + + + + + + + +
Maltose + + + + + + + +
Xylan + + + + + + + +
Starch + + + + + + + +
Xylose + + + + + + + +
Lactose + + + + ND ND + +
Galactose + + + + ND + ND 4-
Sucrose + + + + + + + +
Cellulose - - - - - - - -
Mannitol + + + - ND ND ND +
Cellobiose + + + + + + + +
Fructose + + + + ND ND ND ND
Mannose + N D + + ND + ND ND

aIsolate B6A-RI (this study).

bIsolate LX-11 (this study).

cThermoanaerobacter strain 86A (14, 24, 29).
dThermoanaerobacter ethanolicus strain JW 200 (32).
eClostridium thermohydrosulfuricum strain 39E (36).
1{C thermohydrosulfuricum strain E100—69 (neotype) (7).
8Thermoanaerobium brockii strain HTD4 (37).

hClostridium thermosulfurogenes 4B (25).
ND = Not determined.

52

TABLE 3. Comparison of xylanase activity from various thermoanaerobes.

Strains Growth Final Activity Protein Specific
(A560) pH (U/ ml) conc. Activity
(mg/ ml) (U / mg)

B6A-RI 1 .40 5.1 0.108 1.66 0.065
LX-11 1.30 4.2 0.067 2.00 0.033
39E 1 .15 5.8 0.029 1 .98 0.015
48 1.05 5.5 0.023 2.25 0.010
IW 200 1.25 5.9 0.017 2.99 0.006
HTD4 0.85 5.5 0.006 2.40 0.003
E100 1.02 5.1 0.005 1.85 0.003
LQRI 0.92 6.5 0.206 1 .50 0.137

B6A-RI = New isolate.

LX-11 = New isolate.

39E = Clostridium thermohydrosulfuricum strain 39E.

48 = Clostridium thermosulfurogenes strain 48.

JW 200 = Thermoanaerobacter ethanolicus strain JW 200.
HTD4 = Thermoanaerobium brockii strain HTD4.

E100 = C. thermohydrosulfuricum strain E100-69 (neotype).

LQRI = C. thermocellum strain LQRI.

The strains were cultivated in TYE media containing 0.5% xylose except C.
thermocellum (GS medium containing 0.5% cellobiose). Xylanase activity
was assayed by incubating the enzyme at 65°C for 30 min. in sodium acetate
buffer (50 mM, pH 5,5) contining 1% (w/v) xylan (oat spelt, Sigma). The
reducing sugar formed was determined by dinitrosalicyclic acid (DNS)
method. One unit of xylanase activity is defined as the amount of enzyme
that released 1 umol of xylose equivalent per minute.

5 3
Molecular taxonomic analyses

In order to characterize strain LX-11 and B6A-RI further, and to
compare these two new isolates with other xylanolytic thermoanaerobic
eubacteria, further studies were made by analyzing G+C content, DNA-DNA

hybridization and total protein content.

DNA base composition

The G+C content of both of the new isolates BéA-RI and LX-11 was 36
mol% (Table 4). This was the same for C. thermosulfurogenes 4B and C.
thermohydrosulfuricum E100-69. For the other organisms the G+C content
was very similar ranging from 34 to 35 mol%, with the exception of C.

thermocellum LQRI with a G+C content of 43 mol% (Table 4).

DNA-DNA hybridization

In order to determine the relatedness of these xylanolytic
thermoanaerobes DNA—DNA hybridization studies were performed (Table 4).
The results clearly show that there is little relatedness between C.
thermocellum LQRI and the other thermoanaerobes, as the relative amount
of DNA-DNA homology was 13% or lower, thus C. thermocellum LQRI was
placed in a separate group. The high level of binding within the remaining
organisms gave rise to two other groups. Significant similarity was formed
between LX-11 and C. thermosulfurogenes 48, with 89% homology which
together with the new isolate B6A-RI formed group II. C .
thermohydrosulfuricum 39E and T. ethanolicus JW200 were very similar,
with 96% homology, and formed group HI together with T. brockii HTD4 and
C. thermohydrosulfuricum E100-69.

54

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5 5
Electrophoretic analysis of whole-cell proteins

Cellular protein patterns obtained by gel electrophoresis are useful for
differentiation of species and have a strong relationship to the degree of
DNA-DNA homology. To get comparable protein patterns for all strains, the
organisms were cultivated in the same medium (TYE medium) except for C.
thermocell um (GS medium) and were harvested at the end of the
exponential growth phase. The soluble protein patterns of the eight strains
are shown in Fig. 3. A considerable difference in protein banding patterns
was evident between C. thermocellum LQRI and the other organisms.
Proteins from the new isolates B6A-RI and LX-ll and C. thermosulfurogenes
48 were all similar, as were proteins from T. brockii HTD4, T. ethanolicus

JW200 and C. thermohydrosulfuricum 39B and E100-69.

 

 

A B C D E F G H
-- 200
_~-— -— ——-~_. 116
93—_=:: __ ~97
66 " -..=“_. ‘22—" 66
“- ‘4 --
.3 --
4s 33:. Iii-r 4s
32 —- -
21 ’
14 I

Figure 3. Electrophoretic comparison of cellular proteins from

thermoanaerobic strains that display xylanase activity.

Lane A, Clostridium thermocellum LQRI; lane B, Thermoanaerobacter
strain B6A-RI; lane C, new isolate strain LX-ll; lane D, Clostridium
thermosulfurogenes 4B; lane E, Thermoanaerobium brockii HTD4; lane F,
Thermoanaerobacter ethanolicus JW200; lane G, Clostridium
thermohydrosulfuricum 39E; lane H, Clostridium thermohydrosulfuricum
E100-69. Numbers indicate the size (kDa) of the molecular weight markers.

DISCUSSION

This study describes two new thermophilic anaerobic bacteria, B6A-RI
and LX-ll isolated from Frying Pan Springs in Yellowstone National Park. A
comparison was made of these organisms with other xylanolytic
thermoanaerobes in order to address their taxonomic affiliation.
Traditionally, morphological and biochemical characteristics have been
considered important in the classification of bacteria. At present, spore
formation is considered a valuable taxonomic criterion to distinguish
between organisms, but often sporulation can only be observed under very
specialized laboratory conditions and is often misleading for taxonomic
assessment of anaerobic bacteria as some organisms originally thought to be
non-sporeformers have been found to sporulate (15).

Although classification of bacteria based on similarities in phenotypic
characteristics has been successful in the past, this approach is not precise
enough for differentiating between superficially similar organisms or for
determining phylogenetic relationships among the bacteria within a group.
More and more organisms are being classified based on differences at the
molecular level with respect to G+C content, 16S ribosomal RNA sequences
and DNA-DNA hybridization, all characteristics which cannot be influenced
by conditions of growth in the laboratory.

In this present paper we have compared the new isolates B6A-RI and
LX-ll with other thermophilic, xylanolytic anaerobes. Strain B6A-RI
appeared identical in metabolic and morphological properties to
Thermoanaerobacter strain 86A (29) but differed from strain LX-ll which
formed spores. The physiological and biochemical properties of these

saccharolytic thermoanaerobic strains indicated that the current taxonomic

57

 

 

The

5 8
assignments and nomenclatures of these strains based on phenotypes were

not adequate and should be provided with new taxonomic assignments.
Thus, it is intended here to re-examine the taxonomic position of xylanolytic
Clostridium species in comparison with Thermoanaerobacter and
Thermoanaerobium species based on differences at the molecular level.

The G+C values of all the organisms were very similar and did not
allow for group separation. Based on DNA-DNA homology data, protein
banding patterns and metabolic studies, we place the strains under study in 3
different groups and propose new taxonomic assignments in which
sporeformers and non-sporeformers are placed in the same genus and species
(Table 5). Clostridium thermocellum strain LQRI stands out as an organism
that is least related to the other 7 strains. This culture forms group I and it is
suggested that it retains its original taxonomic assignment without any
change.

Clostridium thermosulfurogenes strain 48 and the new xylanolytic
isolates LX-ll and B6A-RI are closely related and fall in group II for which a
genus name Thermoanaerobacterium gen. nov. is given. Strain LX-ll is
given a taxonomic assignment as Thermoanaerobacterium xylanolyticum sp.
nov. due to its xylanolytic properties. Strain B6A-RI is named and classified
as Thermoanaerobacterium saccharolyticum sp. nov. Both strain B6A-RI and
Thermoanaerobacter strain 36A behave like true saccharolytic strains
producing amylases while growing on starch as well as on other substrates
(14, 24). Thermoanaerobacterium thermosulfurigenes strain 48 is the type
species for the genus Thermoanaerobacterium.

Group III includes Thermoanaerobacter ethanolicus strain IWZOO,

Clostridium thermohydrosulfuricum strain 39B and strain E100-69, and

Thermoanaerobium brockii strain HTD4 due to their close relatedness. This

 

59

 

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60
group is termed Thermoanaerobacter. Thermoanaerobium brockii strain

HTD4 is renamed Thermoanaerobacter brockii comb. nov., Clostridium
thermohydrosulfuricum strain E100-69 is renamed as Thermoanaerobacter
thermohydrosulfuricus comb. nov. C. thermohydrosulfuricum strain 39B
described by Zeikus et al., (36) is nearly identical to T. ethanolicus strain
JW200 and, thus both strains are considered to belong to the same species.
Perhaps, T. ethanolicus strain IWZOO is an asporogenous mutant of strain 3912.
Therefore, strain 39B is renamed as Thermoanaerobacter ethanolicus strain
39B, and Thermoanaerobacter ethanolicus strain IWZOO is the type strain. A
taxonomic key for preliminary identification of saccharolytic, ethanologenic
thermoanaerobes is outlined in Table 6.

This proposed taxonomic arrangement of these organisms is supported
by previous studies by other workers. I. Wiegel first suggested that T. brockii,
T. ethanolicus, and C.“ thermohydrosulfuricum 3913 could be close taxonomic
relatives because they display a similar substrate range and temperature range
for growth and a biphasic growth curve (30). Kondratieva et al. (10)
recognized the similarity of Thermoanaerobium lactoethylicum to both T.
brockii and T. ethanolicus and suggested that the genus name
Thermoanaerobium, and not Thermoanaerobacter, be used for these species.
Bateson et al. (1) have reported that the 16S rRNA sequences of T. brockii, T.
ethanolicus and C. thermohydrosulfuricum were nearly identical but
significantly different from C. thermosulfurogenes. This is supported by 165
rRNA cataloging studies of Cato and Stackebrandt (4) in which it was found
that sporeforming Clostridia do not form one phylogenetic homologous
family but six sublines which embrace both sporeforming and non-
sporeforming species as well.

The genera Thermoanaerobacter (32) and Thermoanaerobium (37)

61

TABLE 6. Taxonomic key for preliminary identification of saccharolytic,
ethanologenic thermoanaerobes.
A. Does not reduce thiosulfate, ferments cellulose.

1. Clostridium thermocellum

B. Does reduce thiosulfate, does not ferment cellulose.

1. Reduces thiosulfate to elemental sulfur
Thermoanaerobacterium thermosulfurigenes
Thermoanderobacterium saccharolyticum
Thermoanaerobacterium xylanolyticum

2. Reduces thiosulfate to H28
Thermoanaerobacter brockii
Thermoanaerobacter ethanolicus

Thermoanaerobacter thermohydrosulfuricus

6 2
were used to describe the first thermophilic anaerobic rod-shaped, Gram-

positive, non-sporeforming bacteria that produce ethanol and lactate as
principal saccharide fermentation products. Thermoanaerobacter is listed
under irregular, non-sporeforming Gram-positive rods in Bergey's Manual of
Systematic Bacteriology (30). At present, the genus Thermoanaerobacter is
comprised of two described species, T. ethanolicus (32), and T. finnii (26).
Strain IWZOO has been designated the neotype strain of Thermoanaerobacter
ethanolicus.

The genus Thermoanaerobium is also comprised of only two described
species, T. brockii (37) and T. lactoethylicus (10), with T. brockii strain HTD4 as
the neotype strain. Previously organisms belonging to the genus
Thermoanaerobacter and Thermoanaerobium were considered to be strict
anaerobes, motile or non-motile rods, Gram-positive to variable that were
non-sporeforming, except for T. finm’i (26) and formed chains of cells
comprised of uneven lengths including mini cells. Recently, T. brockii was
shown to form spores (5). All species were saccharolytic and degraded starch
but not cellulose and formed ethanol and/ or lactate as major end products
with lower levels of H2/ C02, and acetate. Consequently, it was not possible to
adequately distinguish between Thermoanaerobacter versus
Thermoanaerobium strains based on these morphological, cellular and
nutritional properties alone. Using the criteria detailed in this paper of
distinguishing between organisms based on differences at the molecular
level, we propose the following classification for Thermoanaerobacter and

Thermoanaerobium.

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Description of 'I‘hermoanaerobacterium gen. nov.

Ther.mo.an.ero.bac.teri.um. Gr. n. thermos hot; Gr. pref. annot;

Gr.n.aerair; Gr.n. bacterion a small rod; M.L. neut. n.

Thermoanaerobacterium, rod which grows in absence of air at high

temperatures.

Cellular characteristics. Straight rods 0.5 x > 15 um. Forms long
filaments, cells motile, peritrichous flagella. Gram negative cell wall.
Catalase negative. Reduces thiosulfate to elemental sulfur. Deposits
elemental sulfur on cells when grown with thiosulfate. Spores may be
detected. Surface colonies are about 0.5-5 mm in size. The optimum
temperature for growth is 60°C. The G+C content of the DNA is 25-40 mol%.
The principal products of carbohydrate fermentation are ethanol, acetate,

lactate, H2, and C02. The type species is Thermoanaerobacterium

thermosulfurigenes comb. nov.

Description of Thermoanaerobacterium thermosulfurigenes (25)
ther.mo.sul.fur.i'ge:nes. Gr. adj. thermos hot. L.n. sulfur brimestone,

Gr. suffix-genes born form; N.L. neut. adj. thermosulfurigenes

releasing sulfur in heat.

Cellular characteristics. Straight rods 0.5 x > 2 pm. Stains Gram-
negative; exponential phase cells motile by peritrichous flagellation. Forms
long filaments and deposits yellow elemental sulphur on cells and in the
medium when grown with thiosulfate. Neither sulfite nor sulfide is
detected. Swollen, white refractile, spherical endospores formed. No outer
wall membranous layer present in thin sections. Sporulation occurs in
xylose- or pectin-containing media. Agar embedded colonies are fluffy, 0.5-1.5

mm in diameter, and not pigmented. DNA base composition of 32.6 (+0.04)

 

0f Cafb(

polygal
galacto'

aesculii
methar
lactate
Sulfi te

hydrol

VOIcan

Natior
33743,

DeScri

6 4
mol%G+C. Cytochromes undetectable; catalase negative.

growth characteristics. Optimum temperature for growth is 60°C,
maximum 75°C, minimum 35°C, pH range for growth; optimum 5.5-6.5
minimum 4.0, maximum <7.6. Obligate thermophile and anaerobe.
Complete growth inhibition by penicillin, streptomycin, cycloserine,
tetracycline, chloramphenicol (each at 100 ug ml'l), sodium azide (500 pg
ml-I) or 02 (0.203 x 105 Pa). Growth is inhibited by 2% NaCl and by sulfite.

Metabolic characteristics. Chemoorganotroph. Utilizes a wide variety
of carbohydrates as energy sources including pectin, arabinose, cellobiose,
polygalacturonose, rhamnose, amygdalin, salicin, inositol, mannitol, xylose,
galactose, glucose, mannose, maltose, starch, melibiose, sucrose, trehalose or
aesculin. No growth on Hzt/COZ, lactose, cellulose, tartrate, lactate, pyruvate,
methanol or glycerol. Fermentation products of glucose are ethanol, H2, C02,
lactate and acetate. Methanol and isopropanol formed on pectin. Sulfate,
sulfite and nitrate not reduced. Pectin methylesterase and polygalacturonase
hydrolase are produced.

Habitat. Algal-bacterial mat ecosystems associated with thermal,
volcanic springs. This strain was isolated from Octopus Spring, Yellowstone
National Park, USA.

Type strain. Thermoanaerobacterium thermosulfurigenes 4B (ATCC
33743, DSM 2229).

Description of Thennoanaerobacterium xylanolyticum sp. nov.
xy.lan.o.ly'ti.cum. Gr. n. xylanosum, xylan; Gr. adj. lyticus, dissolving;
N. L. adj. xylanolyticum, xylan dissolving.
Cellular characteristics. Rods approximately 0.8-1.0 um by 2.0-7.0 um.

Spores are spherical and terminal. Cells are motile. The organism is Gram-

 

6 5
negative. Surface colonies are circular with smooth edges, surface texture

rough, cloudy to white and about 2—5 mm in size. The mol% G+C of the

DNA is 36.1. Catalase negative.

Growth characteristics. Anaerobic. Can initiate growth in a Nz-gassed

media without addition of a chemical (cysteine or NaZS) reducing agent. pH

optimum for growth is about 6.0. The optimum temperature for growth is
60°C with a growth temperature range between 45-70°C. Growth is inhibited
by penicillin G (200 jig/ml), neomycin sulfate, ampicillin, streptomycin
sulfate, rifampicin, polymyxin B, erythromycin, tetracycline, acridine orange
(all at 100 ug/ ml). Yeast extract stimulates growth. Growth is inhibited even
by 1% NaCl.

Metabolic characteristics. Utilizes mannose, sucrose, cellobiose,
arabinose, rhamnose, galactose, fructose, maltose, pyruvate, lactose and
glucose. Cellulose, melibiose, melezitose, xylitol, ribose, raffinose and lactate
are not fermented. Fermentation end products include ethanol, acetic acid, H2
and C02. Lactate not detected in fermentation broth.

Habitat. Geothermal areas of Yellowstone National Park, Wyoming.

Type strain. Thermoanaerobacterium xylanolyticum strain LX-ll
(ATCC 49914).

Description of Thermoanaerobacterium saccharolyticum sp. nov.
sac.cha.ro.ly'ti.cum. Gr. n. sacchar, sugar; Gr. adj. lyticus, dissolving;
N.L. neut. adj. saccharolyticum, sugar-dissolving.
Cellular characteristics. Rods approximately 0.8-1.0 by 3.0-15 um with
some cells occasionally being as long as 30 um. Elongated cell morphology
during nutrient limitation or stationary phase. The cell walls contain three

electron-dense layers of 5 nm thickness, alternating with electron light layers

66
of similar thickness. Gram-negative but a distinct outer membrane

characteristic of many Gram-negative bacteria is not observed. Cell division
proceeds by formation of well-defined division septa, often producing
daughter cells of unequal length. Cells motile, peritrichous flagella. Cells
survived heating at 85°C for 15 min, but not at 90°C for 5 min. Cells contain a
plasmid of 1.5 Md. Surface colonies on agar plates are soft, tan, circular,
convex with hollow centers ("donut" shape). Colony sizes range from 0.5-4.0

mm after 4 days growth at 55°C. The mol% G+C of the DNA is 29-31 (Tm).

Catalase negative.

Growth characteristics. Anaerobic, will initiate growth in a Nz-gassed
media without the addition of chemical reducing agents. Addition of
reducing agents (cysteine or N a25) did not generally stimulate growth. pH
optima for growth is 6.0, and minimum and maximum pH for growth
initiation is 3.5 and below 9.0, respectively. The temperature data for growth
are Tmax 68-70°C, TOP, 60°C, and Tmin 45°C. Growth is inhibited by penicillin
G (200 ug/ ml), chloramphenicol (100 ug/ ml), neomycin (100 ug/ ml), or 02 (0.2
atrn). The culture is resistant to 2% NaCl. Yeast extract stimulates growth.

Metabolic characteristics. Ferments a wide variety of carbohydrates
including glucose, fructose, mannose, galactose, maltose, cellobiose, sucrose,
lactose, trehalose, xylose, starch, rhamnose, raffinose and xylan. Cellulose,
ribose, melibiose, melezitose, xylitol, or sorbitol were not fermented. No
growth occurs in absence of a fermentable carbohydrate. Fermentation
products from either glucose or xylan include ethanol, acetic acid, lactic acid,
H2 and C02. L-Rhamnose is fermented to equimolar amounts of 1,2-
propandiol, plus a mixture of ethanol, acetic acid, lactic acid, H2 and C02.

Habitat. Geothermal sites in Wyoming (Yellowstone National Park

and thermopolis areas) and Nevada (Steamboat area).

 

6 7
Type strain. Thermoanaerobacterium saccharolyticum strain B6A-RI

(ATCC 49915).

Description of Thermoanaerobacter brockii (37) comb. nov.

brock-i.i M.L. gen. no. brockii of Brock; named after Thomas Dale

Brock, who pioneered studies on physiological ecology of extreme

thermophiles.

Cel_l_ul_ar chm'__agteristics. Rods measuring 1.0 um by 2-20 pm. Cells
frequently uneven in length (mini-cells) and occurring in chains, pairs and
filaments. Gram positive and round, terminal endospores detected. DNA
base composition of 30.0 - 31.4 (i 1) mol% G+C. Cytochrome pigments and
catalase absent. Mono-layered cell wall architecture without an outer wall
membrane.

Cplony Characteristics. Colonies are uniformly round, mucoid, non-
pigmented, flat, and grow to a diameter of 0.2 - 0.3 cm in 48 h.

Growth Characteristics. Obligately anaerobic. Optimum temperature
for growth 65-70°C, maximum < 85°C and minimum > 35°C. pH optimum
for growth ~7.5, no growth above 9.5 or below 5.5.

Metabolic characteristics. Chemoorganotroph. Utilizes a variety of
saccharides as energy sources including starch, maltose, glucose, lactose,
sucrose and cellobiose. Growth inhibited by air, penicillin, cycloserine,
streptomycin, tetracycline and chloramphenicol. Fermentation end products
are ethanol, lactic acid, acetic acid, hydrogen and carbon dioxide. Reduces
thiosulfate to hydrogen sulfide.

Habitat. Anaerobic thermal features associated with volcanic activity
including springs, decomposing photosynthetic biomass and sediments.

Type strain. Thermoanaerobacter brockii HTD4 (ATCC 33075, DSM

6 8
1457). This strain was isolated from a Washburn thermal springs edge

sediment located in Yellowstone National Park, U.S.A.

Description of Thermoanaerobacter thermohydrosulfuricus comb. nov.
ther.mo.hy.dro.sul.fur'i.cus. M.L. masc. n. thermos hot; M.L. masc. adj.
hydrosulfuricum pertaining to hydrogen sulfide; M.L. masc. adj.
thermohydrosulfuricum indicating that the organism grows at high
temperatures and reduces sulfite to H25.

Cellular characteristics. Rods approximately 0.3-0.6 um by 2.0—13.0 um
Rods are single or in short chains, in some strains long filamentous group of
cells. Cells are motile and peritrichous. Spores are spherical, terminal,
sporangia swelling the cell. Sporulating cultures showing thinner, more
elongated cells than non-sporulating cultures. Cells are Gram-variable. Cell
wall is composed of two layers. The outermost layer consist of hexagonally
shaped particles. The center-to-center distance between adjacent particles is
13.5 nm; they are composed of glycoprotein containing glucose, galactose,
mannose and rhamnose, which have a molecular weight of approximately
140,000. The outermost layer completely covers the cell and is resistant to
digestion by proteolytic enzymes. Cell walls contain meso-DAP. The mol%
of G+C is 35-37 (Tm). Catalase negative.

Growth charJacteristics. Obligately anaerobic. The optimum
temperature is 67-69°C; the maximum temperature at which growth occurs is
76-78°C. Growth at 37°C is poor with no growth at 28°C. Growth occurs at pH
5.5-9.2; optimum growth from 6.9-7.5. Growth is inhibited by H2 in the gas
phase and by lactate.

Metabolic characteristics. Hz-COZ are produced in the media containing

liver infusion. H25 is produced from tryptophan, peptone, and yeast extract.

6 9
Sulfite and thiosulfate are reduced to H25; sulfate is not reduced. Acetyl

methyl carbinol is not produced. Fructose, galactose, glucose, mannose,
xylose, cellobiose, maltose, sucrose, trehalose, pectin, esculin and salicin are
fermented. Fermentation of dextrin, potato starch, mannitol, dulcitol and
sorbitol, and coagulation of litmus milk are variable. Inositol, erythritol,
glycerol, lactate, tartrate and cellulose are not fermented; nitrite but not
nitrate is reduced, coagulated albumin is not hydrolyzed and indole is not
produced. Products of metabolism in PYG broth are acetate and lactic acids,
ethanol, C02 and H2; formic, butyric, isovaleric, and isocaproic acids, and
propanol and isopropanol may be detected. Methanol is a major metabolic
end product if grown in pectin. Reduces thiosulfate to hydrogen sulfide.

Habitats. Isolated from extraction juices of beet sugar factories; from
mud and soil; from hot springs in Utah and Wyoming and from a sewage
plant in Georgia.

Neotype strain. Thermoanaerobacter thermohydrosulfuricus E100-69

(DSM 567 = NCIB 10956).

IO.

LITERATURE CITED

Bateson, M. M., J. Wiegel, and D. M. Ward. 1989. Comparative analysis
of 16S ribosomal RNA sequences of thermophilic fermentative bacteria
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12:1-7.

Biely, P. 1985. Microbial xylanolytic systems. Trends Biotechnol. 3:286-
290.

Brock, T. D. 1986. Introduction: an overview of the thermophiles, p. 1-
16. In T. D.; Brock (ed.), Thermophiles: general, molecular, and applied
microbiology-1977. John Wiley 8: Sons, New York.

Cato, E. P., and E. Stackebrandt. 1989. Taxonomy and phylogeny, p. 1-26.
In N. P. Minton and D. J. Clark (ed.), Clostridia. Plenum Press, New
York.

Cook, G. M., P. H. Jansson, and H. W. Morgan. 1991. Endospore
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Goodwin, 8., and J. G. Zeikus. 1987. Physiological adaptations of
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in Sarcina ventriculi. J. Bacteriol. 169:2150-2157.

Hollaus, P., and U. Sleytr. 1972. On the taxonomy and fine structure of
some hyperthermophilic saccharolytic Clostridia. Arch. Microbiol.
86:129-146.

Hyun, H. H., and J. G. Zeikus. 1985. General biochemical
characterization of thermostable pullulanase and glucoamylase from
Clostridium thermohydrosulfuricum. Appl. Environ. Microbiol.
49:1168-1173.

Johnson, J. L. 1981. Genetic characterization, p. 450-472. In P. Gerhardt,
R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg,
and G. B. Phillips (eds.), Manual of Methods for General Bacteriology,
American Society for Microbiology, Washington, DC.

Kondratieva, E. N., E. V. Zacharova, V. I. Duda, and V. V. Krivenko.

1989. Thermoanaerobium Iactoethylicum spec. nov. a new anaerobic

bacterium from a hot spring of Kamchatka. Arch. Microbiol. 151:117-
12.

7O

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

71

Laemmli, U. K. 1970. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature (London) 227:680-685.

Lamed, R., E. Bayer, B. C. Saha, and J. G. Zeikus. 1988. Biotechnological
potential of enzymes from unique thermophiles, p. 371-383. In G.
Durand, L. Bobichon, and J. Florent (eds.), Proc. 8th Intl. Biotechnol.
Symp., Paris.

Lamed, R., and J. G. Zeikus. 1980. Ethanol production by thermophilic
bacteria: Relationship between fermentation product yields of and
catabolic enzyme activities in Clostridium thermocellum and
Thermoanaerobium brockii. J. Bacteriol. 144:569-578.

Lee, C., B. C. Saha, and J. G. Zeikus. 1990. Characterization of
Thermoanaerobacter glucose isomerase in relation to saccharidase
synthesis and development of single-step processes for sweetener
production. Appl. Environ. Microbiol. 56:2895-2901.

Lowe, S. E., H. S. Pankratz, and J. G. Zeikus. 1989. Influence of pH
extremes on Sporulation and ultrastructure of Sarcina ventriculi. J.
BaCteriol. 171:3775-3781.

Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951.
Protein measurement with the Folin phenol reagent. J. Biol. Chem.
193:265-275.

Mandel, M., and J. Marmur. 1968. Use of ultraviolet absorbance-
temperature profile for determining the guanine plus cytosine content
of DNA, p. 195-206. In L. Grossman and K. Moldave (eds.), Methods in
enzymology, vol. XII, part B, Academic Press, New York.

Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic
acid from micro-organisms. J. Mol. Biol. 3:208-218.

Miller, G. L. 1959. Use of dinitrosalicylic acid reagent for
determination of reducing sugars. Anal. Chem. 31:426-428.

Ng, T. K., A. Ben-Bassat, and J. G. Zeikus. 1981. Ethanol production by
thermophilic bacteria: fermentation of cellulosic substrates by

cocultures of Clostridium thermocellum and Clostridium
thermohydrosulfuricum. Appl. Environ. Microbiol. 41:1337-1343.

Paice, M. G., R. Bernier, Jr., and L. Jurasek. 1988. Viscosity-enhancing
bleaching of hardwood kraft pulp with xylanase from a cloned gene.
Biotechnol. Bioeng. 32:235—239.

24.

7.6.

27.

29.

31.

Heath-44 HALJA

.mf‘mfi-ya

24.

26.

27.

29.

30.

31.

7 2
Paice, M. G., and L. Jurasek. 1984. Removing hemicellulose from
pulps by specific enzyrnic hydrolysis. J. Wood Chem. Technol. 4:187-
198.

Pfennig, N., and S. Wagener. 1986. An improved method of preparing
wet mounts for photomicrographs of microorganisms. J. Microbiol.
Methods. 4:303-306.

Saha, B. C., R. Lamed, C.-Y. Lee, S. P. Mathupala, and J. G. Zeikus. 1990.
Characterization of an endo-acting amylopullulanase from
Thermoanaerobacter strain B6A. Appl. Environ. Microbiol. 56:881-886.

Schink, B., and J. G. Zeikus. 1983. Clostridium thermosulfurogenes 5p.
nov., a new thermophile that produces elemental sulphur from
thiosulphate. J. Gen. Microbiol. 129:1149-1158.

Schmid, U., H. Giesel, S. M. Schoberth, and H. Sahm. 1986.
Thermoanaerobacter finnii spec. nov., a new ethanologenic
sporogenous bacterium. Syst. Appl. Microbiol. 8:80-85.

Smibert, R. M., and N. R. Krieg. 1981. General characterization, p. 409-
443. In P.; Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W.
A. Wood, N. R. Krieg, and G. B. Phillips (eds.), Manual of methods for
general bacteriology. American Society for Microbiology, Washington,
DC.

Weimer, P. J. 1985. Thermophilic anaerobic fermentation of
hemicellulose and hemicellulose-derived aldose sugars by
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Weimer, P. J., L. W. Wagner, S. Knowlton, and T. K. Ng. 1984.
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H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (eds.) Bergey's
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Wiegel, J., L. H. Carreira, C. P. Mothershed, and J. Puls. 1983.
Production of ethanol from biopolymers by anaerobic, thermophilic,
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ethanolicus JW200 and its mutants in batch cultures and resting cell
experiments. Biotechnol. Bioeng. Symp. 13:193-205.

32.

33.

35.

36.

37.

73

Wiegel, J., and L. G. Ljungdahl. 1981. Thermoanaerobacter ethanolicus
gen nov., spec. nov., a new, extreme thermophilic, anaerobic
bacterium. Arch. Microbiol. 128:343-348.

Wiegel, J., L. G. Ljungdahl, J. R. Rawson. 1979. Isolation from soil and
properties of the extreme thermophile Clostridium
thermohydrosulfuricum. J. Bacteriol. 139:800-810.

Wiegel, J., C. P. Mothershed, and J. Puls. 1985. Differences in xylan
degradation by various noncellulolytic thermophilic anaerobes and
Clostridium thermocellum. Appl. Environ. Microbiol. 49:656-659.

Zeikus, J. G. 1979. Thermophilic bacteria: ecology, physiology, and
technology. Enzyme Microbiol. Technol. 1:243-252.

Zeikus, J. G., A. Ben-Bassat, and P. W. Hegge. 1980. Microbiology of
methanogenesis in thermal, volcanic environments. J. Bacteriol.
143:432-440.

Zeikus, J. G., P. W. Hegge, and M. A. Anderson. 1979.
Thermoanaerobium brockii gen. nov. and sp. nov., a new
chemoorganotrophic, caldoactive, anaerobic bacterium. Arch.
Microbiol. 122:41-48.

Chapter III

Regulation and characterization of xylanolytic enzymes of

Thermoanaerobacter strain B6A-RI

74

 

Du
produced
and the l
non-Iimi
predomi
content!
extracell
arabinot
respecti
activity
Purified
With x}
sodium
area of
multip;
apPTOx
Weight

ABSTRACT

During growth on xylan and xylose Thermoanaerobacter strain B6A-RI
produced endoxylanase, B-xylosidase, arabinofuranosidase and acetyl esterase
and the first three activities appeared to be produced coordinately. Under
non-limiting xylan growth conditions these enzyme activities were
predominanatly cell associated, however, during growth on limiting
concentrations of xylan the majority of endoxylanase activity was
extracellular rather than cell associated. Endoxylanase, B-xylosidase and
arabinofuranosidase activities were induced by xylan, xylose and arabinose
respectively. Acetyl esterase activity was constitutive and endoxylanase
activity was catabolite repressed by glucose. Endoxylanase activity was
purified using gel filtration with Sephacryl S-300 and affinity chromatography
with xylan coupled to Sepharose CL-4B preequilibrated to 45°C with 50 mM
sodium acetate buffer (pH 4.0) and eluted with 0.1% soluble xylan. A single
area of activity was identified on the zymogram which was composed of
multiple bands, and one major protein complex with a molecular weight of
approximately 160 kDa, and a minor protein complex with a molecular
weight of approximately 130 kDa on SDS-PAGE. The endoxylanase activity
stained with Schiff's reagent indicative of glycoproteins. The purified
endoxylanase displayed a specific activity of 41 units/ mg protein on xylan,

and a pH and temperature optima of 6.0 and 70°C respectively.

75

B‘
ofaHla
xykurh
forZOt.

l
severa
enzyn~
and B
thnv
Short
subst
3 ant
Clear
and
“on;
(Slu
arak
dea

from

ther

flex-O

SUbS

INTRODUCTION

B-1,4—xylans are heterogeneous polysaccharides found in the cell walls
of all land plants and in almost all plant components (35). After cellulose,
xylan is the most abundant renewable polysaccharide in nature, accounting
for 20 to 30% of the dry weight of woody tissue.

Complete breakdown of a branched acetyl xylan requires the action of
several hydrolytic enzymes (6, 25, 39). The most important microbial
enzymes involved in xylan degradation are endo-1,4-B-D-xylanase (EC 3.2.1.8)
and B-D-xylosidase (EC 3.2.1.37). Endo-1,4-B-D-xylanase acts on chains of
xylans, arabanoxylans and 4-MeO-glucuronoxylans. B-xylosidase breaks down
short oligosaccharides from the nonreducing end to xylose, and has
substantial transferase activity (25). A number of other glycosidases may play
a more minor role in polysaccharide degradation, including a-L-fucosidase
cleaving terminal L-fucoside from xyloglucans and rhamnogalacturonans
and a-L-rhamnosidase (EC 3.2.1.40) releasing L-rhamnose from the
nonreducing end of partially degraded rhamnogalacturonans. oc-D-
Glucuronidase releases D-glucuronic acid from xylan and a-L-
arabinofuranosidase (EC 3.2.1.55) removes arabinosyl side chains. Often
xylans are present in the partially acetylated form, which come under attack
from acetyl esterases (EC 3.1.1.6).

These xylan degrading enzymes are produced by a wide variety of
microorganisms including aerobic and anaerobic mesophiles and
thermophiles (6, 32). The majority of the studies have been carried out in
aerobic, meSOphilic fungi and bacteria (6, 25, 32, 39).

Enzymatic hydrolysis products from xylan, namely xylose can be

subsequently converted into liquid fuel, single-cell protein, solvents and

76

other cher
Such biocc
residues a:
may be per
cellulose p
(6). Anoth
manufactur
of fruit ji
modificatio

The 1
fr0m hemic
the enzyme
in inexPer

Characterist

7 7
other chemical products using selected fermentative microorganisms (6, 8).

Such bioconversion processes are particularly attractive for the elimination of
residues and wastes produced by agriculture and forestry. Furthermore, it
may be possible to use xylanases in biopulping processes for the preparation of
cellulose pulp, bio-bleaching and purification of fibers for rayon manufacture
(6). Another potential application of xylanases may be in the food and feed
manufacture such as improving baking with high fiber materials, clarification
of fruit juices and wine, improved animal feed properties by fiber
modification and production of food thickeners.

The feasibility of using xylanase for cellulose bleaching or purification
from hemicellulose will be highly dependent on the intrinsic selectivity of
the enzymes, the absence of cellulolytic activity and the availability of enzyme
in inexpensive bulk quantities. Stability is also a highly desirable
characteristic for industrial enzymes, and potential sources for thermostable
xylanases include several thermophilic microorganisms (5, 10, 23, 31).

Thermophilic anaerobes produce active saccharidases that enable rapid
hydrolysis of polymers such as cellulose and starch (41). Detailed
characterization of xylanolytic enzyme complexes present in
thermoanaerobes has not yet been reported. Clostridium thermocellum
produces a cellulosome which organizes cellulases for rapid degradation of
cellulose (2). Little is known about the hemicellulases from thermophilic
anaerobic bacteria that grow rapidly on insoluble xylan. Wiegel et al. (36, 37)
studied a variety of thermophilic, anaerobic, saccharolytic bacteria,
including Thermoanaerobacter ethanolicus,Thermobacteroides
acetoethylicus, Thermoanaerobium brockii, and Clostridium thermocellum,
which all ferment xylan albeit at a very slow rate. Studies were also

conducted on Thermoanaerobacter strain B6A an organism isolated from an

 

algal ma
shown tc
86A was
glucosei

R1
Yellows:
(l9). 1
xylanolj
cellulolj
the kim
the reg

and thi

7 8
algal mat present in Big Spring, Thermopolis, Wyoming (34) which was

shown to extensively degrade xylan (35). Later Thermoanaerobacter strain
B6A was shown to possess a number of saccharidases including amylase,
glucose isomerase, and high levels of endoxylanase (17).

Recently we isolated two xylanolytic species from Frying Pan Springs in
Yellowstone National Park, Wyoming, a thermal spring with a pH below 6.0,
(19). One of the isolates,Thermoanaerobacter strain B6A-RI, is a model
xylanolytic thermoanaerobe because it has very active xylanases but it is not
cellulolytic. The purpose of the present study was three fold: first to identify
the kinds and complexity of xylanase activities present; second to determine
the regulation of xylanolytic enzymes with respect to the growth substrate,

and thirdly, to partially purify and characterize the endoxylanase complex.

Chemical

All
Sigma Cl
used we

furnaces

Organis
T.

as descr

Measuy
(
Xylose

reagen

(Mom.-

MATERIALS AND METHODS

Chemicals and gases

All chemicals were reagent grade or better and were obtained from
Sigma Chemcal Co., St. Louis, Mo., or Mallinckrodt, Inc., Paris, Ky. All gases
used were at least 99.9% pure and were passed over copper-filled Vycor

furnaces (Sargent Welch Scientific Co., Skokie, Ill.) to remove oxygen.

Organism and culture conditions
Thermoanaerobacter strain B6A-RI was grown on TYE medium at 60°C

as described elsewhere (19).

Measurement of growth and end products

Cell growth was determined by measuring optical density at 660 nm.
Xylose was measured as reducing sugar with dinitrosalicyclic acid (DNS)
reagent (21). Ethanol and acetate were determined by flame ionization gas
chromatography. Cell free culture media (1 ml) was acidified with 100 ml of
10 N H3PO4, and 2 ml were injected into a Hewlett Packard HP 5890A gas
chromatograph equipped with a Chromosorb 101 (80/ 100 mesh) column
(Supelco). The column was operated at 200°C, and the detector and injector

temperatures were 250°C and 190°C, respectively.

Enzyme production and batch culture studies

Thermoanaerobacter strain B6A-RI was grown in 750 ml fermentation
vessels (New Brunswick Sci., New Brunswick, NJ) containing 500 ml of
medium and 1.0%(w/v) oatspelt xylan, which was added before autoclaving.

The vessel was rendered anaerobic by the addition of 0.01%(w/v) Nags. The

79

vessel was
(95:5), and
(3 ml) wer
fermentatic
culture w
microcentr
cells were
1.5 ml of 1

measure c

Enzyme a
All

dEtermjm
50 mM St
The rea‘
addjfiOn‘

Th
XylopyI-a.
Volume '
terminate

410 nm.

8 0
vessel was agitated at 200 rpm, sparged with nitrogen and carbon dioxide

(95:5), and the temperature maintained at 60°C, without pH control. Samples
(3 ml) were taken at intervals for determination of growth, reducing sugar,
fermentation products and enzyme activities. For enzyme activities, 1.5 ml of
culture was centrifuged at 16,000 x g for 5 min in an Eppendorf
microcentrifuge, and the supernatant taken as the extracellular fraction. The
cells were washed twice in 50 mM MOPS buffer (pH 6.0) and resuspended in
1.5 ml of the same buffer. These cells were sonicated for 2 min and used to

measure cellular, i.e., both soluble and cell bound activity.

Enzyme activities

All enzyme assays were incubated at 65°C. EndoxylanaSe activity was
determined using oat spelt xylan. The assay consisted of 1%(w/v) xylan with
50 mM sodium acetate (pH 5.5) and enzyme to give a final volume of 0.2 ml.
The reaction mixture was incubated for 2 h and xylose quantitated by
addition of 0.8 ml DNS reagent (21) and absorbance measured at 640 nm.

The B-xylosidase assay consisted of 1 mM p-nitrophenyl B-D-
xylopyranoside, 50 mM sodium acetate (pH 5.5) and enzyme to give a final
volume of 0.4 ml. The reaction mixture was incubated for 10 min and
terminated by the addition of 0.8 ml of 0.5 M sodium carbonate and read at
410 nm.

Arabinofuranosidase activity was determined in a reaction mixture
containing 2 mM p-nitrophenyl a-L-arabinofuranoside, 50 mM sodium-
acetate buffer (pH 5.5) and enzyme to give a final volume of 0.5 ml. The
reaction was incubated for 30 min and terminated by the addition of 0.33 ml

of 1 M sodium carbonate and read at 410 nm.

 

The
MES (pH
incubated
1 M SOdlL‘
shortly t
temperati
linear ove
activities
(intemati.
determin.
End0xyla

eqUivaler

SYMhesi:

Sy
PrOCedm
Chemica
LKB Big
in a 500
the mixt
bath. Tt

IQmOve

8 l
The acetyl esterase assay consisted of 1 mM a-napthylacetate, 50 mM

MES (pH 6.0) and enzyme to give a final volume of 0.5 ml. The reaction was
incubated for 25 min and then 0.5 ml of 0.01% (w/v) Fast Corinth V salt in
1 M sodium acetate buffer (pH 4.3) containing 10% (v/v) Tween 20 (prepared
shortly before use) was added, and the solution incubated at room
temperature for 10 min before being read at 535 nm. All enzyme assays were
linear over the period of the assays. B-xylosidase and arabinofuranosidase
activities were expressed as umoles of p-nitrophenol released per 'min
(international units) per ml of enzyme source, and acetyl esterase activity was
determined as umoles of a-napthol per min (U) per ml of enzyme source.
Endoxylanase activities were expressed as umoles of reducing sugar (xylose

equivalents) released per min (U) per ml of enzyme source.

Synthesis of xylan-coupled Sepharose CL-4B and purification procedure
Synthesis of xylan-coupled Sepharose CL-4B was based on the
procedure described by Sundberg and Porath (29). 5 g oat spelt xylan (Sigma
Chemical Co.) was mixed with 35 g suction dried Sepahrose CL-4B (Pharmacia
LKB Biotechnology, Piscataway, NJ) in 90 ml 0.5 M sodium carbonate (pH 13)
in a 500 ml flask. 10 ml epichlorohydrin (Sigma Chemical Co.) was added to
the mixture and incubate at 60°C for 2 h with vigorous shaking in a water
bath. The mixture was washed with excess water on a glass filter-funnel to

remove uncoupled reagents and suspended in distilled water.

Purification of endoxylanases
Thermoanaerobacter strain B6A-RI was grown on TYE medium with
0.5% xylose to mid exponential phase, centrifuged and the culture

supernatant used as a source of endoxylanases. The culture supernatant was

82
concentrated by ultrafiltration using YM 100 membrane with a 100 kDa

molecular weight cut-off (Amicon Co., Danverse, MA), and loaded on
Sephacryl S-300 column ( 2.5 x 50 cm, Pharmacia) prequibrated with 50 mM
NaCl and 50 mM sodium acetate buffer (pH 5.5) and eluted with the same
buffer. The active fractions were pooled and concentrated by ultrafiltration
using a YM 30 membrane with a 30 kDa molecular weight cut-off (Amicon).

Using the procedure described by Rozie et al. (26), cross linked xylan
was prepared as an affinity adsorbent. However, the flow rate with this
material was very slow due to its gelatinous nature. To improve the flow
rate, oat spelt xylan was coupled to Sepharose CL-4B by the modification of
method of Sundberg and Porath (29). For affinity chromatography a
Pharmacia column (1.5 x 33 cm) fitted with a thermostatic jacket connected to
a water bath for temperature control was filled with xylan-coupled Sepharose
CL-4B. The column was equilibrated with 50 mM sodium acetate buffer (pH
4.0) at room temperature and protein sample was loaded once the column
had equilibrated to 45°C. After washing with 50 mM sodium acetate buffer
(pH 5.5) at room temperature the xylanases were eluted with soluble xylan
(0.1%) in 50 mM sodium acetate buffer (pH 5.5) at room temperature. The
active fractions were pooled and incubated at 60°C for 2 - 4 h to digest xylan
coeluted with enzyme and the hydrolysis products were removed by dialysis

against 50 mM sodium acetate buffer (pH 5.5)

Electrophoresis and zymograms

SDS-polyacrylamide gel electrophoresis (PAGE) was performed in 7.5%
gels by the method of Laemmli (13). The gels were stained for endoxylanase
activity in a modification of the cellulase assay described by Béguin (4). A

suspension (0.1% final concentration) of soluble xylan was incorporated into

83
the separating gel before the addition of ammonium persulfate and

polymerization. Protein samples were prepared by heating for 5 min at
100°C in the presence of sample buffer (3% SDS, 10% glycerol, 5% B-
mercaptoethanol in 31 mM Tris hydrochloride (pH 6.8), and then applied to
the gel.

Upon completion of electrophoresis, the gel was washed twice for 30
min in 50 mM sodium acetate buffer (pH 5.5) containing 25% isopropanol and
once in 50 mM sodium acetate buffer for 15 min. The gel was then incubated
in 50 mM sodium acetate buffer for 5 min at 60°C. The gel was stained in a
0.2% (w/v) Congo red solution for 10 min at room temperature with gentle
shaking and destained with 1 M NaCl until excess stain was removed from
the active band. To facilitate photographic documentation, the gel was rinsed
in 5% acetic acid which caused the background to turn dark blue, emphasizing
the bands of activity. Proteins were stained with Coomassie brilliant blue

R250, destained and photographed.

Regulati

Ti
sources
arabinot
levels 0
with the
culture
most sf
xylose ;
activity

cOnstitt

Crowt}
'1
xleSe
prOduC
determ
arabinC
were (I
gTOWth

not in t;

RESULTS

Regulation of xylanolytic enzyme activity levels

Thermoanaerobacter strain B6A-RI was grown on various carbon
sources and endoxylanase, B-xylosidase, acetyl esterase and
arabinofuranosidase were compared with respect to activity (Table 1). High
levels of B-xylosidase were produced during growth on all of the substrates
with the exception of glucose, however, addition of xylose to a glucose grown
culture resulted in enzyme synthesis (Table 1). Endoxylanase activity was
most significant during growth of Thermoanaerobacter strain B6A-RI on
xylose and xylan. Arabinose although the end product of arabinofuranosidase
activity acted as an inducer for this enzyme, and acetyl esterase activity was

constitutive.

Growth and xylanolytic enzyme synthesis

Thermoanaerobacter strain B6A-RI grew readily in batch culture on
xylose with a doubling time of 1.13 h (Fig. 1). Although this organism
produces acetate, ethanol, lactate, C02 and H2, only acetate and ethanol were
determined under these conditions. B-xylosidase, acetyl esterase and
arabinofuranosidase activities were produced during growth on xylose and
were detected in cells. Endoxylanase activity was also synthesized during
growth on xylose, but most activity was detected in the culture supernatant,

not in the cells.

Growth of Thermoanaerobacter strain B6A-RI on xylan was much slower
than on xylose and a significantly longer lag time was observed (Fig. 2). An
increase in reducing sugars occurred during xylan fermentation with the
maximum amount formed during the early phase of growth (i.e. before

84

85

Table 1. Effect of growth substrate on xylanolytic enzyme levels of

Thermoanaerobacter strain B6A-RI.

 

Enzyme activitya

 

 

Substrate 0D.
8- xylosidase endoxylanase arabinosidase acetyl esterase
(m U/ml) (mU/ml) (mU/ml) (mU/ml)

Glucose 1.04 1 1 3 5 4 l
Xylan N. D. 2 7 2 5 7 7 4 3
Maltose l .00 5 8 3 S 3 7
Xylose 0.96 87 2 6 l 1 4 6
Arabinose 0.90 73 6 107 3 5

 

All substrates were present at 0.5%(w/v) except xylan which was

present at 1%(w/v).

aEnzyme unit is expressed as umoles of reducing sugar produced

per min.

Enzyme activities comprise of the cellular fraction from cells in the
late exponential phase.

86

Figure 1. Fermentation and xylanolytic enzyme synthesis time course

of Thermoanaerobacter strain B6A-RI during growth on xylose.

DH

7.0

SES

6()

5.5

5.0

415

4()

Cellular acttvtty (mU/ml)

Extracellular actlvtty (mU/ml)

 

0.0. 660nm

350

300

250

200

ISO

IOO

50

500

450

400

350

300

250

200

ISO

l00

50

 

" i I v v I 1 v 1 v I V ' I V v
o 0.0. ‘

O

. . ethanol
“S .

. xylose >- " . ‘
q
acetate 1

 

 

 

_
.
.-
p
b
r
b
,
y-
l.
.-
p
b
b
.-
p ‘ I
h
.—
p
-
b
-
h
j-
p
.-
D
h
l'
h
p
l-

 

;‘jipow r
~ I .
.~~.\.~AP, }.N .

 

B- xylosidase

acetyl esterase arablnosldase

0 ..

endoxylanase

 

”.4

Tlme (h)

1

t

r-._ “4.3.. m ......... 5:: q
1 ~40 ‘

endoxylanase

J

14 l

A

acetyl esterase

 

35

3O

25

20

Xylose and fermentation products (mm

88

Figure 2. Fermentation and xylanolytic enzyme synthesis time course

of Thermoanaerobacter strain B6A-RI during growth on xylan.

Cellular activity (mU/ml)

Extracellular activity (mU/ml)

Fermentation products (mm

 

 

SO

40-

20"

 

pH

acetate

ethanol

reducing
sugar

 

 

 

400
350 ~
'300 -
250 -
200 -
ISO -
l00 ~
.50 ~

400

 

350

300

V l V V l I' V

250

200

150

U U I V ' l U ' I I

l00

50

 

acetyl esterase

arablnosldge

 

endoxylanase

p— xylosidase

 
   
 

 

 

 

endoxylanase

 

1
I
-
1
1
-
1
1
d
1
1
-
1
1
q
q
1

 

Time (h)

 

H
O

7.0

6.5

6.0

5.5

5.0

4.5

4.0

DH

 

Reducing sugar (mg/ml)

90

significant amounts of fermentation products were formed), and then was
consumed during the exponential phase. All xylanlolytic enzyme activities
were synthesized during growth. Interestingly, although similar levels of
endoxylanase are produced when Thermoanaerobacter strain B6A-RI is grown
on xylan or xylose, the activity was mainly cell associated during growth on
the insoluble substrate in comparison to the predominantly extracellular
activity found during growth on xylose. The possibility that xylanase is
adsorbed onto xylan, therefore reducing the amount of activity that can be
detected in the culture supernatant, cannot be excluded, but high levels of
activity observed tend to suggest that at least some of the enzyme is
nonadsorbed.

Acetyl esterase activity was mainly cell associated, with lower levels
occurring in the supernatant and intracellular fractions. Only low levels of
cell bound arabinofuranosidase activity could be detected, and this may reflect
the fact that oatspelt xylan is particularly low in arabinoside side chains,

consisting mainly of xylose units, thus, not inducing this enzyme.

Cellular location of endoxylanase

The cellular location (i.e. cell bound versus excreted) of cellulases,
xylanases and amylases can be affected by substrate availability. Consequently,
experiments were performed such that the concentration of xylan was varied
during growth of Thermoanaerobacter strain B6A-RI in batch culture, and
levels of endoxylanase activity associated with the cell versus the culture
supernatant were determined (Table 2). At low concentrations of xylan,
endoxylanase was predominantly extracellular and with increasing

concentrations of the substrate, activity was predominatly cell associated.

91

Table 2. Effect of xylan concentration on cell bound versus excreted

endoxylanase activity in Thermoanaerobacter strain B6A-RI.

Xylan Extracellular Cellular Ratio of
concentration activity activity extracellular /
%(w/ v) (mU / ml) (mU / ml) cellular activity
0.05 10 2 5

0.10 24 8 3

0.15 40 26 1.6

0.20 46 38 1.2

0.30 62 74 0.8

Thermoanaerobacter strain B6A-RI was grown in 10 ml pressure tubes on
TYE medium with different concentrations of xylan. Cells were harvested

at midexponential phase and endoxylanase activity determined.

9 2
Purification of endoxylanase activity by affinity chromatography

Extracellular endoxylanase existed as a high molecular weight complex
(greater than 1000 kDa) which could not be separated by standard methods
e.g. ammonium sulfate precipitation, anion exchange chromatography and
cation exchange chromatography using a number of different buffer systems
at different pH values. When analyzed using SDS-PAGE and zymograms,
endoxylanase was composed of multiple bands, including proteins without
endoxylanase activity.

In the purification scheme employed, gel filtration was used to elute a
high molecular weight fraction which was pooled and concentrated. The
results showed that most activity was associated with the high molecular
weight fraction. This partially purified endoxylanase was further purified by
affinity chromatography using xylan-coupled Sepharose CL-4B.

Initially binding of the high molecular weight endoxylanase to the
affinity column material did not occur at 4°C or at room temperature. As
this procedure relies on interaction of the enzyme active site with the
substrate, the temperature of the column was increased to 45°C, in an attempt
to facilitate this interaction. Initially the pH of the column was 5.5, however,
there was limited binding of endoxylanase at this pH, and the pH was lowered
to 4.0 with a significant increase in affinity of the enzyme for the column
matrix. This approach was successful and a single peak of activity eluted,
which appeared on SDS-PAGE as a major band of 160 kDa and a minor 130
kDa band (Fig. 3a).

The in situ zymogram technique has been used for analysis of
thermostable cellulases and xylanases from C. thermocellum (22, 27), and
proved to be a very convenient method for preliminary characterization of

these endoxylanase activities from Thermoanaerobacter B6A-RI. To

93

A B
koam123 m123

200

116
97

66

43

 

Figure 3. SDS-PAGE (A) and a zymogram (B) of endoxylanase purification
from Thermoanaerobacter strain B6A-RI grown on xylose.

10 ug of protein were applied to each lane. Lane m, size marker; lane 1,
extracellular fraction from the culture grown on xylose; lane 2, after gel
filtration using a Sephacryl S-300 column; lane 3, after affinity

chromatography using xylan-coupled Sepharose CL-48.

94
determine that the protein bands seen in SDS-PAGE had endoxylanase

activity, a zymogram was performed by renaturing the endoxylanase after
SDS-PAGE and detection in situ (Fig. 3b). From the zymogram, multiple
endoxylanase activities with high molecular weight were detected indicating
that more than one enzyme with the ability to hydrolyze xylan was present.
PAS (Periodic Acid, Schiff's reagent) staining indicated that the
endoxylanases from Thermoanaerobacter B6A-RI are glycoproteins (data not

shown).

Characterization of the endoxylanase activity

Using the endoxylanase complex eluted from the affinity column,
temperature and pH optima were determined. The pH optimum for the
endoxylanase was 6.0 (Fig. 4) and the temperature optimum was 70°C (Fig. 5).
The specific activity of the endoxylanase complex on xylan was 41 units/ mg
protein. The products of xylan hydrolysis by the affinity purified
endoxylanase complex were mainly xylobiose and xylotetriose as determined

by TLC (data not shown).

95

 

 

 

 

120
A 110 -
o\°
v
> 100 -
.2:
OZ
‘6
a 90 -
2 so -
a:
.‘3
d9 ..
a: 70
60 L
50 i l A l i l i l 1 l
2 3 4 5 6 7 8

Figure 4. The pH profile of partially purified endoxylanase from
Thermoanaerobacter B6A-RI.

96

 

 

 

 

120
y-
A 100 -
o\°
V
> 80 t-
r:
> b
'5, .
a 60 ~
g t
3.: 40 -
52 .
0
a:
20 -
o A l 4 l A l A l A J n J A
30 40 SO 60 70 80 90 100

Temperature (°C)

Figure 5. The temperature profile of partially purified endoxylanase from
Thermoanaerobacter B6A-RI.

DISCUSSION

These data represent the first detailed characterization of the
xylanolytic activities present in Thermoanaerobacter strain B6A-RI. This
organism degrades xylan by the coordinate production of a complex of
hydrolyzing enzymes which act together in a concerted manner to effectively
degrade the substrate. This organism produces the highest level of xylanases
reported for a non-cellulytic producing thermoanaerobe. During growth on
insoluble xylan, cells of Thermoanaerobacter strain B6A-RI produce a cell
bound endoxylanase complex comprised of multiple proteins which is
excreted when the insoluble growth substrate is limiting. In addition,
intracellular B-xylosidase, acetyl esterase and arbinofuranosidase are
produced. Synthesis of the xylanolytic outfit is regulated by pentose
containing substrates.

Acetyl esterase activity was constitutive in Thermoanaerobacter strain
B6A-RI. Xylose and xylan acted as inducers for B-xylosidase and
endoxylanase, arabinose induced arabinofuranosidase. Xylan and xylose are
also required to induce xylanase and B-xylosidase production in Butyrivibrio
fibrisolvens (28), and xylose induces xylanase, xylanopyranosidase and
arabinofuranosidase activity in Clostridium acetobutylicum (18), and
xylanase and B-xylosidase in Bacillus pumilus (24).

In most xylanolytic bacteria endoxylanase activity appears to be mainly
extracellular during growth on xylan, as found in B. fibrisolvens (11, 28),
Thermoanaerobacter ethanolicus (36), and C. acetobutylicum (18). [3-
xylosidase appears to be mainly cell associated in B. fibrisolvens (11, 28) and
equal levels were found in the extracellular fraction and cell bound fraction

in cultures of C. acetobutylicum (18). This differs from the findings in this

97

9 8
study where highest levels of all of the xylan degrading enzymes were found

associated with the cell during growth on xylan, except under conditions of
xylan limitation in which endoxylanase becomes predominantly
extracellular. Similar findings have been reported for pullulanase from
Clostridium thermohydrosulfuricum which was predominantly cell bound
during growth of the organism on 0.5% starch (1). However, when the
organism was grown in continuous culture with growth-limiting amounts of
starch, pullulanase and tit-amylase were overproduced and a partial
disintegration of the cell surface occurred, which was associated with the
formation of membrane "blebs" and extracellular vesicles (1).

In Clostridium thermocellum a cell surface glycoprotein complex,
termed the cellulosome, containing enzymes necessary for the degradation of
cellulose has been demonstrated. This complex exists in cell surface bound
and cell-free forms, and has been shown to be responsible for cellular
adherence to cellulose and for the degradation of cellulose to cellobiose by the
intact organism (2, 16). The cellulosome constitutes the majority of the
endoglucanase activity (~70%) and about one third of the total extracellular
protein, and possesses the major proteins so far reported for the entire
cellulolytic apparatus in this organism (15). Recently, xylanase activity has
also been found to be localized in the cellulosome (22). Our studies in
progress include examining Thermoanaerobacter B6A-RI for the presence of a
xylanosome comprised of endoxylanases, xylan binding proteins and other
xylanolytic enzymes.

Many anaerobic cellulolytic bacteria possess high molecular weight,
multisubunit cellulases which are often found associated with the cell surface
(38) or sedimentable membranous fragments (9). Bacteroides succinogenes

produces CMCase, B-glucosidase, xylanase and B-xylosidase during growth on

99
cellulose, and approximately 50% of each of the released enzymes was

associated with sedimentable subcellular membrane vesicles which adhered
to cellulose (7). In these cellulolytic anaerobes enzyme complexes analogous
to the cellulosome may be present. In Thermoanaerobacter strain B6A-RI, the
finding of predominantly cell bound endoxylanase activity, a strong affinity of
the cells for the substrate and the presence of cell surface protrubances (Lee,
Lowe, Pankratz & Zeikus, manuscript prepared for publication) suggests the
presence of a xylanosome cell surface structure analogous to the cellulosome
in C. thermocellum.

The genes encoding the major endoxylanase and B-xylosidase activities
in Thermoannerobacter strain B6A-RI have been cloned (Lee and Zeikus,
manuscript prepared for publication), and the endoxylanase gene contains a
leader sequence whereas the B-xylosidase gene does not, confirming the
findings from this present study that endoxylanase activity is capable of
excretion from the cell, whereas B-xylosidase intracellular in cell location.

Although there has been great interest in the purification of xylanases,
relatively few attempts have been made using affinity chomatography (26, 30).
This approach has been successful in this study with the finding that
Thermoanerobacter B6A-RI produces multiple endoxylanases. In addition,
endoxylanase activities were associated in a high molecular weight protein
complex as evidenced from gel filtration, and it was difficult to separate this
complex into individual components. The zymogram study showed broad
activity bands due to the presence of multiple endoxylanases of similar
molecular weight. PAS staining indicated that these proteins may be
glycoproteins, therefore, this multiplicity may arise from different
glycosylation of single parent enzyme. However, it can not be ruled out that

this may be the result of proteolysis or RNA processing.

100
Thermoanaerobacter B6A-RI endoxylanase is active at high

temperature and has a temperature optimum similar to other purified
endoxylanases including those from some Bacillus sp. (12, 23), and
Clostridium stercorarium (5). Interestingly the endoxylanase could only bind
to the xylan-coupled Sepharose CL-4B at temperatures above 45°C and
purification attempts at 4°C and at room temperature were not succesful.
This suggests that the substrate binding site of the thermophilic endoxylanase
from Thermoanaerobacter B6A-RI may open at high temperatures. It has
been postulated that thermostable enzymes are compact molecules and need
high temperatures for proper configuration for substrate binding and

catalysis, and the findings in this study support this hypothesis.

10.

11.

LITERATURE CITED

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Bayer, E. A., and R. Lamed. 1986. Ultrastructure of the cell surface
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Bayer, E. A., E. Setter, and R. Lamed. 1985. Organization and
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Béguin, P. 1983. Detection of cellulase activity in polyacrylamide gels
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Groleau, D., and C. W. Forsberg. 1981. Cellulolytic activity of the
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Gruninger, H., and A. Fiechter. 1986. A novel, highly thermostable D-
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102

Honda, H., T. Kudo, Y. Ikura, and K. Horikoshi. 1985. Two types of
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Laemmli, U. K. 1970. Cleavage of structural proteins during the
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Lamed, R., R. Kenig, and E. Setter. 1985. Major characteristics of the
cellulolytic system of Clostridium thermocellum coincide with those
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Lamed, R., E. Setter, R. Kenig, and E. A. Bayer. 1983. The cellulosome: a
discrete cell surface organelle of Clostridium thermocellum which
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activities. Biotechnol. Bioeng. Symp. 13:163-181.

Lee, C., B. C. Saha, and J. G. Zeikus. 1990. Characterization of
Thermoanaerobacter glucose isomerase in relation to saccharidase
synthesis and development of single-step processes for sweetner
production. Appl. Environ. Microbiol. 56:2895-2901.

Lee, S. P., C. W. Forsberg, and L. N. Gibbins. 1985. Xylanolytic activity
of Clostridium acetobutylicum. Appl. Environ. Microbiol. 50:1068—1076.

Lee, Y.-E., M. K. Jain, C. Lee, S.E. Lowe and J. G. Zeikus. 1992.
Taxonomic distinction of saccharolytic thermoanaerobes: Description
of Thermoanaerobium gen. nov., xylanolyticum sp. nov. and
Thermoanaerobium saccharolyticum sp nov. Reclassification of
Thermoanaerobium brockii,Clostridium thermosulfurogenes and
Clostridium thermohydrosulfuricum E100-69 as Thermoanaerobacter
brockii comb. nov., Thermoanaerobacterium thermosulfrigenes comb.
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Thermoanaerobacter ethanolicus. Manuscript submitted to the
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21.

24.

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6105.

Okazaki, W., T. Akiba, K. Horikoshi, and R. Akahoshi. 1985.
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Panbangred, W., E. Fukusaki, E. C. Epifanio, A. Shinmyo, and H.
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Hollaender (ed.), Trends in the Biology of Fermentations for Fuels and
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Schwarz, W. H., K. Bronnenmeier, F. Grabnitz, and W. L.
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31.

32.

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38.

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40.

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Tan, L. U. L., P. Mayers, and J. N. Saddler. 1987. Purification and
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Weimer, P. J., L. W. Wagner, S. Knowlton, and T. K. Ng. 1984.
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41.

105

Zeikus, J. G., C. Lee, Y.-E. Lee, and B. C. Saha. 1991. Thermostable
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Chapter IV

Cloning, sequencing and biochemical characterization of

endoxylanase from Thermoanaerobacter strain BGA-RI

106

ABSTRACT

A genomic library of Thermoanaerobacter B6A-RI was constructed in
Escherichia coli HB101 using the cosmid vector pHC79. A recombinant
plasmid, pXDM1, contained a DNA insert of 28 kb, and gave rise to clones
which had both endoxylanase and B-xylosidase activities. Subcloning of the
plasmid revealed that the two genes were clustered within a 20 kb DNA
fragment. The gene encoding for B-xylosidase (xynB) was located upstream of
the endoxylanase (xynA) gene. xynB was found in a 2.5 kb fragment and xynA
in a 5 kb fragment. A putative 33 amino acid signal peptide was present in
xynA, which corresponded to the N-terminal amino acids. An open reading
frame (ORF) of 3471 bp, corresponding to 1157 amino acid residues, was
found. The molecular weight of the endoxylanase calculated from the
deduced amino acid sequence was 130 kDa. xynA from Thermoanaerobacter
B6A-RI, had strong similarity to genes from family F B—glycanases grouped by
hydrophobic cluster analysis. The distribution of endoxylanase activity was
approximately equal between the periplasm and cytoplasm in the clones
containing xynA. The apparent pH and temperature optimum for the
activity of the cloned endoxylanase was 70°C and 5.5 respectively. The cloned
endoxylanase A was stable at 75°C with no detectable loss of activity over 60
min, and displayed a specific activity of 23 units/ mg protein on oatspelt xylan.
The cloned xylanase was an endo-acting enzyme able to cleave xylan and

xylooligosaccharides, but not xylobiose.

107

INTRODUCTION

Hemicelluloses are the second most abundant polysaccharides in
nature, and they are closely associated with cellulose and lignin in plant cell
walls. The major component of hemicellulose is xylan, a heteropolymer with
a backbone of [HA-linked D-xylose residues and short side chains of arabinose
and methylglucuronic acid (2).

Biodegradation of xylan involves the action of two major xylanolytic
enzymes, namely endoxylanase (1,4-B-D-xylan-xylanohydrolase; B.C. 3.2.1.8)
and B-xylosidase (1,4-B-D-xylan-xylohydrolase; B.C. 3.2.1.37). The endoxylan-
ase randomly cleaves the internal xylosidic linkages of the xylan backbone,
yielding xylooligosaccharides of various lengths which are further hydrolyzed
to xylose molecules by B-xylosidase. The hydrolysis of B-1,4-xylan plays an
important role in the conversion of renewable resources such as straw and
wood into easily fermentable products.

Some possible applications for xylanases in the pulp and paper industry
would require cellulose—free xylanase preparations to prevent cellulose
degradation (36). Thermophilic xylanases could have significant advantages

over meSOphilic xylanases because of their thermostability and catalytic
efficiencies at high temperatures (9). Enzyme catalyzed xylan hydrolysis
depends on the thermostability of xylanase in order to obtain higher reaction
rates and greater solubility of reactants at high temperatures. Fungi such as
Thermoascus aurantiacus (48) and Humicolosa grisea (27), and bacteria such
as Bacillus sp. (37), Clostridium stercorarium (1), Clostridium thermolacticum

(8) and Clostridium thermocellum (28) have been reported to produce

thermostable xylanases.

108

109
Xylanases from various microorganisms have been cloned and

expressed in E. coli (11, 20, 24, 25, 33, 41, 46, 47), but there have been very few
reports on thermostable xylanases from thermophilic anaerobic bacteria (15,
24).

A number of thermoanaerobic bacteria have been known to have
xylanase activity including Thermoanaerobacter ethanolicus, Thermobacter-
aides acetoethylicus, Thermoanaerobium brockii, (42, 43) and C.
thermocellum (28) which all ferment xylan albeit at a very slow rate, and
Thermoanaerobacter strain B6A (39) which was shown to extensively degrade
xylan (38). However, comparatively little is known about the molecular and
biochemical properties of the enzymes from these strains.

Thermoanaerobacter strain B6A-RI is an non-cellulolytic thermophilic
anaerobic bacterium capable of degrading a wide range of carbohydrates
including xylan (23). We have previously shown that the synthesis of
endoxylanase and B-xylosidase in Thermoanaerobacter strain B6A-RI is
induced by xylan and xylose. In this bacterium the degradation of xylan to
xylooligosaccharides is catalyzed by extracellular or cell-bound endoxylanases
followed by hydrolysis to D-xylose by B—xylosidase.

In order to investigate the molecular mechanism of xylan degradation
by Thermoanaerobacter strain B6A-RI and to overproduce thermophilic
xylanases, we have constructed a genomic library of this organism using the
cosmid vector pHC79, from which we isolated a cosmid clone which had both
the endoxylanase and B-xylosidase activities. In this report we describe the
Cloning and expression of Thermoanaerobacter xylanase genes in E. coli, and

the biochemical and molecular properties of a recombinant endoxylanase

Which was purified to homogeneity.

MATERIALS AND METHODS

Bacterial strains and plasmids

The xylanolytic thermoanaerobic bacterium used in this study is
Thermoanaerobacter strain referred to as strain B6A-RI (23). Escherichia coli
strains HB101 (F‘mch mrr hsd520[r3‘m3'] recA13 supE44 aral4 galK2 lach
proA2 rpsL20[Sm'] xylS A: leu mill) and DH50: (F- ¢80dIacZ A[lacZYA-
argF]U169 deoR recAl endAl hst17[rK', mx’r] supE44 A— thi-l gyrA96 relAl)
were used as the recipient strains for recombinant plasmids. Cosmid vector
pHC79 was used for the construction of a genomic library, and plasmid pUC18

or pUC19 was used for subcloning.

Chemicals, media and culture conditions

All chemicals were of reagent grade and were of the highest purity
available. Thermoanaerobacter strain B6A-RI was grown in TYE medium
(50) containing 0.5% xylose at 60°C. LB medium (10 g of tryptone, 5 g of yeast
extract, and 5 g of N aCl per liter) was used to grow the E. coli strains. For agar
plates 1.5% (w/v) Bacto-agar (Difco Laboratories, Detroit, MI) was included.
The medium was supplemented with ampicillin (50 ug/ ml ) for E. coli strains

carrying a vector or recombinant plasmids.

Plate assay for the detection of thermostable endoxylanase

LB agar plates containing 0.2% (w/v) 4-O—methyl-D-glucurono-D-xylan-
Remazole Brilliant Blue R (RBB-xylan), and methylumbelliferylxyloside (10
111M) (Sigma Chemical Co., St Louis, M0) were used for screening of the

endoxylanase and B-xylosidase activity respectively. E. coli was plated onto

110

l l 1
these media and incubated overnight at 37°C. The plates were transferred to

60°C for 2 to 4 hr to detect the enzyme activities.

Preparation of DNA

Thermoanaerobacter strain B6A-RI was grown anaerobically to early
stationary phase at 60°C, and chromosomal DNA was isolated by a
modification of the Marmur method (26) as described previously. Plasmid
DNA was purified from the cleared lysate by the procedure described by
Clewell (7), followed by ultracentrifugation in a cesium chloride-ethidium
bromide density gradient. For screening of recombinant plasmids in
transformants, the rapid alkaline extraction procedure of Birnboim and Doly

(5) was used.

Construction of genomic library of Thermoanaerobacter B6A-RI and cloning
procedure in E. coli

The basic procedure for cloning was conducted according to the method
of Sambrook et al. (32). Chromosomal DNA of Thermoanaerobacter strain
B6A-RI was partially digested with Sau3A and 30 to 40 kb fragments were
isolated from an agarose gel by electroelution, using an electroelution
apparatus, Elutrap (Schleicher & Schuell, Inc. Keene, NH). The cosmid vector
pHC79 was linearized with BamHI and dephosphorylated with Calf intestine
phosphatase. The partially digested chromosomal DNA fragments were
ligated with linearized dephosphorylated pHC79 at a molar ratio of 1:2. The
ligated DNA was packaged with a lambda in vitro packaging kit as described
by the manufacturer (Amersham Co., Arlington Heights, IL). E. coli H8101
was infected with the recombinant phage preparation and plated onto LB agar

plates containing ampicillin. The resulting transformants were screened on

l l 2
LB agar plates containing 0.2% (w/v) RBB-xylan and ampicillin (50 ug/ ml).

Xylanase activtiy was detected as a clear halo around colonies on RBB-xylan

plates after incubation at 60°C.

Restriction enzyme mapping and subcloning of the endoxylanase gene (xynA)

After digestion with the appropriate restriction endonucleases the
desired fragments were eluted from the agarose gel by electroelution using
the Elutrap, and subcloned into the plasmid pUC18. Each subclone was
transformed into E. coli DHSoc, spread on an LB agar plate containing
ampicillin, and incubated overnight at 37°C. The endoxylanase and B-
xylosidase activity was determined using RBB-xylan plates and LB agar plates
containing methylumbelliferylxyloside, respectively and recombinant clones
were identified by the restriction pattern of the plasmid DNA isolated by rapid

alkaline extraction.

Southern hybridization

Chromosomal DNA from Thermoanaerobacter strain B6A-RI and E.
coli HB101 were digested completely with restriction enzymes,
electrophoresed through agarose gels and stained with ethidium bromide.
For hybridization with radiolabed probes, the DNA was transferred to a Zeta-
Probe blotting membrane (Bio-Rad Laboratories, Richmond, CA) by the
method of Southern (34). Gel-purified DNA fragments were labeled with a
random prime DNA-labeling kit (Boehringer Mannheim, Indianapolis, IN)
and a-32P dATP (3,000 Ci/mmol, New England Nuclear, Wilmington, DE)

according to the manufacturers instructions. Hybridization of the DNA was

by the method of Sambrook et al. (32).

l l 3
DNA sequencing

Double-stranded DNA was denatured by the method of Zhang et a1.
(51) and was sequenced in both directions using the dideoxy-chain
termination method and Sequenase version 2.0 kit (United States
Biochemical Co. Cleveland, OH) according to the manufacturer's instructions.
Additional sequencing primers were synthesized by the Michigan State
University Macromolecular Facility.

DNA sequences were analyzed by using the GENEPRO software
package (Hoefer Scientific Instruments, San Francisco, CA) and the University

of Wisconsin Genetics Computer Group GCG package (version 7) (10).

Localization of endoxylanase in E. coli transformants

Cells were subjected to osmotic shock for release of periplasmic
proteins as described previously (44). Following osmotic shock, the cells were
suspended in 50 mM sodium acetate buffer, pH 5.5, and sonicated for five
cycles of 20 seconds with 1 min intervals, keeping the material on ice. Cell

debris was removed by centrifugation at 12,000 X g for 10 min, and the

supernatant was retained as the cytoplasmic fraction.

Purification of cloned endoxylanase

Unless otherwise stated, all operations were performed at room
temperature.
6) Preparation of cell extract: One liter of an overnight culture of E. coli
DHSOL (pZEP12) grown in LB medium containing ampicillin (50 ug/ ml) was
centrifuged at 5,000 X g for 20 min. Cells were resuspended in 40 ml of 50
mM Sodium actate buffer, pH 5.5, and disrupted by passage through a French

pr eSSure cell (American Instrument Co., Inc., Silver Spring, Md.) at 20,000

1 14
lb / inz. The cell extract was centrifuged at 12,000 X g for 20 min to remove cell

debris, and the supernatant was used as the crude enzyme preparation.

(ii) Heat treatment: The cell extract of E. coli DHSa (pZEP12) was heated at
75°C for 10 min in a shaking water bath, and chilled in ice. The denatured
proteins were removed by centrifugation at 12,000 X g for 20 min, and the
soluble fraction used for endoxylanase purification. The heat-treated soluble
fraction was concentrated by centrifugation on an Amicon YM 30 membrane
with a 30 kDa molecular weight cut-off (Amicon Co., Danvers, MA).

(iii) Anion exchange column chromatography: The concentrated sample
was loaded on a column (2.5 cm x 18 cm) of Q-Sepharose (Pharmacia,
Piscataway, NJ) previously equilibrated with 50 mM sodium acetate buffer
(pH 5.5). The column was washed with the same buffer and eluted with a 500
ml linear NaCl (0 to 1.0 M) gradient in the same buffer. The active fractions
were collected and pooled.

(iv) Affinity column chromatography: The active fractions were pooled and
concentrated by ultrafiltration using a YM 30 membrane (Amicon). For
affinity chromatography a Pharmacia column (1.5 cm x 33 cm) fitted with a
thermostatic jacket connected to a water bath, for temperature control was
filled with xylan-coupled Sepharose CL-4B (prepared as described in Chapter
III). The column was equilibrated with 50 mM sodium acetate buffer (pH 4.0)
at room temperature and the protein sample was loaded once the column
had equilibrated to 45°C. After washing with 50 mM sodium acetate buffer
(pH 5.5) at room temperature the xylanases were eluted with soluble xylan
(0.1%) in 50 mM sodium acetate buffer (pH 5.5) at room temperature. The
active fractions were pooled and incubated at 60°C for 2-4 h to digest xylan
coeluted with enzyme, and the hydrolysis products were removed by dialysis

against 50 mM sodium acetate buffer (pH 5.5).

115

Enzyme assays and protein determination

Endoxylanase activity was measured by the release of reducing sugars
from oat spelt xylan (Sigma Chemical Co., St. Louis, Mo.). B-xylosidase
activity was determined by the formation of p-nitrophenol from p-
nitrophenyl-B-D-xyloside (Sigma). The assays were performed as described
previously (Lee. Lowe & Zeikus, manusrcipt prepared for publication).

One unit of activity was defined as the amount of enzyme that
produced 1 umol of reducing sugar per min for endoxylanase, and 1 umol of
nitrophenol per min for B-xylosidase.

Protein concentration was determined by the dye-binding assay of
Bradford (6) with the Bio-Rad protein assay reagent (Bio-Rad Laboratories,

Richmond, CA) with bovine serum albumin (Sigma) as the standard.

Electrophoresis and molecular weight determination

SDS-PAGE was performed as described previously (22) with 7.5%
acrylamide using a Mini-PROTEAN II system (Bio-Rad Laboratories,
Richmond, CA). Proteins bands were visualized by Coomassie brilliant Blue
G-250 staining. The molecular weight of the denatured protein was
estimated by SDS-PAGE with high-range molecular weight standards (Bio-
Rad Lab. Richmond, CA) including myosin (200,000), B-galactosidase (116,250),
phosphorylase (97,400), BSA (66,200) and ovalbumin (43,000).

N-terminal amino acid sequence determination
The protein was prepared by washing the purified endoxylanase with
double-distilled water five times with a Centricon-30 (Amicon) filtration

device to remove contaminating salts and buffer from the solution. The N-

l 1 6
terminal amino acid sequence was identified by a protein sequencer model

477A (Applied Biosystems, Foster City, CA) with an on-line
phenylthiohydrantoin analyzer (Applied Biosystems) in the Macromolecular

Structure Facility, Department of Biochemistry, Michigan State University.

Analysis of products of xylan hydrolysis by thin layer chromatography

The xylan and xylooligosaccharides were dissolved in 50mM sodium
acetate buffer (pH 5.5) and incubated at 60°C with approximately 1.5 U of
purified enzyme. Samples were withdrawn periodically and the hydrolysis
products were analyzed by Whatman HP-K high performance silica gel plates
(10 x 10 cm) using the following solvent system: butanol/ acetic acid/ distilled
water (2:1:1, by vol.), and were visualized using an orcinol spray reagent (100

mg orcinol in 100 ml 20% sulphuric acid in methanol).

RESULTS

Construction of a genomic library and cloning of xylanolytic genes

In order to detect xylanolytic genes, a genomic library of
Thermoanaerobacter B6A-RI was constructed in E. coli HB101 using the
cosmid vector pHC79. Approximately 800 transformants were obtained.
Xylanase activity was detected directly on the RBB-xylan plates. Two colonies
produced clear halos, indicating the presence of xylanase activity. E. coli
HB101 harboring pHC79, lacking cloned xylanolytic genes, did not clear RBB-
xylan plates. When plasmid DNA was isolated from these positive clones
and subjected to restriction enzyme analysis, both plasmids were found to
contain a DNA insert of ca. 36 kbp with identical restriction patterns. This
recombinant plasmid was designated as pZXAl and its physical map was
constructed (Fig. 1).

Genes involved in hemicellulose degradation have been found to be
clustered in a number of bacteria. Due to the coordinate regulation during
growth of Thermoanaerobacter B6A-RI on xylan and xylose (Lee, Lowe &
Zeikus, manuscript in preparation), the hemicellulase genes may be closely
situated on the chromosome. To investigate the xylanolytic activities
expressed in the cosmid clone, E. coli HB101 containing the recombinant
plasmid pZXAl was grown on LB medium and assayed for various enzymatic
activities. High levels of B-xylosidase and endoxylanase activity but relatively
weak acetyl-esterase and arabinosidase activities were detected in the crude
extract of the clone (data not shown).

The growth of E. coli HB101 harboring the recombinant plasmid
pZXAl was poor and some colonies lost the ability to give clear halos on RBB-

Xylan plates indicating that the cosmid clone was not stable. After several

117

118

pZXAl

 

 

E E
2? : E g E
x 8 8 x (E
I 1

31th

l__1

Figure 1. Linear restriction map of plasmid pZXAl which carries the
Thermoanaerobacter B6A-RI DNA insert expressing thermostable

endoxylanase.

The open box represents Thermoanaerobacter chromosomal DNA

insert and closed box represents cosmid vector pHC79.

l l 9
transfers on LB medium supplemented with ampicillin, colonies were

screened on RBB-xylan plates for endoxylanase activity and on LB agar plates
containing methyl-umbelliferylxyloside and ampicillin for B-xylosidase
activity in order to isolate stable clones having both activities. One colony
which consistently gave both activities was isolated and the plasmid DNA
obtained. This plasmid, designated as pXDM1 (Fig. 2), had a smaller size of
DNA insert (28 kb) than the original clone indicating that the insert had

undergone a partial deletion during propagation in the E. coli host.

Subcloning of the endoxylanase A gene

To localize the functional domain of the endoxylanase (xynA) and [3-
xylosidase (xynB) genes, several subcloning experiments were performed
using plasmid pUC18. E. coli DHSa cells were transformed with subcloned
plasmid DNA, and plated on RBB-xylan plates, and LB agar containing
methyl-umbelliferylxyloside, to detect subclones expressing xylanase and B-
xylosidase activity, respectively. Clones exhibiting endoxylanase activity were
visualized directly as clear halos around the colonies due to the diffusion of
the R88 dye released by cleavage. Clones exhibits B-xylosidase activity were
visualized under ultraviolet as colonies having a blue-white fluorescence,
due to the diffusion of the umbelliferone released by cleavage. The
subcloning strategy and the resulting enzyme activities are summarized in
Fig. 2. The two genes were clustered within a 20 kb DNA fragment, but not
next to one another. The gene encoding for B-xylosidase (xyn B) was located
upstream of the endoxylanase (xynA) gene. Using restriction deletions, xynB
was found to be a 2.5 kb fragment and xynA within a 5 kb fragment.

To confirm the chromosomal origin of the DNA insert in pZEP12

Southern hybridizartion was performed on chromosomal DNA from E. coli

120

Figure 2. Physical map of plasmid pXDM1 and functional mapping
of the endoxylanase and B-xylosidase gene domain.

The 28 kb Thermoanaerobacter DNA insert was subjected to
subcloning at various restriction sites (B, BamHI; E, EcoRI; H, HindIII;
P, PstI; S, SalI; X, XbaI). Deleted areas are indicated with dotted lines.
Solid lines indicate subcloned fragments. The hatched areas represent
the location of the genes. The relative amounts of enzyme activities

found in E. coli carrying the DNA fragments is denoted by + or -.

121

+++

.9338
3382?-n

.++++

+

.338“
353320

mcax

 

 

 

122

Figure 3. Southern hybridization analysis of Thermoanaerobacter
BéA-RI and E. coli genomic DNA digests with random-primed DNA
synthesized from the 1.2 kb HincIl-Pstl fragment from pZEP12.

(A) Photograph of 0.8% agarose gel stained with ethidium
bromide. (B) Autoradiogram of DNA transferred to membrane and
hybridized with the 32P-labeled insert fragment. Lane 1, molecular
weight markers; lane 2, E. coli chromosomal DNA digested with PstI;
lane 3, 4 8: 5, Thermoa naerobacter B6A-RI chromosomal DNA
digested with PstI, EcoRI and XbaI, respectively.

123

A B

,(kb) 12345 ‘12345

21.8

2%
3.4 .
2.0
1.9

-
1.7
1.3

 

124
and Thermoanaerobacter B6A-RI digested with EcoRI, PstI and XbaI. An

internal 1.2 kb HincII-Pstl fragment from pZEP12 was used as a probe for
endoxylanase (xynA). The results are shown in Fig. 3. The fragment did not
bind to E. coli chromosomal DNA but did hybridize to Thermoanaerobacter
BéA-RI genomic DNA, indicating that the gene originated from

Thermoanaerobacter B6A-RI.

xyn A sequence analysis

A deletion plasmid containing xynA, designated as pZEP12, was
constructed from pXDM1, and the detailed physical map of xynA is shown in
Fig. 4. The nucleotide sequence of a EcoRI-PstI fragment in pZEP12,
determined by the dideoxy-chain termination method, is shown in Fig. 5.
The sequence is consistent with the observed restriction map. An open
reading frame (ORF) of 3471 bp, corresponding to 1157 amino acid residues,
was found. The molecular weight of the enzyme calculated from the deduced
amino acid sequence was 130 kDa. The N-terminal amino acid sequence of
the endoxylanase, purified from E. coli (pZEP12), is shown as underlined in
Fig. 5. The N-terminal sequence was preceded by a putative 33 amino acid
signal peptide in xynA (boxed area in Fig. 5). The endoxylanase activity of
Thermoanaerobacter BéA-RI was excretable (Lee, Lowe & Zeikus, manuscript
prepared for publication), therefore it was expected that the endoxylanase had
a signal peptide. Hydrophobicity analysis (data not shown) of the deduced
amino acid sequence present in this putative signal peptide also supports the
proposed function for this 33 amino acid sequence. The putative initiation
codon, ATG, was found in two separate places upstream of the cleavage site of
the signal peptide. The putative ribosome-binding sequence, S'-AGGGAGG-

3', was observed 5 bp upstream of the first intiation codon, ATG.

125

 

 

DZEPIZ G.Skb
l__l

Egg E§§§§§§=§§§z
mil: E 31355355 8355 .3 13 d:
If I \I Ill 1/ I I l |
-

xgnA ‘
l

 

 

* ’/////////////////// *

Figure 4. Physical and genetic map of the deletion plasmid pZEP12 containing
xynA contructed from pXDM1.

The cloned Thermoanaerobacter B6A-RI DNA insert is indicated by the
open bar, and pUC18 by the closed bar. The location of the endoxylanase

(xynA) and the location of transcription is shown by the arrow. Shaded bar

indicates the DNA fragment used as a probe.

126

Figure 5. Nucleotide sequence and translated amino acid sequence for
the endoxylanase A gene from Thermoanaereobacter B6A-RI.

The boxed area represents the putative signal peptide. The
arrow denotes the cleavage site of the signal peptide. The dashed
region denotes the N-terminal amino acid sequence as determined
from the purified recombinant endoxylanase. The numbers beneath
the sequence denotes the amino acid number after cleavage of the

signal peptide.

 

61

121

181

241

301

361

421

481

541

601

661

721

781

841

127

CACATTACAGTATGAA‘ITGCAGAAAG‘ITTTGCAGTCAACACAGCAATATACACAGCAAGC

ATcamcaiamococmimmmrrcrnmchofiacacc'rcmocarncacc
'rc;cramaciaracmccasmmcmirecacmrfimmsccmcai
mmrmmmaéumcmamrammarimmmé
GGGGANMRMTAMM-rmncamiacrcméammccrcmmmr

AATTTTTCAATAATTAAITTTACACATGTTACACAGGGAGGTATTATGATGAAAAAEAAT
5.0. HetMetLysAsnAsn

 

 

GTAGATAGGATTGTATCTATTGTTACAGCTTTAATAATGATTTTTGGAGCATCATTATTT

ValAspArgI1eVaISerI1eVa1ThrA1aLeuIleMetIlePheGlyXIaSerLeuPhe

TCCCCGCCAATAAGAGTTTTTGCTGATGACACTAATATAAATCTGGTTTCTAATGGGGAC'

 

SerProProIleArgValPheAl pAspThrAsnIIeAsnLeuvaISerAsnclyAsp
. ?&r"'"""'”"""”'“-""'

TTTGAATCAGGCACAAITGATGGCTGGATTAAGCAAGGTAATCCGACATTAGCAGTAACG
PheGluSerGlyThrIIeAspclyTrpIleLysGInGIyAsnProThrLeuAIavalThr
2° .

ACTGAGCAAGCAATTGGGCAAIACAGTATGAAAGTTACTGGTRGAACACAGACAmATGAA
ThrGluGlnAlaIloclyclnTyrSerHetLysvalThrGIyArgThrGlnThrTyrGIu
40

GGACCTGCATATAGCTTTTTAGGAAAAATGCAGAAAGGTGAATCATATAGTGTATGGCTT
G1yProAlaTyrSerPheLeuigyLysuetGlnLysGIyGluSerTyrServa18erLeu

AAAGTTAGACTTGTTTCTGGACAAAATTCATCTAATCCTTTGATTACCGTGACTATGTTT
LysValArgLeuVa189:6lyfifipAsnSerSerAsnProLeuI1eThrVa1ThrKetPhe

AGAGAAGATéACAATGGCAAGCKTTACGAQACAAIAGTTTGGCAAAAACAAGTTTCTGAA
ArgGluAspAspAsnG1yLys¥égTyrAspThrIleValTrpclnLysGInVa18er61u

GATTcATGGACTACTGTAAGcGGAACTTATAcATTAGATTATATTGGAACATTGAAAACA
AspSerTrpThrThrVa1SerGlyThrTerhrLeuAspTyrIleGlyThrLeuLysThr
120

TTATATATGTATGTAGAATCACCCGATCCAACGCTGGAATACTATATTGATGATGTTGTA

LeuTeretTera161uSerfi:gAspProThrLeuGluTeryrI1eAspAspVa1Va1

60

120

180

240

300

360

420

480

540

600

660

720

780

840

900

901

961

1021

1081

1141

1201

1261

1321

1381

1441

11501

11561

Il621

128

GTAACAACACAAAATCCAATTCAAGTAGGGAACGTAATTGCCAATGAAACTTTTGAAAAT
ValThrThrGlnAsnProIlecanalclyAanaIIIeAIaAsncluThrPhecluAsn
160

GGAAATACTTCTGGATGGATTGGAACAGGTTCATCTGTTGTTAAGGCAGTGTATGGAGTG
GlyAsnThrSerGlyTrpI1eG1yThrGlySerSerValvalnyshlavalTyrclyval
180 ,

GCTCATAGCGGAGATTATAGCTTATTGACGACAGGGAGAACAGCTAATTGGAATGGTCCT

AlaflisSerGlyhspTyrSerLeuLeuThrThrGIyArgThrhlaAsnTrpAsnGlyPro
200

AGCTATGATTTGACTGGCAAAATAGTGCCTGGTCAACAATACAATGTTGATTTTTGGGTG
SerTyrAspLeuThrGlyLysageValProGlyG1nGlnTyrAsnvalAspPheTrpva1
O

AAATTTGTTAATGGCAACGATACAGAACAAATAAAGGCTACTGTTAAAGéGACTTCTGAé
LysPheva1A3nGlyAsnAspgagcluclnIleLyshlaTbrvalLysAIaThrSerAsp

AAAGACAATTATATAGAAGTTAATGATTTTGCAAATGTAAAQAAAGGAGAATGGACAGAA
LysAspAsnTyrIlecanalhsnAspPhehlaAanalhsnLysclycluTrpThrGIu
260

ATAAAAGGCAGTTTTACTTTACCTGTTGCAGATTAGAGCGGTATTAGCATCTATGTAGAA
IleLysG1ySerPheThrLeu356Va1A1aAspTyrSerGlyI1eSer11eTera1G1u
0

TCTCAAAATCCTACTTTAGAGTTTTACAITGAIGATTTTTCTGTAATIGéTGAAATTTCi
SorelnlsnProThrLouGIugggryrIIeAspAspPheSerValI1eG1yGIu11eSer

AATAATCAGATTACGATACAAAATGACATTccAGATTTAIATTCAGTAETTAAAGATIHT
AsnAsnGlnIleThrIleclnggghspIleProAspLeuTyrSerVa1PheLysAspTyr

TTTCCTATAéGCGTTGCAGTTGATCCAAGéAGATTAAATGATGCTGATCOACATGCTCAA
PheProIleclyVa1A1aValgggProserArgLeuAsnAspAlaAspProB1shlaGln

TTGACGGCTAAACATTTTAATATGCTTGTTGCAGAAAAOGCCATGAAACCGGAAAGCTTG
LeuThrAlaLysHisPheAsngggneuVa1A1aGluhsnhlanetLyaProGluSerLeu

CAACCTACAéAAGGGAACTTTACCTTCGATAATGCTGATAAAATTGTTGACTATGCAKTA
GlnProThrGluGlyAsnPhegrfipheAspAsnAlahspLysIleVa1AspTyrA1alle
. 8

GCACATAATATGAAAAT6AOAGGTCATACTTTACTTTGGCATAATCAAGTTCCAGATTGG
A1aHisAsnMetLysMetArgclyaisThrLeuLeuTrpHisAsnGanalProAspTrp
400

960

1020

1080

1140

1200

1260

1320

1380

1440

1500

1560

1620

1680

1681

1741

1801

1861

1921

1981

2041

2101

2 161

12221

12281

2341

2401

129

TTTTTCCAAGATCCATCTGACCCATCcAAGTCTGCTTGGAGAGATTTACTGCTTCAALGA
PhePheGInAspProSerAspPrOSerLysSerAlaSerArgAspLeuLeuLeuG1nArg
420 .

TTAAAAACGCATATAACAKCTGTGTTAGACCATTTTAAAACAAAGTATGGTTCTCAAAAT
LeuLysThrHisIleThrThrvalLeuAspHisPheLysThrLysTyrGlyserclnhsn
440

CCAATAATCGGATGGGAIGTTGTAARTGAGGTTCTTGATGATAATGGCAKTTTAAGAAAT
ProI1e!1eG1yTrpAspVa132333nG1uva1LeuAspAspAsnGlyhsnLeuArgAsn

TCTAAGTGGTTGCAGAEIATAGGACCTGHTTATATAGAAAAAGCTTTTGANTNTGCACAT

SerLysTrpLeuGInI1eIleGlyProAspTyrI1eGluLysAlaPheGluTyrhlafiis
480

GAAGCAGATCCATCTflTGAAATTGTTTATTAATGACTATAACATTGAAAKTAATGGTGTT

GluA1aAspProSerfletLysLeuPheIlehsnAspTyrAsnIlecluAsnAsnclyval
500

AAGACGCAGGCTATGTATGACTTAGTGAAAAAATTAAAGAGTGAAGGTGTGCCAATDGAI
LysThrGlnAlaHetTyrAspLeuVa1LysLysLeuLysSerGluGlyva1ProIlahsp
520

GGAATAGGCATGCAAATGCACATAAATATAAATTCAALTATTGACAkrkéhAAAGCTTCT
GlyIlealyuetclnuetfiis§1333n1lensnSerAsnlleAspAsnIleLyshlaSQr
4

ATAGAAAAACTTGCTTCATTAGGTGTGGAAAIACAAGrAACTGAATmAGAINTGAACare
IleGlunysLeuALaSerLeuclyva1G1uIleGlnvalThrGIuLeuAspuatAsnlbt
- 560

AACGGTAAIATATcmAACGAAGCAITGcrihhAcAAGCTAGAITGIATAAGCANTTGTTi
AsnGlyAsnIleSCrAsnGlugiaLeuLeuLysGInAlaArgLeuTerysGlnLeuPhe
o

GAITTQITTIAAGCTGAGAAACAGrATAILACTGCTGTAGTTTTTTGGGGAGTTTCAGAT
AspLeuPheLysAlaGluLysGlnTyrIleThrAlaVa1Va1PheTrpGlyVa18erAsp
600

GAICTkACTTGGCTTAGTRAGCCAAATGCTCCGCTGCTTTTTGATTCAAAATTGCAGGCG

AspVa1ThrTrpLeuSerLyszgoAsnA1aProLeuLeuPheAspserLysLeuGlnhla
0

AAGCCAGCATTCTGGGCAGTAGTmGATCCAAGCAAAGCTATACCTGATATTCAATCTGCA
LysProAlaPheTrpAlaValaidhspproSerLysAlaIlePrOAspIleclnSerAIa
0

AAAGCTTTAéAAGGCTCACéGACGAITGGAGCAAATGTTGATAGTTCTTGGAAACTTGTA
LysAlaLeuG1uGlySerProThrIleclyAIaAanaInspSerSerTrpLysLeuval
660

1740

1800

1860

1920

1980

2040

2100

2160

2220

2280

2340

2400

2460

2461

2521

2581

2641

2701

2761

.2821

2881

2941

3001

3061

3121

3181

13()

AAACcATTGinTGTAAATAéTTATGTAGAAGGAAcGGTCGGAGCAACTGCTACTGTTAAG
LysProLeuTera1AsnThrTyrvalcluclyThrVa161yAIaThrA1aThrVa1Lys
680 '

TcTATGTGG8AmACTAAAAACTTGTATTTéTTAGTACAAGTTTCAGACAAIACTCCATCT
8eruetTrpAspThrLysAsnLeuTereuLeuVaIGInVa18erAspAsnThrProSer
- 700 '

AATmATGATGGTHTTGAGAITTTTGTAGAIAAGAATGACGATEAATCTACTTCTTATGAA
AsnAsnAspGlyIleGIuIlephevaInspLysAsnAspAspLysSerThrSerTyrclu
720

ACTGATGAIGAAOGTTATACAKTTAAGAGGGATGGTACAGGGAGCTCAGATKTTACCAAA
ThrAspAspGluArgTerhrIIeLysArgAspclyThrG1y8erSerAspIlerhrnys
_ 74

TATGTGACRTCTAATGCTGACGGATATGTAGCACAGCTAGCTATTCCAATTGAAGAIHTC
TeralThrSerAsnAIaAspclyTyrva1A1aGlnLeuA1aIleProIlecluhsplle
. 760 '

AGTCCTGCAGTTAATGATmAAATTGGATTTGACATTAGAATAAACGATGAIAAAGGTAKT
SerProAIaVaIAsnAspLyaIleclyPheAspI1eArgIlehsnhsphspnysGlyAsn
780

GGTAAAAIAGATGCAATAACGGTTTGGAATGATTATACCAACAGTCAAAATACTAATACA
GlyLysIlehsphlalleThrValTrpAsnAspTerhrAsnSerGlnAsnThrAsnThr
800

TCGTIETTTOGAGAIL1TGTIITGTCAAAATCTGCACAAAICGCAACAGétNTNENTGGé
SerTerheGlyAspI1eVa1Le:SerLysSerA1aG1nI13A1aThrA1aIleTyrGIy
82

ACACCAéTTATTGATGGAAAAGTaGAIGAiaITTGGAATAATGTTGAACCTATTTCGACA
ThrProValI1eAspGlyLysVa1AspAspIleTrpAsnAsnvalcluProI1eSerThr
' 840

AATACAIGGATTTTGGGTTCAAATGGTGCTACTGCGACAéAGAAAATGATGTGGGAOGRT
AsnThrTrpIleLeuGIySerzgnG1yA1aThrA1aThrGlnLysHetMetTrpAspAsp
o

AAGTACCTTTATGTTTTGGCGGATGTTACAGATTCAAATCTGAACAAATéCAGTAITAH2
LysTereuTera1LeuA1aAspvalThrAspSerAsnLeuAsnLysSerSerIIeAsn
880

CCATATGAACAAGATTCTGTAGAAGTTTTTGTAGATCAGAATAATGAIAAGACAACTTKé
ProTyrGluGlnAspServaIGIuValPheva1AspGlnhsnAsnAspLysThrThrTyr
900

TATGAGAATGATGACGGCCAAIATAGAGTCAACTATGACAATGAACAGAéTTTTGGGGGA

TyrGluAsnAspAspGlyGlnTyrArgVa1AsnTyrAspAsnGIuG1nSerPheG1yGly
920

2520

2580

2640

2700

2760

2820

‘2880

2940

3000

3060

3120

3180

3240

3241

3301

3361

3421

3481

3541

3601

3661

3721

3781

3841

3901

3961

131

AGCACCAATTCAAATGGGTTTAAGTCAGCAACAAGTCTTACACAAAGTGGATATATTGTA
SerThrAsnSerAsnG1yPheLysSerA1aThrSerLeuThrG1n8erGlyTyrIleval
940 .

GAAGAAGCCATTCCTTGGAéGAGTATCACTCCGTCAAATGGCACTATCATRGGAITTGAC
GluGluAlaIleproTrpThrSerI1eThrProSerAsnGlyThrIleIleclyPheAsp
960

TTGCAAGTTAATAATGCAGATGAAAAIGGTAAGAGGACAGGTITTGTCACATGGTGCGAT
LeuGanalbsnAsnhlaAspGluAsnclyLyaArgThrclyIlevalrhrTrp0ysAsp
980

cccaccccciamcamcimammcrscammacmmacaccruc
ProSarGlyAsnSerTrpGInAspThrSerclyPheGlyAsnLeuLeuLeuThrclyLys

1000
CCATCTGGTéCCCTTAAAAAAGGTGTGACATTTGATGACATAAAGAATAGTTGGGCAAAA

ProSerGlyAlaLeuLyaLysGfiyyalThrPheAspAspIleLysAsnSerTrphlaLys
10

GACGCAATAéAAGTATTAGCATCAAGGCATATAGTAGAAGGAATGACAGACACTCAGTAT

AsphlallecluVaILeuAIaSerArgfiisIlevalcluclyfletrhrAspThrclnmyr
1040

GAACcAAACAAGACAGTGACAAGAGCAGAATTCACAGCAATGATACTGAGGCTTTTAAAC
GluProAsnLysThrvalThrArgnlaGluPheThrAIaMetIleneuArgLeuLeuAsn
1060

AIAAAAGAAGAGCAAmACAGTGGAGAATTTAGCGATGTAAATIGTGGAGACTGGTATGCA
IleLyscluGluclnTyrSerclyGluPheSerAspVa1&5n8erGlyAapTrpTyrA1a
1080

AATGCAATAGAAGCAGCAEATAAAGCGGGAATAATCGAAGGTGAOGGAAAAAATGCAAGé
AsnAlaI1e61uA1aA1aTerysAlaGlyIleIlecluG1yAspGlynysAsnA1aArg
1100

CCTAATGACAGCATAACAAGAGAAGAGATGACGCAATAGCCATGAGGGCATACGAGATGé
ProAsnAspSerIleThrArgGIucluHatThrGInEnd
1120

TGACACAGTACAAAGAAGAAAACATAGGTGCAACATCATTTAGCGATGACAAATCCAIAA

GCGACTGGGCAAAGAATGTAGTGGCAAATGCGGCAAAACTAGGAATAGTAAATGGAGAGC

CAAATAACATGTTTGCACCTAAAGATATAGCTACGAGAGC 4000

3300

3360

3420

3480

3540

3600

3660

3720

3780

3840

3900

3960

l 3 2
Comparison of the deduced amino acid sequence of xynA from

Thermoanaerobacter BéA-RI with GenBank and EMBL sequence libraries (see
Table 1) indicated that xynA has a strong similarity to genes from family F B—

glycanases, grouped by hydrophobic cluster analysis (12, 19).

Localization and biochemical characterization of the purified endoxylanase A.

To determine the location of the endoxylanase A produced in E. coli,
cells were grown to early stationary phase and subjected to osmotic shock to
isolate periplasmic enzymes, and sonicated to isolate intracellular enzymes
(data not shown). The distribution of endoxylanase activity was
approximately equal between the periplasm and cytoplasm in the clones
containing xynA. These data suggest that the signal peptide of
Thermoanaerobacter BéA-RI is recognized and processed by E. coli cells,
because of the periplasmic location of the enzyme.

Thermoanaerobacter B6A-RI endoxylanase A expressed in E. coli was
purified to homogeneity (Table 2). Heat treatment of cell extracts at 75°C for
10 min was a very efficient purification step for the enzyme from E. coli as
most of the thermolabile E. coli proteins could be removed by this procedure.
After heat treatment, the purification yield increased 7 fold, without loss of
endoxylanase activity. After additional steps of purification by anion-
exchange chromatography and affinity chromatography with xylan-coupled
Sepharose CL-4B (Lee 8: Zeikus, manuscript prepared for publication), the
enzyme was purified to homogeneity as demonstrated using SDS-
polyacrylamide gel electrophoresis (Fig. 6). The purified enzyme consisted of
a single band with a molecular weight of 130,000. The faint bands of lower
molecular weight observed may be proteolytic digests of the enzyme, since

they displayed endoxylanase activity. The apparent pH and temperature

133

Table 1. Comparison of the translated amino acid sequence of endoxylanase

(xynA ) from Thermoanaerobacter B6A-RI with other endoxylanases. I

Organism Gene Similarity Identity Family
(%) (%)
Bacillus circulans xynA 44.1 22.7 G
Bacillus pumilus xynA 49.7 24.6 G
Bacillus subtilis xynA 42.6 22.5 G
Butyrivibrio fibrisolvens xynB 53.5 30.2 F
Bacillus sp. C-125 xynA 68.4 47.6 F
Caldocellum saccharolyticum xynA 66.2 42.8 F
Clostridium thermocellum xynZ 53.8 30.1 F
Clostridium acetobutylicum xynB 46.9 20.6 G
Pseudomonas fluorescens xynA 51.6 29.7 F
Pseudomonas fluorescens xynB 44.9 23.4 F

134

Table 2. Summary of purification of endoxylanase from E. coli (pZEP12)

Purification Total Total Specific Purification

step activity protein activity (fold) Yield
(units) (mg) (Units/ mg) (%)

Cell-free extract 1298 820 1.58 1.0 100

Heat treatment 1283 109 11.77 7.4 99

Q-Sepharose 651 41 15.88 10.0 50

Affinity 166 0.73 227.4 143.9 12.8

chromatography

135
optimum for the activity of the cloned endoxylanase was 5.5 and 70°C

respectively (Fig. 7 8r 8). The cloned enzyme was stable at 75°C, with no
detectable loss of activity over 60 min, but was less stable at higher
temperatures with a half-life of approximately 35 min at 80°C. The specific
activity of the purified endoxylanase A was 23 units/ mg protein on oatspelt

xylan.

Hydrolysis of xylan and xylooligosaccharides

The time course of hydrolysis of the soluble fraction of oat spelt xylan
and xylooligosaccharides by the recombinant endoxylanase is shown in Fig. 9.
Several different sizes of xylooligosaccharides were released from oat spelt
xylan. After 30 min of incubation an increase in xylobiose and xylotriose was
observed with a concommitant decrease in intermediary products of
hydrolysis such as xylotetrose and xylopentose. When the incubation was
prolonged to 5 hr, the main products were xylobiose and xylotriose with small
amounts of xylose and xylopentose. Endoxylanase A was not active on
xylobiose. Xylotriose was cleaved to xylobiose and xylose, and xylopentose
was initially cleaved to xylotriose and xylobiose, but on prolonged incubation
a small amount of xylose was formed. These results indicate that the cloned
xylanase A was an endo-acting enzyme, able to cleave xylan at random, and

having no activity on xylobiose.

136

kDaM 12 34MkDa

‘200......_. -. .. 200

I116 ----- --- - 116
97 “H *' 97

66 --' ~- 66

45 M-E .-45

Figure 6. SDS-PAGE analysis of recombinant endoxylanase purification from
E. coli (pZEP12).

Lane M, molecular weight markers; lane 1, crude E. coli (pZEP12)
extract (20 ug protein); lane 2, following heat treatment (10 pg protein); lane 3,

after anion exchange chromatography (5 pg protein) and lane 4, after affinity

chromatography (3 ug protein).

137

 

 

 

 

120

100 "
’o‘
23
>. 80 r-
3:
.2
H
0 60 '-
a
d)
.2 4o —
out
2 b
a)
D:

20 P

0 + l l 4 l 1 l l

2 3 4 5 6 7 8

Figure 7. The effect of pH on the activity of recombinant endoxylanase from
E. coli (pZEP12).

138

 

 

 

 

120
100 '-
°\O I-
V
> 80 "
3.:
.2
75
CU 60 '-
Q)
.2 4o -
u
g r
0
m
20 "'
0 1 1 1 1 1 1 1 1 1 1 1 1 1
3O 40 50 60 70 80 90 100

Temperature (°C)

Figure 8. The effect of temperature on the activity of recombinant

endoxylanase from E. coli (pZEP12).

139

Figure 9. Time course of hydrolysis of the soluble fraction of oat spelt
xylan and xylooligsaccharides by the recombinant endoxylanase.
Oligosaccharides of xylose (xylobiose, xylotriose and xylopentose)
and oat spelt xylan were incubated with purified endoxylanase and
analyzed using thin layer chromatography. M = size markers of xylose
(X1), xylobiose (X2), xylotriose (X3) and xylopentose (X5). Samples were
taken at time 0 (0), 15 min (1), 30 min (2), 1 hour (3) and 5 hours (4).
Hydrolysis of xylan (Xn), xylobiose (X2), xylotriose (X3) and xylopentose

(X5) were determined.

 

140

o::.

chvaHc—z

 

:a:~n=o:_

.\

vmuficzwmuacz

.111

 

DISCUSSION

Previous studies in our laboratory have shown that endoxylanase and
B-xylosidase appear to be coordinately regulated during growth of
Thermoanaerobacter B6A-RI on xylan and xylose (Lee, Lowe & Zeikus,
manuscript prepared for publication) and the findings here demonstrate that
the genes encoding for these two enzymes are closely situated on the
chromosome, thus, facilitating coordinate expression and production of
enzymes for efficient hydrolysis of xylan.

Initially we intended to clone the gene encoding endoxylanase since it
cleaves the B-1,4-linkage of B-1,4-xylan, which is a major component of the
hemicellulose. However, conversion of xylan to D-xylose requires at least
two enzymes; the endoxylanase converts xylan to its small molecular weight
oligosaccharide form, then B-xylosidase is used to convert the
xylooligosaccharides to D-xylose. It has been demonstrated that genes
encoding xylanolytic enzymes are clustered on the chromosome in many
bacteria including Bacteroides ovatus (40), Bacillus pumilus (29) and
Caldocellum saccharolyticum (24). The genes encoding endoxylanase and
B-xylosidase are the first two genes to be cloned from Thermoanaerobacter
B6A-RI. They were isolated together from the genomic library of this
organism by virtue of their close proximity on the chromosome and
expression as active enzymes in E. coli.

Most enzymes of family F have xylanase activity. For example, the
specific activity of C. thermocellum xynZ towards carboxymethyl cellulose
(CMC) is less than 0.5% of the activity towards xylan (15). However,
Cellulomonas fimi exoglucanase in spite of having high xylanase activity, has

an even higher activity towards CMC (13). In this regard, it is not too

141

l 4 2
surprising that xynA from Thermoanaerobacter B6A-RI should show strong

similarity in its catalytic domain to enzymes within this group.

The similarity of xynA with other genes in family F seemed to have
little bearing on the similarity of the organisms carrying the genes. xynA was
most similar to the endoxylanase (xynA) from the aerobic mesophile Bacillus
sp. strain 0425 (18), and xynA from the anaerobic thermophile Caldacellum
saccharolyticum (24). Slightly lower homology was observed between xynZ
from C. thermocellum (16), xynA from Butyrivibrio fibrisolvens (25) and
xynA from Pseudomonas flourescens (17). Interestingly, with the exception
of endoxylanase A from Pseudomonas flourescens, and xynA from
Butyrivibrio fibrisolvens for which data was not available, all of the genes
showing high homology with xynA from Thermoanaerobacter B6A-RI
encode for thermostable endoxylanases.

Multiple xylanases have been reported in several microorganisms (1,
30, 35, 40, 45), and Thermoanaerobacter B6A-RI is also known to have several
endoxylanases of different molecular weight (Lee, Lowe & Zeikus, manuscript
prepared for publication). Southern hybridization analysis, using the cloned
xynA as a probe indicated that any additional endoxylanase genes, if present
in Thermoanaerobacter B6A-RI, must have limited homology with the
cloned endoxylanase gene, as hybridization of the probe did not occur with
other fragments of the chromosomal DNA.

The endoxylanase activity from Thermoanaerobacter B6A-RI was
excretable, with increasing levels becoming extracellular under xylan
limitation (Lee, Lowe 8: Zeikus, manuscript prepared for publication). The
distribution of endoxylanase activity was approximately equal between the

periplasm and cytoplasm in E. coli containing the intact endoxylanase gene.

1 4 3
This finding suggests that the putative signal peptide identified in the gene is

recognized and the protein is processed by E. coli cells.

The mode of action of endoxylanase A from Thermoanaerobacter
B6A-RI was similar to other described endoxylanases from Cryptococcus
albidus (2, 3), Aspergillus niger (14) and C. stercorarium (1). These enzymes
cleave xylotriose, therefore requiring at least three xylose residues for catalytic
activity. Xylan is hydrolyzed randomly, giving xylobiose and xylotriose, and
these enzymes have no activity against xylobiose. The endoxylanase A was

similar in its mode of action to these enzymes.

10.

11.

LITERATURE CITED

Berenger, ].-F., C. Frixon, J. Bigliardi, and N. Creuzet. 1985. Production,
purification, and properties of thermostable xylanase from Clostridium
stercorarium. Can. J. Microbiol. 31: 635-643.

Biely, P., M. Vrsanska, and Z. Kratky. 1980. Complex reaction pathway

of acryl B-xylosidase degradation by B-xylanase of Cryptococcus albidus.
Eur. J. Biochem. 112:375-381.

Biely, P., M. Vrsanska, and Z. Kratky. 1980. Xylan-degrading enzymes of
the yeast Cryptococcus albidus, identification and cellular location. Eur.
J. Biochem. 108:313-321.

Biely, P. 1985. Microbial xylanolytic systems. Trends Biotechnol.
3:286-290.

Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction
procedure for screening recombinant plasmid DNA. Nuc. Acids
Res. 7:1513-1523.

Bradford, M. M. 1976. A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the principle
of protein-dye binding. Anal. Biochem. 72: 248-254.

Clewell, D. B. 1972. Nature of Col E1 plasmid replication in Escherichia
coli in the presence of chloramphenicol. J. Bacteriol. 110:667-676.

Debeire, P., B. Priem,. G. Strecher, and M. Vignon. 1990. Purification and
properties of an endo-1,4,-xylanase excreted by a hydrolytic thermophilic
anaerobe, Clostridium thermolacticum. Eur. J. Biochem. 187:573-580.

Dekker, R. F. H., and G. N. Richards. 1976. Hemicellulases: their
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Chapter V

Genetic organization, sequence and biochemical characterization of

recombinant B—xylosidase from Thermoanaerobacter strain B6A-RI

149

ABSTRACT

A deletion plasmid, pXPH3, was constructed from cosmid clone
pXDM1 which contained both endoxylanase (xynA) and B-xylosidase (xynB)
genes. The nucleotide sequence of a PstI-HindIII fragment in pXPH3 that
contained xynB was determined. An open reading frame (ORF) of 1,500 bp,
corresponding to 500 amino acid residues, was found. The molecular weight
of the enzyme deduced from the DNA sequence was 55 kDa. Another Open
reading frame (ORF1) of unknown function was found 21 bp downstream
from the first stop codon of xynB. xynB, ORF] and xynA had the same
direction of transcription. Southern blots confirmed the chromosomal
origin of the DNA insert in pXPH3 and that xynA and xynB were closely
situated on the chromosome of Thermoanaerobacter B6A-RI. The xynB from
Thermoanaerobacter B6A-RI exhibited 45% amino acid similarity with 18%
amino acid identity to xynA of Thermoanaerobacter B6A-RI and 61%
similarity and 37% identity with B-xylosidase gene from Caldocellum
saccharolyticum. Recombinant B-xylosidase was purified from E. coli (pXPH3)
cells. The enzyme was a monomer with a molecular mass of 55 kDa. The
specific activity and pH and temperature optima for hydrolysis of p-
nitrophenyl-B-D-xylopyranoside (PNPX) was 5.53 U/mg, 5.5 and 70°C,
respectively. The B-xylosidase was stable at 65°C, but lost activity at 85°C. The
purified enzyme had hydrolytic activity towards xylopentose, xylotriose,
xylobiose and PNPX, but had no activity toward xylan. The activity of the

purified enzyme was also tested on a number of different sunstrates.

150

INTRODUCTION

Xylan is the second most abundant renewable polysaccharide in nature
and is comprised of a B-1,4-linked D-xylose polymer with arabinofuranose,
glucuronic acid and methylglucuronic acid side groups (27). Complete
breakdown of xylan requires the action of several hydrolytic enzymes of
which endoxylanase (EC 3.2.1.8) and B-xylosidase (EC 3.2.1.37) are the most
important (3). Endoxylanase randomly cleaves the [34,4 bonds in the xylan
backbone to yield oligosaccharides of varying chain length. The resulting
xylooligosaccharides are attacked by B-xylosidase from the non-reducing end
(29).

Microbial B-xylosidases occur in both bacteria and fungi, with the latter
group being the most studied. Relatively little work has been done on B-
xylosidases from thermophilic bacteria (1, 9, 18). B-xylosidase genes from
Bacillus pumilus (19, 30), Bacillus polymixa (23), Bacillus subtilis (2),
Butyrivibrio fibrisolvens (25), and Caldocellum saccharolyticum (14) have
been cloned into Escherichia coli.

Thermoanaerobacter strain B6A-RI, a thermophilic anaerobic
bacterium isolated from Yellowstone National Park, grows actively on xylan
as a sole carbon source and does not have cellulase activity (13). In this
organism the degradation of xylan to xylooligosaccharides is catalyzed by
excreted or cell-bound endoxylanases followed by hydrolysis to xylose by
intracellular B-xylosidase. Both enzymes were regulated together as their
production were induced by xylan or xylose but not glucose (Lee, Lowe 8:
Zeikus, manuscript prepared for publication).

To isolate genes encoding for xylanolytic enzymes a genomic library of

Thermoanaerobacter strain B6A-RI was constructed using the cosmid vector

151

1 5 2
pHC79. One cosmid clone, pZXAl, which has a 36 Kbp DNA insertion was

found to have both endoxylanase and B-xylosidase activities. The
endoxylanase (xynA) and B-xylosidase (xynB) genes were closely linked
within 20 kb on the chromosome and the xynA has been subcloned and its
complete nucleotide sequence determined (Lee 8: Zeikus, manuscript
prepared for submission). The purpose of the present report is to describe the
complete nucleotide sequence of the xynB gene, its genetic organization with
xylanase A (xynA) and the biochemical properties of the Thermoanaerobacter

B-xylosidase expressed in E. coli.

MATERIALS AND METHODS

Bacterial strains

Thermoanaerobacter strain B6A-RI was isolated from Yellowstone
National Park as described previously (13). Escherichia coli DHSa (F'
080dlacZAM15 AllacZYA'argFlU169 deoR recAl endAl hst17[rK', mfi]
supE44 1‘ thi-l gyrA96 relAl) was used as the subcloning host and was
purchased from BRL (Life Technologies, Inc., Gaithersburg, MD).

Chemicals, media and culture conditions

All chemicals were of reagent grade and were of the highest purity
available. Xylose, xylobiose, PNPX, and nitrophenyl derivatives of sugars
were purchased from Sigma Chemical Co. (St. Louis, Mo). Xylotriose and
xylopentose were kindly given by Dr. P. Reilly at Iowa State University.
Restriction enzymes, T4 DNA ligase and calf intestinal alkaline phosphatase
were obtained from BRL or Boehringer Mannheim Biochemicals (BMB,
Indianapolis, IN). Thermoanaerobacter strain B6A-RI was grown in TYE
medium (31) containing 0.5% xylose at 60°C. LB medium (10 g Bacto tryptone,
5 g Bacto yeast extract, 5 g NaCl per liter) suplemented with ampicillin (50

ug/ ml) was used to grow E. coli strains harboring recombinant plasmids.

Preparation of chromosomal DNA from Thermoanaerobacter B6A-RI
Chromosomal DNA of Thermoanaerobacter strain B6A-RI was
prepared from cells grown in TYE medium containing 0.5% xylose at 60°C by
the method of Marmur (16). Plasmid DNA harbored in E. coli cells was
prepared from cleared lysates by the procedure described by Clewell (6). For

153

1 5 4
screening of recombinant DNA in transformants, the rapid method using the

alkaline extraction procedure of Birnboim and Doly (4) was used.

Subcloning procedure

The basic procedure was performed according to the method of
Sambrook et al. (22). Plasmid pXDM1 (Lee 8: Zeikus, manuscript prepared for
submission) which contained both xynA and xynB was digested with the
restriction endonucleases PstI and HindIII. A 2.0 kb PstI-HindIII fragment
which contain xynB was isolated by electroelution, using the Elutrap
(Schleicher 8: Schell Inc., Keene, NH). This fragment was ligated into pUC18
digested with PstI and HindIII, and the resulting recombinant plasmid was
named pXHP3. Transformation of E. coli DHSa was performed by the
Hanahan method as described by Perbal (20). The transformed colonies were
replica plated onto LB agar medium containing ampicillin and screened on
LB agar plates containing 10 ug/ ml of the fluorogenic substrate 4-
methylumbelliferyl-B-D-xylopyranoside (Sigma), by incubating the colonies
overnight at 37°C and then transferring to 60°C. The plates were examined
under ultraviolet light and the positive clones identified by a blue-white
fluorescence due to the diffusion of the umbelliferone released by cleavage of

the substrate.

Southern hybridization

Chromosomal DNA from Thermoanaerobacter strain B6A-RI and E.
coli HB101 were digested completely with restriction enzymes,
electrophoresed through agarose gels and stained with ethidium bromide.
For hybridization with radiolabed probes, the DNA was transferred to) a Zeta-

Probe blotting membrane (Bio-Rad Laboratories, Richmond, CA) by the

1 5 5
method of Southern (26). Gel-purified DNA fragments were labeled with a

random prime DNA-labeling kit (Boehringer Mannheim, Indianapolis, IN)
and a-32P dATP (3,000 Ci/mmol, New England Nuclear, Wilmington, DE)
according to the manufacturers instructions. Hybridization of the DNA was

by the method of Sambrook et al. (22).

DNA sequencing

Double-stranded DNA was sequenced in both directions by using the
dideoxy-chain termination method (24) and Sequenase (United States
Biochemical Co. Cleveland, OH) according to the manufacturer's instructions.
Additional sequencing primers were synthesized by the Michigan State
University Macromolecular facility. DNA sequences were analyzed using the
GENEPRO software package (Hoefer Scientific Instrument, San Francisco, CA)
and the University of Wisconsin Genetics Computer Group GCG package
(Version 7) (8).

Enzyme assays

The activity of B-xylosidase was measured with p-nitrophenyl B-D-
xylopyranoside (PNPX) in 50 mM sodium acetate buffer, pH 5.5. The reaction
mixture, composed of 1 mM PNPX, 50 mM sodium acetate (pH 5.5), and
diluted enzyme in 0.4 ml reaction volume, was incubated at 60°C for 10 min.
The reaction was stopped by the addition of 0.8 ml of 0.5 M NazCO3 and the
absorbance of the nitrophenol released was measured at 410 nm. 1 unit of
enzyme was defined as the amount of enzyme capable of releasing 1 umol
nitrophenol in 1 min. Other nitrophenol derivatives of sugars were also

tested as substrates under the same conditions.

1 5 6
Purification of B-xylosidase

Unless otherwise stated, all purification steps were carried out at room
temperature.
(i) Preparation of cell extract: B—xylosidase was purified from E. coli DHSo:
harboring plasmid pXPH3. Cells grown on 1 L LB medium supplemented
with ampicillin (50 ug/ ml) were suspended in 40 ml of 50 mM sodium acetate
pH 5.5, and disrupted by passage through a French pressure cell (American
Instrument Co., Inc., Silver Spring, Md.) at 20,000 lb/inz. The supernatant
obtained by centrifugation ( 12,000 x g, 20 min) was used as the crude enzyme
preparation.
(ii) Heat treatment: The extract of E. coli was heat treated at 75°C for 15 min
in a shaking water bath, and chilled on ice. The denatured proteins were
removed by centrifugation at 12,000 x g for 20 min.
(iii) Anion exchange column chromatography: The supernatant from the
heat treatment step was loaded onto a column (2.5 cm x 18 cm) of Q-
Sepharose (Pharmacia, Piscataway, NJ) equilibrated with 50 mM sodium
acetate buffer (pH 5.5). The enzyme was eluted with a linear gradient (0 - 1.0
M) of sodium chloride in a total volume of 500 ml. The active fractions were
pooled and concentrated by ultrafiltration with a YM 30 membrane (Amicon
Co. Ltd., Danvers, MA).
(iv) Gel filtration chromatography: The concentrated sample was further
purified using a Waters system FPLC (Division of Millipore, Milford, MA)
with a Superose-12 column (Pharmacia) equilibrated with 50 mM sodium

acetate buffer (pH 5.5) containing 50 mM sodium chloride.

1 5 7
N-terminal amino acid sequence determination of B-xylosidase

The protein was prepared by washing the purified B-xylosidase with
double-distilled water five times with a Centricon-30 (Amicon) filtration
device to remove contaminating salts and buffer from the solution. The
samples were hydrolyzed in a gas phase for 24 h using 5.7 M-HCl. Amino acid
composition analysis was performed with a Pico—Tag amino acid analyzer
(Waters Associates, Milford, MA). The N-terminal amino acid sequence was
identified by a protein sequencer model 477A (Applied Biosystems, Foster
City, CA) with an on-line phenylthiohydrantoin analyzer (Applied
Biosystems) in the Macromolecular Structure Facility, Department of

Biochemistry, Michigan State University.

Protein measurement and polyacrylamide gel electrophoresis

Protein concentration was estimated by the method of Bradford (5)
using Protein assay reagent from Bio-Rad with bovine serum albumin
(Sigma) as a standard. SDS-PAGE was performed as described previously (11)
with 12% acrylamide. Proteins bands were visualized by Coomassie brilliant
Blue G-250 staining. The molecular weight of the denatured protein was
estimated by SDS-PAGE with low-range molecular weight standards (Bio-
Rad) including phosphorylase (97,000), BSA (66 000), ovalbumin (43 000),
carbonic anhydrase (31,000), soybean trypsin inhibitor (21,500) and lysozyme
(14,400).

Analysis of products of xylan hydrolysis
The xylan and xylooligosaccharides were dissolved in 50 mM sodium
acetate buffer (pH 5.5) and incubated at 60°C with approximately IU of

purified enzyme. Samples were withdrawn periodically and the hydrolysis

1 5 8
products were analyzed by Whatman HP-K high performance silica gel plates

(10 x 10 cm) using the following solvent system: butanol/ acetic acid/ distilled
water (2:1:1, by vol.), and were visualized using an orcinol spray reagent (100

mg orcinol in 100 ml 20% sulphuric acid in methanol).

RESULTS

Subcloning and DNA sequencing

A deleted cosmid clone, pXDM1, consisting of the 28 kb chromosomal
DNA of Thermoanaerobacter strain B6A-RI and pHC79, containing the
endoxylanase (xynA) and B-xylosidase (xynB) genes is shown in Fig. 1. The
complete nucleotide sequence of xynA and its flanking regions have been
determined previously (Lee 8: Zeikus, manuscript prepared for submission).
A deletion plasmid, pXPH3, containing xynB, was constructed from pXDM1
and the physical map of xynB is shown in Fig. 2.

The nucleotide sequence of a PstI-HindIII fragment in pXPH3,
determined by the dideoxy-chain termination method, is shown in Fig. 3.
The sequence is consistent with the observed restriction map. An open
reading frame (ORF) of 1,500 bp, corresponding to 500 amino acid residues,
was found. The molecular weight of the enzyme deduced from the DNA
sequence was 55 kDa.

The putative ribosome-binding sequence, 5'-GGGGGG-3', was observed
7 bp upstream of the intiation codon, ATG. Another open reading frame
(ORF1) of unknown function was found 21 bp downstream from the first
stop codon of xynB. Interestingly xynB, ORFI and xynA have the same

direction of transcription (Fig. 1).

Southern hybridization analysis

To confirm the chromosomal origin of the DNA insert in pXPH3 and
to determine that xynA and xynB were closely situated on the chromosome of
Thermoanaerobacter B6A-RI, rather than occurring together as a result of

rearrangement during the cloning, Southern blots were performed on

159

160

 

 

pXDM1
Skb.
l_____J
s H a P E x
HP HEE H H EE H EEP PHHH
111 \ll 11 “1111 (III
-,_|.|f -
1:91;) (2:4)
xynBORFI xgnA

Figure 1. Physical map of cosmid clone pXDM1 which contains the 28 kb
chromosomal DNA of Thermoanaerobacter strain B6A-RI expressing the
endoxylanase (xynA) and B-xylosidase (xynB) genes. I

The open area denotes the 28kb insert and the closed area represents
the pHC79. The DNA was cut at various restriction sites (B, BamHI; E, EcoRI;
H, HindIII; P, PstI; S, San; X, XbaI). Arrows indicate the location and

transcriptional orientation of the genes.

161

pXPH3

 

0.2kb

z i : a 3:3? 75;" 3 E

31’. E 1% 13 flagx 13m 13 "f

l l l I \ll 1 \l l I
f - _

 

*- ’/////////////////////////////////////, il-

Figure 2. Physical and genetic map of the deletion plasmid pXPH3 containing
the B—xylosidase (xynB) from Thermoanaerobacter B6A-RI.

The location and transcriptional orientation of xynB is indicated by

arrow. The shaded bar represents the DNA fragment used as aprobe for
Southern hybridization.

162

Figure 3. The nucleotide sequence and deduced amino acid sequence of
B-xylosidase (xynB) from Thermoanaerobacter B6A-RI and its flanking
region.

The under lined region denotes the N-terminal amino acids
determined from the purified enzyme and the start sites of correct

reading frame for xynB and ORFI are indicated by arrows.

 

 

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GGTAGGAGATGAGGTAAAGCWACAAMTACCTATANGACAGGAWACTE:
1Va161yAspGluVaILysProPheTyrAsnPheThr‘ryrI IeAspArgI lePheAspSe

AMAGAMTCGGAATAAGGCCAHTGTGGAMTOGGATNATGCCIAMAMNAGé
rPheLeuG luI 1eG lyI IeArgProPheVa 1G1uI leGlyPheMetProLysLysLeuAI

. . . . 0 . LITA .
TCTGGTACACAGACAGTATTTTmTTGGGAGGGGAATGTCACTCCTCCCAAGG TGA
:SerclyThrGInThrVa lPhe'rerrpGluGIyAanalThrProProLysAsp'ryrGl

O . O O O A A
AAAGTGGAGCGATCTTGTCAAAGCGGTTTTGCATCACTTTATTTCTCGATATGGG TTG
uLysTrpSerAspLeuVa 1LysA1aVa1LeuHisH18PheI 1eSerArg‘l‘yrG 1yI 1:261

60

120

180

240

300

360

420

480

540

600

660

720

780

840

900

901

961

1021

1081

1141

1201

1261

1321

1381

1441

1501

1561

1621

164

AGAAGTCTTGAAGTGGCCATTTGAGATATGGAATGAACCAAACTTAAAAGAGTTTTGGAA
uGluValLeuLysTerroPheGIuIleTrpAsncluProAsnLeuLysGluPheTrpLy

AGATGCTGATGAGAAAGAATATTTTAAGCTGTACAAGGTTACTGCAAAGGCTATAAAGGA
sAspAlaAspGluLysGluTerheLysLeuTerysValThrAIaLysAIaIleLysGI

AGTAAATGAAAATTTGAAGGTAGGAGGGCCTGCTATATGéGGTGGTGCTGACTACTGGAE
uValAsnGluAsnLeuLysVa1G1yGlyProA1aI1eCysGlyGlyA1aAspTerrpI1

AGAAGATTTTTTGAATTTCTGCTATGAAGAAAATGTTCCTGTAGATTTTGTATCGCGACA
eG1uAspPheLeuAsnPheCysTyrG1uGluAsnva1Prova1AspPheVa18erArgH1

CGCTACCACATCTAAGCAAGGTGAATATACGCCACATCTCATATACCAGGAGATTATGCC
3A1aThrThrSerLysGInclycluTerhrProHisLeuI1eTyrGlnGluIIeHetPr

ATCTGAATACATGCTAAACGAATTTBAAACAGTGAGAGAGATCATAAAAAACTCACATTT
oSerGluTeretLeuAsnGluPheLysThrva1ArgcluI1eIlenyshsnSerHisPh

TCCGAACCTTCCGTTTCATATAACTGAGTACAATACTTCATATEGTCCTCAAAATCCTGT
eProAsnLeuProPheHisI1eThrGluTyrAsnThrSerTyrSerProGInAsnProVa

ACACGATAC8CCATTTAATGCTGCCTATATTGCCAGGATTTTAAGCGAAGGCGGAGATTA
181sAspThrProPheAsnAlallaTyrIleAIaArgIleLeuSercluclyclyAspTy

TGTTGATTCATTTTCTTACTGGACGTTTAGCGACGTTTTCGAAGAAAGAGATGTGCCGCG
rVa1Asp8erPheSerTerrpThrPheSerAspva1PheGluGluArgAspVa1ProAr

ATCCCAATTCCATGGAGGATTTGGACTTGTGGCATTGAATETGATACCAAAGCCTACCTT
gSerGlnPheHisGlyGlyPheGlyLeuVa1A1aLeuAsnMetIleProLysProThrPh

TTACACATTTAAATTCTTTAATGCTATGGGAGAGGAAATGCTTTATAGAéATGAGCACA1
eTerhrPheLysPhePheAsnAlafietGlyGluGluHetLeuTyrArgAspGluflisxe

GCTTGTGACGAGGAGAGATGATGGCTCTGTTGCACTCATAGCTTGGAATéAAGTTATGGA
tLeuValThrArgArgAspAspclyServa1A1aLeuIleAlaTrpAsnGIuValuetAs

TAAGACTGAAAATCCAGATGAAGATTATGAAGTCGAGATACCAGTTAGATTCAGAGATGT
pLysThrGluAsnProAspGluAspTyrcluVaIGIuI1ePrdVa1ArgPheArgAspVa

960

1020

1080

1140

1200

1260

1320

1380

1440

1500

1560

1620

1680

1681

1741

1801

1861

1921

1981

2041

2101

2161

2221

2281

2341

2401

1(55

GTTTATTAAAAGACAATTGATTGATGAAGAACATGGCAATCCATGGGGAACGTGGATACA
1PheIlenysArgGInLeuIlehspGIuGIuHisGlyAsnProTrpGlyThrTrpIlefli

CATGGGAAGGCCGAGGTATCCTAGCAAAGAACAGGTAAATAQATTGAGAGAAGTTGCAAA
sMetGlyArgProArgTerroSerLysGluG1nVa1AsnThrLeuArgGIuva1A1aLy

GCCAGAGATTATGACAAGTCAGCCTGTTGCGAATGACGGATACTTAAATCTAAAGTTTAA
sProGluI1eHetThrSerG1nProVa1A1aAsnAspGlyrereuAsnLeuLysPheLy

ATTAGGTAAAAATGCAGTTGTATTGTATGAATTGACTGAGAGAATTGATGAATCAAGCAC
sLeuGlyLysAsnAlaVa1ValLeuTyrGIuLeuThrGIuArgI1eAspGluSerSerTh

ATITATAGGACTTGATGAIAGCAAGATAAATGGATATTGATGCACATTAGGAGGGATTGA
rTyrI1eG1yLeuAspAspSerLysI1eAsnGlyTyrEnd

ORFI

TATGGGACTTTTTGACATGCCACTGCAAAAGCTTAGAGAATACACTGGTACAAATCCATG
MetGlyLeuPheAspMetProLeuG1nLysLeuArgGluTerhrclyThrAsnProCy

cccTGAAGATTTcGArGasrATTcGGAmaGGGCTTTAGATGAGATGAGGTCAGTTGarcc
sproGluAspPheAspG1nTerrpAspArgA1aLeuAspclunetArgSerVa1Asppr

CAAAATTAAAATGAAAAAAAGTAGCTTTCAAGTGCCTTTTGCAGAGTGCTACGATTTGTA
oLysI1eLysHetLysLysSerSerPheG1nVa1ProPheA1aGluCysTyrAspLeuTy

CTTTACAGGTGTTCGTGGTGCCAGAAITCATGCAAAGTATATAAGACCTAAGACAGAAGé
rPheThrGlyVa1ArgclyA1aArgIlenisAIaLysTyrI1eArgProLysThrGluGl

GAAACATCCAGCGTTGATAAGATTTCATGGATATTCGTCAAATTCAGGCGACTGGAACGA
yLysHisProAlaLeuIleArgPheHisGlyTyrSerSerAsnSerGlyhspTrpAsnAs

CAAATTAAATTAOGTAGCGGCAGGCTTTAOCGTTGTGGCEATGGATGCAAGAGGTCAA68
pLysLeuAsnTera1A1ah1aGlyPheThrva1ValAlaHetAspAlaArgGlyGlnGl

AGGGCAGTCTCAAGATGTTGGCGGTGTAAATGGGAACACTTTAAATGGGCATATTAIAAG
yGlyG1nSerGlnAspVa161yGlyVa1A5nGlyAsnThrLeuAsnGIyHisI1eI1eAr

AGGGTTAGACGATGATGCTGACAACATGCTTTTTAGGCATATCTTCTTAéATACTGCCCA
gGlyLeuAspAspAspAlaAspAsnuetLeuPheArgHisIlepheLeuAspThrAIacl

1740

1800

1860

1920

1980

2040

2100

2160

2220

2280

2340

2400

2460

2461

'2521

2581

2641

2701

2761

2821

2881

2941

3001

3061

3121

3181

1(56
GTTGGCTGGAATAGTTATGAATATGCCAGAAATCGATGAAGATAGAGTTGCAGTCATGGG
nLeuAlaGlyI1eVa1MetAsnMetProG1uI1eAspGluAspArgVaIAIaValuetcl

ACCTTCTcAAGGTGGAGGTTTGTCATTAGCGTGTGCAGCATTGGAACCCAAGATAOGCAA
yPrOSerG1nGlyclyG1yLeuSerLeuA1aCysAlaAIaLeuGluProLysIIeArgLy

AGTAGTATCAGAGTACCCATTCTTGTCTGATTATAAAAGAGTTTGGGATTTAGACCTTGC
sVa1Va18erGluTerroPheLeuSerAspTerysArgValTrpAspLeuAspLeuAI

GAAAAATGCTTACCAAGAGATTACGGACTATTTTAGGCTTTTTGATCCAAGGCATGAAAG
aLysAsnAlaTyrGlnGluI1eThrAspTyrPheArgLeuPheAspProArgnisGluAr

AGAAAATGAGGTGTTTACTAAGCTTGGCTATATAGATGTTAAGAATCTGGCGAAGAGGAI
gGluAsnGluVa1PheThrLysLeuGlyTyrIIeAspValLysAsnLeuAIaLysArgI1

AAAAGGCGAEGTGTTAATGTGCGTTGGGCTTATGGATCAAGTATGTCCGCCATCAACTGT
eLysGlyAspValLeuMetCysVa161yLeuMetAspG1nVa1CysProProSerThrva

CTTTGCAGCCTACAACAACATACAGTCAAAGAAGGATATAAAAGTGTATCCTGATTATGG
1PheA1aA1aTyrAsnAsnIleclnSerLysLysAspIleLysValTerroAspTyrGI

ACATGAGCCTATGAGAGGATTTGGAGAITTRGCGATGCAGTTTNTGTTGGAACEATKTTC

yHisG1uProMetArgGlyPheGlyAspLeuAlaMetGlnPheMetLeuGluLeuTyrSe.

ATAAGGAGATGCAAAGTGCATCTCTTAACTGTATTCTTTATTAATTTTTTATATTCGCTT
rEnd

ACGTTACGTTTAAGCCTGTGTATTTTTTAAAACACGATGAAAAATAATTTGGGTCTTGTA
TGCCTACAGACTTGGCGATTTCAAATGACTTCATGTTTTTTTCTTTAATAAGGCTTATGG
CTTATGGCTTTTTCCATTCGCTTATTTGTCAGGTATTCTATGAAGTTTATGCCTGTCTCC

TTTTTGAATGTTGG 3194

2520

2580

2640

2700

2760

2820

2880

2940

3000

3060

3120

-3180

l 6 7
chromosomal DNA from E. coli and Thermoanaerobacter B6A-RI digested

with EcoRI, PstI and XbaI. An internal 1.2 kb HincII-PstI fragment from
pZEP12 and a 0.9 kb SspI-HindIII fragment from pXPH3 were used as a probe
for endoxylanase (xynA) and B-xylosidase (xynB), respectively. The results
are shown in Fig. 4. Both fragments did not bind to E. coli chromosomal
DNA, but did hybridize to Thermoanaerobacter B6A-RI genomic DNA,
indicating that these genes originated from Thermoanaerobacter B6A-RI.
Both probes hybridized to identical size bands of Thermoanaerobacter B6A-RI
DNA digested with PstI (lane 3) and XbaI (lane 5). This indicates that these
two genes (xynA and xyn B) are clustered on the chromosomal DNA of
Thermoanaerobacter B6A-RI and no apparent rearrangement occurred during

the cloning procedure.

Comparison of xynB with other genes

Comparison of the deduced amino acid sequence of xynB from
Thermoanaerobacter B6A-RI to xynA from the same organism, and [3-
xylosidases from B. pumillus IPO (30), Butyrivibrio fibrisolvens (25) and
Caldocellum saccharolyticum (14) are presented in Table 1. The xynA and
xynB from Thermoanaerobacter B6A-RI exhibited 45% amino acid similarity
to each other with 18% amino acid identity (Table 1). Comparison of xynB
with GenBank and EMBL sequence libraries identified the B-xylosidase gene
from Caldocellum saccharolyticum as being most similar, with 61% similarity
and 37% identity. The translated sequence for xynB did not share a strong
identity with other endoxylanases. No genes were found which showed a

high amino acid similarity to the Thermoanaerobacter B6A-RI ORF1.

168

Figure 4. Southern hybridization analysis of Thermoanaerobacter
B6A-RI and E. coli genomic DNA digests with random-primed DNA
synthesized from the 1.2 kb HincII-Pstl fragment from pZEP12 and
0.9 kb Sspl-Hindlll fragment from pXPH3.

(A) Photograph of 0.8% agarose gel stained with ethidium
bromide. (B) and (C) Autoradiogram of DNA transferred to membrane
andhybridized with the 32P-labeled insert fragment from pZEP12 and
pXPH3, respectively. Lane 1, molecular weight markers; lane 2, E. coli
chromosomal DNA digested with PstI; lane 3, 4 8: 5,
Thermoanaerobacter B6A-RI chromosomal DNA digested with PstI,

EcoRI and XbaI, respectively.

 

 

169

C
12345
_ U

 

170

Table 1. Relative homology between B-xylosidase from Thermoanaerobacter

B6A-RI and other B-xylosidases.

Strain % Similarity % Identity
Bacillus pumilus IPO 40.9 16.0
Butyrivibrio fibrisolvens 45.9 15.5
Caldocellum saccharolyticum 60.5 36.5
Thermoanaerobacter B6A-RI 44.7 17.5

(xynA)

17 1
Purification and characterization of recombinant B-xylosidase

Recombinant B-xylosidase was purified from E. coli (pXPH3) cells to
determine its physicochemical properties. A summary of the purification
procedure is shown in Table 2. Heat treatment was a particularly effective
step, resulting in a 5-fold purification and 81% yield. . SDS-PAGE analysis (Fig.
5) revealed that the enzyme was monomeric with an approximate size of 55
kDa. This is identical to the value (55 kDa) calculated from the deduced
amino acid sequence. The molecular mass of the recombinant enzyme was
estimated to be 60 kDa by FPLC. The N-terminal amino acid sequence of the
recombinant B-xylosidase was Met—Ile-Lys-Val-Arg-Val-Pro-Asp-Phe-Ser-Asp.
This was identical to the N-terminal amino acid sequence deduced from the
DNA sequence.

The pH optimum for hydrolysis of PNPX was approximately 5.5 with
the enzyme showing high activity from pH 5.0 to 6.6 (Fig. 6). The optimum
temperature for hydrolase activity was 70°C, with activity decreasing rapidly
at higher temperatures (Fig. 7). To examine the thermostability of the
enzyme, B-xylosidase was incubated in 50 mM sodium acetate buffer, pH 5.5,
at various temperatures for up to 60 min, and the residual activities were
assayed and compared with the untreated sample. B-xylosidase was stable at
65°C, but lost activity at 85°C. The half-life of the enzyme at 75°C was 55 min
(data not shown).

The purified B-xylosidase had hydrolytic activity towards xylopentose,
xylotriose, xylobiose and PNPX, but had no activity toward xylan. The activity
of the purified B-xylosidase with various para- and ortho-nitrophenyl-
glycosidic substrates was tested (Table 3). The B—xylosidase was highly
specific, as it was only active on p-nitrophenyl-B-D-xylopyranoside and o-

nitrophenyl-B-D-xylopyranoside. The low level of activity observed with p-

172

Table 2. Summary of purification of B-xylosidase from E. coli (pXPH3)

Purification Total Total Specific Purification

step activity protein activity (fold) Yield
(units) (mg) (U nits / mg) (%)

Cell-free extract 165 391 0.422 1.0 100

Heat treatment 134 67 2.6 4.7 81.2

Q-Sepharose 101 21 4.81 11.4 61.2

Gel filtration 83 15 5.53 13.1 50.3

173

43

31

21

Figure 5. SDS-PAGE analysis of the purification of recombinant B-xylosidase

from E. coli (pXPH3) cells.
Lane M, molecular weight markers; lane 1, crude E. coli (pXPH3) extract
(20 ug protein); lane 2, following heat treatment (10 ug protein); lane 3, after

anion exchange chromatography (5 ug protein) and lane 4, after gel filtration

chromatography (3 ug protein).

Relative activity (%)

174

 

120

100 -

80r-

60'-

4or

20F

 

 

 

Figure 6. The pH profile of recombinant B-xylosidase

175

 

 

 

 

120
1.
A 100 "'
o\° .
v
£- 80 -
.2 P
4-0 >-
0
CB 60 T
Q)
.2 '
4 .-
E 0
m h
a:
20 *-
l
O J l 1 l 4_ l 1 I 1 l 1 1 4
30 40 50 60 70 80 90 100

Temperature (°C)

Figure 7. The temperature profile of recombinant B-xylosidase

176

Table 3. Hydrolysis of various nitrophenyl-glycosides by the purified

B-xylosidase

Substrate Specific activity (U/mg)*
p-Nitrophenyl-B—D-xylopyranoside 5.529
o-Nitrophenyl-B-D-xylopyranoside 4.696
p-Nitrophenyl-B-D-fucopyranoside 0.045
p-Nitrophenyl-a-D-glucopyranoside 0.000
p-Nitrophenyl-B-D-glucopyranoside 0.141
p-Nitrophenyl-o:-L-arabinofuranoside 0.073
o-Nitrophenyl-B-D-galactopyranoside 0.000
p-Nitrophenyl-B-D-lactopyranoside 0.000

*These values represent the mean values of three independent experiments.

1 7 7
nitrophenyl-B-D-fucopyranoside, p-nitrophenyl-oc-L-arabinofuranoside and p-

nitrophenyl-B-D-glucopyranoside may not be significant, and no activity was
detected with the other substrates tested.

Oligosaccharides of xylose (xylobiose, xylotriose and xylopentose) and
oat spelt xylan were incubated with purified B—xylosidase and analyzed using
thin-layer chromatography (Fig. 8). All of the xylooligosaccharides (polymers
of two to five xylose residues) were degraded to xylose. Transferase activity
was also detected during the early stages of hydrolysis. No activity could be
detected using oat spelt xylan as the substrate. No B-xylosidase activity was

detected in control extracts from E. coli.

178

Figure 8. Time course of hydrolysis of the soluble fraction of oat spelt
xylan and xylooligsaccharides by the recombinant B-xylosidase.
Oligosaccharides of xylose (xylobiose, xylotriose and xylopentose)
and oat spelt xylan were incubated with purified B-xylosidase and
analyzed using thin layer chromatography. M = size markers of xylose
(X1), xylobiose (X2), xylotriose (X3) and xylopentose (X5). Samples were
taken at time 0 (0), 15 min (1), 30 min (2), 1 hour (3) and 5 hours (4).
Hydrolysis of xylan (Xn), xylobiose (X2), xylotriose (X3) and xylopentose

(X5) were determined.

179

I
0.9
0

mx

2:: 5:23:05

Hozwmuwcz meHc—zvaHo—z

o ., .00....

 

mx
mx

fix

DISCUSSION

The complete nucleotide sequence of the B-xylosidase gene (xynB) of
Thermoanaerobacter B6A-RI was established together with its flanking
regions. In our other studies, the endoxylanase and B-xylosidase activity in
Thermoanaerobacter B6A-RI have been shown to be under coordinate
control, induced by xylose or xylan and the endoxylanase gene (xynA) was
cloned and sequenced (Lee 8: Zeikus, manuscript prepared for submission).

In the cloned chromosomal DNA from Thermoanaerobacter B6A-RI,
xynA and xynB genes were clustered within the 20 kb region, and had the
same transcriptional orientation. The proximity of xynB, ORFI and xynA on
the cloned DNA and their same direction of transcription suggests the
possibility of their being organized together as an operon. This is further
substantiated bt the finding that no terminator or prominent stem-loop was
identified between xynB and ORFl and the downstream region of ORF] by
sequence analysis. However, it still remains to be shown that xynA and xynB
are part of the same operon in Thermoanaerobacter B6A-RI. Studies using
RNA, such as Northern blot analysis and primer extension experiments
would answer this question. Recent studies with Bacteroides ovatus (28),
Bacillus pumilus (17) and Caldocellum saccharolyticum (14) have reported
the clustering of genes involved in hemicellulose degradation in these
organisms.

Confirmation that the cloned xynB is from the Thermoanaerobacter
B6A-RI chromosomal DNA was obtained by Southern blot analysis. A single
PstI and XbaI fragment was hybridized to B-xylosidase probe suggests that only
one copy of the cloned gene is present in the chromosome. However, the

possibility that Thermoanaerobacter B6A-RI has more than one B—xylosidase

180

1 8 1
cannot be ruled out, since multiple B—xylosidases have been reported in B.

pumulis (19) and Caldocellum saccharolyticum (14 ).

Localization studies of the cloned B-xylosidase revealed that the
enzyme was located within the E. coli cell (data not shown), and this finding
is in accordance with the findings of others that bacterial B-xylosidases are
generally intracellular (12, 18). Most of the B-xyosidase activity from
Thermoanaerobacter B6A-RI was intracellular (data not shown).

The deduced amino acid sequence of thermostable
Thermoanaerobacter B6A-RI B-xylosidase exhibited a higher degree of
homology to thermostable B—xylosidase from C. saccharolyticum (36%
identity) (14) than to those of less thermostable B-xylosidases from Bacillus
(30) and Butyrivibrio (25) (less than 16% identity).

The cloned B-xylosidase had a monomeric molecular weight of 55 kDa
which was similar to the size of B-xylosidase from B. fibrisolvens (25) and B.
pumulis (19) each comprising of a single subunit of 60,000 and 62,600
molecular weight, respectively. The B-xylosidase from C. acetobutyricum (12)
is composed of two different subunits with molecular weights of 85,000 and
63,000. Fungal B-xylosidases have relatively larger molecular weight subunits
of 116,000 to 118,00 (10, 15).

The substrate specificity of B—xylosidase from Thermoanaerobacter
B6A-RI for xylooligosaccharides was similar to other bacterial B-xylosidases
characterized (7, 12, 19, 25). Most B—xylosidases have little or no activity
against xylan, and this was also found with B-xylosidase from
Thermoanaerobacter B6A—RI. These enzymes degrade xylooligosaccharides to
xylose and also have transferase activity, which are considered to be the

properties of a B-xylosidase (21).

10.

11.

LITERATURE CITED

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characterization of a thermostable B-xylosidase from
Thermomonospora fusca. J. Gen. Microbiol. 135:293-299.

Bemier, R. Jr., H. Driguez, and M. Desrochers. 1983. Molecular cloning
of Bacillus subtilis xylanase gene in Escherichia coli. Gene 26:59—65.

Biely, P. 1985. Microbial xylanolytic systems. Trends Biotechnol. 3:286-
290.

Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction
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Bradford, M. M. 1976. A rapid and sensitive method for quantitation
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Kitpreechavanichi, V., M. Hayashi, and S. Nagai. 1986. Purification and

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1 8 3
Lee, S. F., and C. W. Forsberg. 1987. Isolation and some properties of a

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Lee, Y.-E., M. K. Jain, C. Lee, S.E. Lowe and J. G. Zeikus. 1992.
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Thermoanaerobium saccharolyticum sp nov. Reclassification of
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nov. and Thermoanaerobacterium thermohydrosulfuricus comb. nov.,
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Thermoanaerobacter ethanolicus. Manuscript submitted to the
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Luthi, B., D. R. Love, J. McAnulty, C. Wallace, P. A. Caughey, D. Saul,
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genes encoding xylan-degrading enzymes from the thermophile
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1024.

Matsuo, M., and T. Yasui. 1984. Purification and some properties of B-
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Moriyama, H., E. Fukusaki, J. Cabrera Crespo, A. Shinmyo, and H.
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xylosidase produced by a newly isolated Bacillus stearothermophilus
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Panbangred, W., O. Kawaguchi, T. Tomita, A. Shinmyo, and H. Okada.

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comparison of their gene products. Eur. J. Biochem. 138:267—273.

Perbal, B. 1988. A practical guide to molecular cloning. 2nd ed., John
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Sandhu, J. S., and J. F. Kennedy. 1984. Molecular cloning of Bacillus

polymixa 1-4-B-D xylanase EC-3.2.1.8 gene in Escherichia coli. Enz.
Microbiol. Technol. 6:271-274.

Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with
chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467.

Sewell, G. W., E. A. Utt, R. B. Hespell, K. F. Mackenzie, and L. 0.
Ingram. 1989. Identification of the Butyrivibrio fibrisolvens xylosidase
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Environ. Microbiol. 55:306-311.

Southern, E. M. 1975. Detection of specific sequences among DNA
fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517.

Whistler, R. L., and E. L. Richards. 1970. Hemicelluloses, p. 447-469. In
W. Pigman and D. Horton (ed.), The carbohydrates-chemistry and
biochemistry, 2nd ed., vol. 2A. Academic Press, Inc., New York.

Whitehead, T. R., and R. B. Hespell. 1990. The genes for three xylan-
degrading activities from Bacteroides ovatus are clustered in a 3.8-
kilobase region. J. Bacteriol. 172:2408—2412.

Wong, K. K. Y., L. U. L. Tan, and J. N. Saddler. 1988. Multiplicity of B-
1,4-xy1anase in microorganisms: functions and applications. Microbiol.
Rev. 52:305-317.

Xu, W.-Z., Y. Shima, S. Negoro, and I. Urabe. 1991. Sequence and

properties of B-xylosidase from Bacillus pumilus IPO: Contradiction of
the previous nucleotide sequence. Eur. J. Biochem. 202:1197-1203.

Zeikus, J. G., A. Ben-Bassat, and P. Hegge. 1980. Microbiology of
methanogenesis in thermal volcanic environments. J. Bacteriol.
143:432-440.

Chapter VI

Cloning, sequencing and biochemical characterization of

xylose isomerase from Thermoanaerobacter strain B6A-R1

185

ABSTRACT

The xylose isomerase gene from Thermoanaerobacter strain B6A-RI
was cloned by complementation using Escherichia coli xyl-S mutant strain
H8101. One positive clone was detected and the recombinant plasmid, pZXI6,
was isolated. The clone contained vector pUC18 and an insert fragment of 4.5
kilobases. The cloned xylose isomerase gene (xylA) was expressed
constitutively in E. coli. The gene contained one open reading frame (ORF) of
1317 base pairs in length which corresponds to 439 amino acid residues. The
predicted molecular weight of 50,474, was calculated from the deduced amino
acid sequence. A putative promotor region (Pribnow box),
TATAATATATAAT, which repeated twice at the -10 region in E. coli, was
found 25 bp upstream of the ribosomal binding site. The deduced amino acid
sequence of Thermoanaerobacter B6A-RI xylose isomerase exhibited very
high homology to those from C. thermosulfurogenes and C .
thermohydrosulfuricum. Codon usage in xynA, xynB and xylA showed a
clear propensity of AT-containing isocodons. The native molecular mass of
the purified recombinant thermostable xylose isomerase was 200kDa, and the
enzyme was a tetramer comprised of identical subunits. The apparent
temperature and pH optimum for the activity of the cloned xylose isomerase

was 80°C and 7.0 to 7.5 respectively.

186

INTRODUCTION

Thermoanaerobacter strain B6A-RI is a thermophilic anaerobic bacteria
capable of actively degrading xylan. Endoxylanase and B-xylosidase degrade
the polymer to D-xylose, which is isomerized to D-xylulose and further
catabolized in the cell. The isomerization of D-xylose is catalyzed by D-xylose
isomerase (EC 5.3.1.5) which is also used industrially to convert D-glucose to
D-fructose syrups (2, 6, 7).

In Bacillus subtilis, the enzymes encoded by the xylose regulon consist
of genes for xylan and xylose utilization, which cooperate in the utilization of
hemicelluloses (14). These genes are negatively regulated by the repressor
(xle) and are induced by xylose (16). In Thermoanaerobacter strain B6A—RI,
the endoxylanase and B-xylosidase activities were induced during growth on
xylan and xylose (Lee, Lowe 8: Zeikus, manuscript prepared for publication).
The synthesis of xylose isomerase from Thermoanaerobacter was also
induced by either xylose or xylan (18).

We have isolated genes encoding endoxylanase (xynA) and B-
xylosidase (xynB) from the genomic library of Thermoanaerobacter strain
B6A-RI, which were found to be organized in a cluster on the chromosome.
To date there is no data regarding the genetic organization of the xylose
isomerase (xyl) genes in Thermoanaerobacter. In this report, we describe the
cloning, sequencing of the xyl gene from Thermoanaerobacter strain B6A-RI

and discuss it 5 relationships to endoxylanase and B-xylosidase.

187

MATERIALS AND METHODS

Bacterial strains and plasmids

Thermoanaerobacter strain B6A-RI (22) was used as the source of the
xylose isomerase gene. E. coli xyl' mutant strain HB101 (F'mch mrr
hstZOIrB’mB'] recA13 supE44 ara14 gaIK2 lacYI proA2 rpsLZOISm'] xyl5 1' (cu
mill) (3) were used as a host strain for cloning by complementation. Plasmid

pUC18 was used as the cloning vector.

Chemicals, media and growth conditions

All chemicals were of reagent grade and were of the highest purity
available. Thermoanaerobacter strain B6A-RI was grown under anaerobic
conditions at 60°C in TYE medium (37) containing 0.5% xylose as a carbon
source. For growth of E. coli strains, Luria broth (10 g of tryptone, 5 g of yeast
extract, and 5 g of NaCl per liter) was used for liquid culture, and solid
medium contained 1.5% agar (Difco Laboratories, Detroit, MI). Ampicillin
(50ug/ ml) was supplemented for the selection of transformants. MacConkey
agar (Difco, Ann Arbor, MI) plates containing 1% xylose were used to detect

xylose isomerase positive clones.

DNA preparation and cloning procedure

Chromosomal DNA of Thermoanaerobacter strain B6A-RI was isolated
by a modification of the Marmur method as described previously (19).
Plasmid DNA was purified in large scale by the procedure described by
Clewell (8), followed by centrifugation in a cesium chloride-ethidium
bromide density gradient. The alkaline denaturation method of Birnboim 8:

Doly (4) was used for rapid extraction of plasmid DNA.

188

l 8 9
Chromosomal DNA from Thermoanaerobacter B6A-RI was partially

digested with restriction endonuclease Ach and 4 to 10 kbp DNA fragments
were isolated from an agarose gel by electroelution, using the elutrap
(Scheicher 8: Schuell Inc., Keene, NH). Plasmid vector pUC18 was completely
digested with Ach and dephosphorylated with Calf intestinal alkaline
phosphatase. Ligation was performed by the procedure of Sambrook et al. (28)
and transformed into E. coli HB101 competent cells prepared by the Hanahan
method as described by Perbal (26). Transformants were selected on

MacConkey agar plates supplemented with 1% xylose and ampicillin.

Sequence determination of the xylA gene

The recombinant plasmid was denatured by the method of Zhang et
al. (38), and the nucleotide sequence was determined by the dideoxy chain
termination method (29), using a Sequenase version 2.0 kit (United States
Biochemical Co. Cleveland, OH). The sequence information was analyzed
using the GENEPRO software package (Hoefer Sceientific Instrument, San
Francisco, CA) and the University of Wisconsin Genetics Computer Group

GCG package (11).

Enzyme assay

Cell extracts prepared by sonication and purified preparations were
used as enzyme sources. Glucose isomerase activity was measured by
incubating a reaction mixture that contained 10 mM MgSO4, 1 mM CoClz,
0.8 M glucose, and the enzyme in 100mM MOPS buffer (pH 7.0) at 65°C. The
xylose isomerase activity assay was performed at 65°C with a reaction mixture
contained 70 mM xylose, 10 mM MnSO4 and enzyme in the same buffer. The

amount of fructose or xylulose formed was estimated by the cystein-carbazole-

1 9 0
sulfuric acid method (12). One unit of activity was defined as the amount of

enzyme which released 1 umol of ketose per min under the assay conditions

described.

Purification of xylose isomerase

Unless otherwise stated all operations were performed at room
temperature and used the method of Lee et al. (21).
(i) Preparation of cell extract: E. coli (pZXI6) cells from a I L overnight culture
were suspended in 40 ml of 50 mM MOPS buffer (pH 7.0) containing MgSO4
(10 mM) and C002 (1 mM). The cells were broken by two passages through a
French pressure cell at 16,000 lb/inz. The cell debris were removed by
centrifugation at 12,000 X g for 20 min, and the supernatant used as the crude
enzyme preparation.
(ii) Heat treatment: The cell extract obtained was heated at 85°C for 15 min
and chilled in ice. The precipitated protein was removed by centrifugation at
12,000 X g for 20 min, and the soluble fraction used for xylose isomerase
purification.
(iii) Anion exchange chromatography: The soluble fractions from the heated
cell extracts were loaded onto a DEAE-Sepharose CL-6B (Pharmacia,
Piscataway, NJ) column equilibrated with 50 mM MOPS buffer (pH 7.0)
containing 5 mM MgSO4 and 0.5 mM CoC12. The enzyme was eluted with a
linear NaCl gradient (0 to 1.0 M) in the same buffer. Active fractions were
pooled and concentrated by ultrafiltration using a YM 30 membrane with a
molecular weight cut-off of 30 kDa (Amicon Co. Danvers, MA).
(iv) Gel filtration chromatography: The concentrated xylose isomerase
sample was applied to a Superose 12 HR 10/ 30 gel filtration column

(Pharmacia LKB Biotechnology Inc., Piscataway, NJ) with a Waters fast-

l 9 1
protein liquid chromatography (FPLC) system (Division of Millipore, Milford,

MA)

Protein determination and SDS-PAGE

The protein amount was determined by the Bradford method using the
Bio-Rad protein reagent (Bio-Rad Laboratories Ltd., Richmond, CA) with
bovine serum albumin (Sigma Chemical Co.) as the standard. Enzyme
fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) in 12% polyacrylamide gels in a Mini-PROTEAN
II system (Bio-Rad). Protein bands were visualized by Coomassie brilliant

blue staining.

N-terminal amino acid sequence determination of xylose isomerase

The protein was prepared by washing the purified xylose isomersae
with double-distilled water five times with a Centricon-30 (Amicon) filtration
device to remove contaminating salts and buffer from the solution. The
samples were hydrolyzed in a gas phase for 24 h using 5.7 M-HCl. Amino acid
composition analysis was performed with a Pico-Tag amino acid analyzer
(Waters Associates, Milford, MA, U.S.A.). The N-terminal amino acid
sequence was identified by a protein sequencer model 477A (Applied
Biosystems, Foster City, CA) with an on-line phenylthiohydrantoin analyzer
(Applied Biosystems) in the Macromolecular Structure Facility, Department

of Biochemistry, Michigan State University.

RESULTS

Cloning of the xylose isomerase gene

The xylose isomerase gene from Thermoanaerobacter strain B6A-RI
was cloned by complementation using E. coli xyl-S mutant strain HBIOI.
Transformants were screened on MacConkey agar plates supplemented with
1% xylose and ampicillin. One positive clone which gave a red color on this
plate was detected and the recombinant plasmid, pZXI6, was isolated. The
clone contained vector pUC18 and an insert fragment of 4.5 kilobases (Fig. 1).
The orientation of the fragment of Thermoanaerobacter B6A-RI
chromosomal DNA in the vector, did not affect the level of xylose isomerase
activity (data not shown) suggesting that the cloned xylA gene from
Thermoanaerobacter B6A-RI was expressed in E. coli by its own promoter.

In Thermoanaerobacter B6A-RI xyose isomerase activity was induced
by D-xylose, whereas in E. coli the cloned xylA was expressed constitutively
and addition of D-xylose to the medium did not increase the production (data

not shown).

Nucleotide sequence of the xyl gene

The nucleotide sequence and deduced amino acid sequences are shown
in Fig. 2. The computer analysis of the nucleotides sequenced revealed one
open reading frame (ORF) of 1317 base pairs in length which corresponds to
439 amino acid residues. This was confirmed as the correct reading frame for
the Thermoanaerobacter B6A-RI xylA gene by comparison of the amino-
terminal amino acid sequence (underlined in Fig. 2), determined from the

cloned xylose isomerase purified from E. coli (pZXI6).

192

193

 

 

 

 

pZXl6
z '2 E 2 5% - -
2 as as E a"; 55 <8:
I I I I II I I
i -
0.5kb
xglA ‘——‘

Figure I. The physical map of pZXI6 containing vector pUC18 with the insert
fragment containing the xylose isomerase gene.

The xylose isomerase gene from Thermoanaerobacter strain B6A-RI
was cloned by complementation using E. coli xyI-S mutant strain HBIOI.

Transformants were screened on MacConkey agar plates supplemented with

1% xylose and ampicillin.

194

 

Figure 2. The nucleotide sequence and deduced amino acid sequences
of the cloned xylose isomerase.

The computer analysis of the nucleotides sequenced revealed
one open reading frame (ORF) of 1317 base pairs in length which
corresponds to 439 amino acid residues. The underlined region
denotes the correct reading frame for the Thermoanaerobacter B6A-RI
xylA gene by comparison of the amino-terminal amino acid sequence
determined from the xylose isomerase purified from

Thermoanaerobacter B6A (Lee 8: Zeikus, 1991).

61

121

181

241

301

361

421

481

541

601

661

721

781

1595

TATTTTAGTATCTTCCCAGAIGTTGGCTAATAGTTGGTTTGACAGGGTCGGTTATTTTTT

 

TTAAAATTTTTTTATTAmonAGAAGGArTTTTrAAarrworcracaammkmrharnrara

2giuuuuuuuxnunuuunmmcsaamncaascAGGAAGCTTTATGAATAAAIATTTTGAGA
' SLIL HetAsnLysTerheGluA

 

ACGTATCTAAAATAAAATATGAAGGACCAAAATCAAATAATCCTTATTCCTTTAAATTTT
analSerLysIleLysTyrcluGlyProLysSerAsnAsnProTyrserPheLysPheT

 

ACAATCCAGAGGAAGTAATCGATGGCAAGACGATGGAGGAGCATCTCCGCTTTTCTATAG
yrAsnProGluGluValI1eAspGlyLysThrHetcluGIuHiBLeuArgPheSerIlea

CTTATTGGCACACTTTTACTGCTCATGGAACAGATCAATTTGGCAAGGCTACTATGCAAA
1aTerrpHisThrPheThrAlaAspGlyThrAspGlnPheclyLysAIaThrHetclnA

GACCATGGAACCACTACACAGATCCTAIGGATATAGCGAAACGAAGGGTAGAAGCAGCAI
rgProTrpAanisTerhrAspProMetAspIlehlaLysArgArgVa1G1uA1aA1aP

TTGAGTTTTTTGATAAGATAAATGCACCTTTCTTCTGCTTCCATGATAGGGATATTGCCC
heGluPhePheAspLysI1eAsnAlaProPhePheCysPheHisAspArgAspI1eA1aP

CTGAAGGAGATACTCTTAGAGAGACAAACAAAAACTTAGATACAATAGTTGCTATGATAA
roGluGlyAspThrLeuArgGluThrAsnLysAsnLeuAspThrI1eVa1A1aMetI13L

AGGATTAcTTAAAGAccAGCAAGACAAAAGTTTTGTGGGGTACOGCAAATCTTTTCTCCA
ysAspTereuLysThrserLysThrLysVa1LeuTrpG1yThrA1aAsnLeuPheSerA

ATCCGAGATTTGTACATGGTGCATCAACATCCTGCAATGCTGACGTTTTTGCATATTCTG
snProArgPheva18isGlyAlaSerThrSerCyaAsnhlaAspVa1PheA1aTyrSerA

CAGCGCAAGTCAAAAAAGCCCTTGAGAITACTAAGGAGCTTGGCCGCGAAAACTACGTAT
lahlacanalLysLyshlaLeuGIuIleThrLysGIuLeuclyArgGluAsnTeraIP

TTTGGGGTGGAAGAGAAGGGTACGAGACGCTTCTCAATACAGATATGGAGTTAGAGCTTS
heTrpGlyGlyArgGIuGlyTyrGIuThrLeuLeuAsnThrAspMetGluLeuGIuLeuA

ATAACTTTGCAAGATTTTTGCACATGGCTGTTGACTATGCAAAGGAAATCGGCTTTGAAG
spAsnPheAlaArgPheLeuHisMetAlaVa1A5pTyrA1aLysGluI1eGlyPheG1uG

60

120

180

240

300

360

420

.480

540

600

660

720

780

840

841

901

961

1021

1081

1141

1201

1261

1321

1381

1441

1501

1561

1596

GTCAGTTCTTGATTGAGCCéAAGCCAAAGSAGCCTACAAAACATCAATACGACTTTGACG
1yG1nPheLeuIlecluProLysProLysGluProThrnysHisGlnTyrAspPheAspv

TGGCAAATGTATTGGCATTCTTGAGAAAATACGACCTTGACAAATATTTCAAAGTAAATA
aInlahanalLeuA1aPheLeuArgLysTyrAspLeuAspLysTyrPheLysva1AsnI

TCGAAGCAAACCATGCGACATTGGCATTCCACGACTTCCAACATGAGCTAAGATACGCCA
1eGluAIaAanisAlaThrLeuAlaPheHisAspPheG1nHisGluLeuArgTyrAIaA

GAATAAACGGTGTATTAGGATCAATTGACGCAAATACAGGCGACKTGCTTTTGGGATGGG
rgIlehsnGIYValLeuclySerIlehspAlaAsnThrGlyhspnetLeuLeuclyTrpA

aracccaccasrrcccTacaskrAracGamnauuummmumccrarcramcaacrcaraa
spThrAspGlnPhePrdThrAspIlehrguetThrThrLeuhlanetTyrGluValIleL

AGATGGGTGGATTTGACAAAGGTGGCCTTAACTTTGATGCAAAAGTAAGACGTGCTTCAT
yahetGlyGlyPheAspLysGlyGlyheuhsnphehsphlanysValArgArgAIaSerP

TTGAGCGAGAAGATCTTTTCTTAGGTCACATAGCAGGAATGGATGCTTTTGCAAAAGGCT
heGluProGIuAspLeuPheLeuGlyfiisIleAIaG1yMetAspA1aPheA1aLysG1yP

TTAAAGTTGCTTACAAGCTTGTGAAAGATEGCGTATTTGACAAGTTCATCGAAGAAAGKT
heLysVa111aTerysLeuValLysAspclyValPheAspLysPheIleclucluhrgr

ACGCAAGCTAGAAAGAAGGCATTGGCGCTGATATTGTAAGCGGTAAAGCTGACTTCAAGA
yrAlaSerTerysGIuclyIleclyAIaAspI1eValSerGIyLysAIaAspPheLyss

GCCTTGAAAAGTATGCATTAGAGCACAGCCAGATTGTAAACAAATCAGGCAGACAAGAGC
orLeuGluLysTyrAlaLeuGlunisSerGlnIIeVa1AsnLysSerG1yArgcln61uL

TATTAGAATCAATCCTAAATCAGTATTTGTTTGCAGAATAATGAAACATGAGGGCGGCTT
euLeuGluSerIleLeuAsnG1nTereuPheAlaG1uEndEnd

CATGCTTCATTAAGGCTGCCCTCAACAAAAATCATGGAGGTAAATGTATGTATTTTTTAG

GGATAGATTTAGGGACATCATCAGTTAAGATAATACTGATG 1601

900

960

1020

1080

1140

1200

1260

1320

1380

1440

1500

1560

1 9 7
The ATG initiation codon is preceded with a spacing of 7 base pairs, by

a putative ribosomal binding sequence AAGGAGG which is complementary
to the 3' end of both the E. coli and B. subtilis 16S ribosomal RNA (25, 33). A
putative -10 promotor region (Pribnow box), TATAATATATAAT, which
repeated twice at the -10 region in E. coli, was found 25 bp upstream of the

ribosomal binding site.

Comparison of amino acid sequences

The deduced amino acid sequence of xylose isomerase from
Thermoanaerobacter B6A-RI was compared to those of Clostridium
thermosulfurogenes (20), Clostridium thermohydrosulfuricum (9), E. coli
(30), Bacillus subtilis (34), Streptomyces violaceoniger (13), Streptomyces
rubiginosus (36) and Thermus thermophilus (10) and the results are shown
in Table 1.

The length of the polypeptide chain encoding for the xylA gene in
Thermoanaerobacter B6A-RI was identical to that of C. thermosulfurogenes
4B (439 amino. acid residues) and was similar to those of C.
thermohydrosulfuricum 39B (438 residues), E. coli and B. subtilis enzymes
(440 residues), whereas the enzymes from S. violaceoniger, Ampullariella,
and Arthrobacter have polypeptides which are shorter by 47 amino acid
residues at the NHz-terminal end. The deduced amino acid sequence of
Thermoanaerobacter B6A—RI xylose isomerase exhibits very high homology
to those from C. thermosulfurogenes and C. thermohydrosulfuricum (98%
and 89% identity, respectively) and their sequence alignment is shown in Fig.

3.

198

Table 1. Relative homology between xylose isomerase from

Thermoanaerobacter B6A-RI and other xylose isomerases.

Strain % Similarity % Identity
B. subtilis 82.5 % 69.6 %
E. coli 64.0 % 45.9 %
T. thermophilus 51.1 % 30.5 %
S. rubiginosus 49.9 % 26.5 %
S. violaceoniger 48.3 % 26.4 %
C. thermosulfurogenes 99.1 % 98.2 %

C. thermohydrosulfuricum ND 90.4 %

199

Figure 3. Comparison of the xylose isomerase amino acid sequence of:
[I] Thermoanaertobacter B6A-RI, [2] Clostridium thermosulfurogenes
4B, [3] Clostridium thermohydrosulfuricum 39E. Stars under the
sequence indicate amino acids which are different in all three

sequences compared.

86A [1]
4812]
391-313]

200

MNKYFENVSKIKYBGPKSNNPYSFKPYNPEEVIDGKTMBEHLRFSIAYWH
MNKYFENVSKIKYEGPKSNNPYSFKPYNPEEVIDGKTMEEHLRFSIAYHH

-MEYFKNVPQIKYEGPKSNNPYAFKFYNPDEIIDGKPLKEHLRFSVAYNH
as: a at t at:

TFTADGTDQFGKATMQRPWNHYTDPMDIAKRRVEAAFBFFDKINAPFFCF
TFTADGTDQFGKATMQRPNNHYTDPMDIAKARVBAAFEFPDKINAPYPCF

TPTANGTDPFGAPTMQRPWDHFTDPMDIAKARVEAAPBLPEKLDVPFFCP
* * kt t x * tn

HDRDIAPEGDTLRETNKNLDTIVAMIKDYLKTSKTKVLHGTANLFSNPRF
HDRDIAPEGDTLRETNKNLDTIVAMIKDYLKTSKTKVLWGTANLPSNPRF
HDRDIAPEGETLRETNKNLDTIVAMIKDYLKTSKYKVLWGTANLFSNPRF

VHGASTSCNADVFAYSAAQVKKALBITKELGRENYVPWGGREGYBTLLNT
VHGASTSCNADVPAYSAAOVKKALEITKELGGENYVFHGGRBGYBTLLNT

VHGAATSCNADVFAYAAAOVKKALEITKELGGQNYVFHGGREGYETLLNT
t t **

DMELBLDNFARFLHMAVDYAKEIGPEGOPLIBPKPKBPTKHQYDFDVANV
DMBPELDNPARFLHMAVDYAKEIGKBGQFLIBPKPKEPTKHQYDPDVANV

DMELELDNLARPLHMAVEYAQEIGPBGQFLIBPKPKEPTKHQYDPDAASV
a t t t :

LAPLRKYDLDKYPKVNIEANHATLAPHDFQHELRYARINGVLGSIDANTG
LAFLRKYDLDKYFKVNIBANHATLAFHDPQHELRYARINGVLGSIDANTG

HAFLKKYDLDKYFKLNIBANHATLAGHDPQHELRYARINNMLGSIDANMG
* tr e

DMLLGWDTDQFPTDIRMTTLAMYEVIKMGGFDKGGLNFDAKVRRASFEPE
DMLLGWDTDQFPTDIRMTTLAMYEVIKMGGFDKGGLNFHAKVRRASFEPE

DMLLCWDTDQYPTDIRMTTLAMYEVIKMGGFNKGGLNFDAKVRRASPEPE
* *

DLFLGHIAGMDAPAKGPKVAYKLVKDGVFDKFIBERYASYKEGIGADIVS
DLFLGHIAGMDAFAKGFKVAYKLVKDRVFDKFIBERYASYKDGIGADIVS

DLFLGHIAGMDAPAKGPKVAYKLVKDGVPDRFIEBRYKSYREGIGAEIVS
* *

GKADFKSLEKYALEHSQIVNKSGROELLESlLNQYLFAE
GKADPKSLEKYALERSQIVNKSGRQELLESILNQYLFAE

GKANFKTLBEYALNNPKIENKSGKQELLESILNQYLFS
* * **** * t

50
50
49

100
100
99

150
150
149

200
200
I99

250
250
249

300
300
299

350
350
349

400
400
399

439
439
438

2 O 1
Codon usage in xynA, xynB and xylA

Table 2 summarizes codon usage in the three genes from
Thermoanaerobacter B6A-RI which are involved in xylan degradation. xynA
and xynB were closely linked on a chromosome of Thermoanaerobacter B6A-
RI (Lee 8: Zeikus, manuscript prepared for publication). Averaged codon
usage for E. coli (1) is included for comparison. The patterns of codon usage
among the three genes were very similar to each other. There is a clear
propensity of AT-containing isocodons, and this probably results from the

low G+C content (36 mol%) of this organism.

Biochemical characterization of cloned xylose isomerase
The thermostable Thermoanaerobacter B6A-RI xylose isomerase
expressed in E. coli was purified to homogeneity (Fig. 4). Heat treatment of
cell extracts at 85°C for 15 min was a very efficient purification step for the
enzyme from E. coli as approximately 90% of E. coli proteins could be
removed by this procedure. After heat treatment, the purification yield
increased 18 fold. After additional steps of purification by anion-exchange
chromatography and gel filtration. Preparations of homogeneous
recombinant xylose isomerase were obtained and its purity was demonstrated
on SDS-polyacrylamide gel electrophoresis (Fig. 4). The purified enzyme
consisted of one type of subunit with a molecular mass of 50 kDa. The
molecular mass of the purified recombinant enzyme was 200kDa, indicating
that the enzyme is a tetramer comprised of identical subunits.
The apparent temperature and pH optimum for the activity of the
cloned xylose isomerase was 80°C and 7.0 to 7.5 respectively.- The enzyme was
stable at 80°C for 60 min, and 0.5 mM C02+ and 5 mM Mg2+ were required for

optimum enzyme activity and thermostability.

202

Table 2. A comparison of the codon usage frequency in the three genes from
Thermoanaerobacter B6A-RI which are involved in xylan degradation.

Frequency (mol%) of codon usage

 

 

 

Amino Codon Themxoanaerobacter B6A-RI E. coli
acid
xynA. xynB ny\
Phe T'IT 2.8 5.6 5.2 1.3
'I'I‘C 0.4 I .0 3.0 2.2
Leu TTA 2.1 1.4 1.6 0.7
TIC 1.7 2.0 1.8 0.9
CIT 1.4 2.0 . 3.0 0.8
CT C 0 0.4 0.5 0.8
CT A 0.1 0.4 0.7 0.2
CFC 0.6 0.2 0 6.8
He ATT 3.3 2.4 1.6 2.2
ATC 0.6 0.8 1.1 3.7
ATA 3.4 3.4 2.3 0.2
Met ATG 1 .8 2.2 2.7 2.8
Val CIT 3.1 2.2 1.1 2.9
GTC 0.3 1.0 0.5 1.2
GT A 2.8 2.4 2.7 1.8
CTG 1.0 1.8 0.5 2.2
Ser TCI‘ 2.4 1.4 ‘ 0.7 1.3
TCC 0.2 0.4 0.7 1.5
TCA 2.0 1.0 1.4 0.4
TCG 0.4 0.2 0 0.6
AGT 1.5 0.4 0 0.3
AGC 1.2 1.2 1.1 1.4
Pro CCT 1.2 2.0 1.4 0.5
CCC 0.2 0.2 0 0.3
CCA 1 .9 2.4 1.1 0.7
CCG 0.4 0.8 0.5 2.5
Thr ACT 2.9 1 .2 1.1 1.1
ACC 0.4 0.6 0.5 2.4
ACA 3.6 2.0 2.7 0.3
ACG 1.1 1.0 0.9 0.8
Ala GCl‘ 2.0 1 .6 2.7 2.6

CCC 0.4 0.4 0.7 2.2

 

203

Table 2. A comparison of the codon usage frequency in the three genes from
Thermoanaerobacter B6A-RI which are involved in xylan degradation (cont.).

Frequency (mol%) of codon usage

 

 

 

Amino Codon Thmnoanaerobacter B6A-RI E. coli
acid
xynA xynB xylA
Ala GCA 3.1 1.2 4.6 2.3
GCG 0.4 0.6 0.7 3.2
Tyr TAT 3.8 3.8 2.3 1.0
TAC 0.7 2.2 2.5 1.5
lfis CAT L0 L4 L4 0]
(ZAC: OJ ‘L0 ‘L4 'L2
Gln CAA 3.1 1.0 1.4 1.0
CAG 1.1 0.8 0.9 3.2
Asn AAT 6.8 4.2 3.0 1.0
AAC 1.3 0.8 2.3 2.8
Lys AAA 4.4 3.2 5.0 4.1
AAG 2.0 3.8 3.4 1.3
Asp CAT 6.3 5.4 4.6 2.5
GAC 2.3 1.4 3.0 3.0
Glu GAA 4.2 6.4 3.9 4.9
GAG 0.7 2.8 3.9 ' 1.8
Cys TGT 0 0.4 0 0.4
TCC 0.1 0.4 0.5 0.5
Trp TGG 2.2 2.2 1.1 0.7
Arg CGT 0.1 0 0.2 3.1
CCC 0 0 0.7 2.0
CGA 0 0.6 0.2 0.2
OGG 0 0 0 0.2
AGA 1.2 3.0 2.5 0.1
AGG 0.4 1.2 0.5 0.1
Gly GGT 2.3 1.0 2.0 3.8
GGC 1.3 1 4 2 5 3 1
GGA 2.8 3 2 1 8 0 4

204

kDa M1234MkDa

97
66

45
31

22

 

 

Figure 4. SDS-PAGE analysis of the thermostable Thermoanaerobacter
B6A-RI xylose isomerase expressed in E. coli (pZXI6).

Lane M, molecular weight markers; lane 1, crude extract; lane 2, after
heat treatment; lane 3, after anion exchange chromatography; lane 4, after gel

filtration.

DISCUSSION

Xylose isomerase is an important industrial enzyme (2, 6, 7) because of
its ability to catalyze the conversion of glucose to fructose. Xylose isomerases
from thermophilic bacteria have high temperature optima and are
thermostable, indicating their potential use for the commercial conversion of
starch to high fructose corn syrup at high temperature with increased fructose
yield.

Xylose isomerases has been isolated and studied from many
microorganisms. xyl genes from E. coli (30), B. subtilis (34, 35), T.
thermophilus (10), S. violaceoniger (24), C. thermohydrosulfuricum (10) and
C. thermosulfurogenes (19) have been cloned and sequenced.

Comparison of the primary structure of xylose isomerases from three
thermophilic anaerobic bacteria, Thermoanaerobacter B6A-RI, C.
thermosulfurogenes and C. thermohydrosulfuricum, indicate extensive
similarities between this enzyme from these organisms. Because these strains
have evolved in thermal hotsprings in Yellowstone National Park, (22, 31)
these organisms might have a common phylogenic origin with conservation
of xylose isomerase, or alternatively gene transfer might have occurred
between these strains.

In several bacteria in which the genetic organization of the xyl genes
has been examined, the xylose isomerase and xylulose kinase genes were
found to be part of one operon (15, 17, 23, 32, 34). Another open reading frame
with the same transcriptional orientation was found downstream of xylA

from the cloned DNA fragment (data not shown) and this may be a xylulose

kinase gene (xle).

205

2 0 6
The predicted molecular weight of 50,474, calculated from the deduced

amino acid sequence, is in good agreement with the subunit molecular
weight of 50,000 reported for xylose isomerase purified from
Thermoanaerobacter (21). The recombinant protein was purified to
homogeneity and its physical and biochemical properties were determined.
The molecular mass of the purified recombinant xylose isomerase was 200
kDa, indicating that the enzyme is a tetramer composed of identical subunits.
This result is consistent with the native xylose isomerase from
Thermoanaerobacter B6A (21). The xylose isomerase expressed in E. coli
displayed identical pH and temperature optimum, thermostability, and metal
ion requirements with the xylose isomerase from Thermoanaerobacter (21).

The base composition of the coding region of xyIA is 39.7 mol% G+C, a
value that is close to the G+C content (36 mol %) of this organism (22). The
low G+C content of Thermoanaerobacter B6A-RI xylA results in an extremely
biased usage of synonymous codons, A or T, which were present in the third
position of all dominant codons examined in this organism.

The high thermostability of Thermoanaerobacter B6A-RI xylose
isomerase made it possible to use heat treatment as one of the most efficient
purification steps of the cloned enzyme produced in the mesophilic host E.
coli. The presence of metal ions (Mg2+ and Co“) and a high protein
concentration in cell extracts were essential for optimal recovery of the
enzyme during heat treatment. The thermostable xylose isomerase produced
by the recombinant plasmid pZXI6 was one of the most abundant protein in
E. coli.

The overall biochemical and physico-chemical properties of the
recombinant xylose isomerase purified from E. coli (pZXI6) were identical to

the native enzyme purified from Thermoanaerobacter B6A (21). The N-

207
terminal amino acid sequences in the recombinant and native xylose

isomerase were identical (21).

The catalytic mechanism for xylose isomerase was originally believed
to involve histidine-directed general base catalysis (27). Lee et al. (20)
identified the active site histidine residues of xylose isomerase from C.
thermosulfurogenes by site-directed mutagenesis, and found that His101 was
the only essential histidine residue involved directly in catalysis. The rate
limiting step in the isomerization reaction was found to be hydrogen transfer
and not substrate ring opening.

At present the molecular mechanism for high thermophilicity (i.e.
temperature optimum for activity at 80°C and thermostability at 85°C) of this
enzyme is not clear. This could be addressed through the use of nested
deletion mutants in order to identify regions on the gene which are

responsible for activity and thermostability.

10.

LITERATURE CITED

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coding strand of the E. coli genome. N uc. Acids Res. 12:2235-2241.

Antrim, R. L., W. Colliala, and B. J. Schnyder. 1979. Glucose isomerase
production of high-fructose syrups, p. 97-155. In L. B. Wingard (ed.),
Applied biochemistry and bioenginerering. Academic Press, Inc., New
York.

Bachmann, B. J. 1972. Pedigrees of some mutant strains of Escherichia
coli K-12. Bacteriol. Rev. 36:525-557.

Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction
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Bradford, M. M. 1976. A rapid and sensitive method for quantitation of
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Bucke, C. 1977. Industrial glucose isomerase. p. 147-171. In A.
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Bucke, C. 1980. Enzymes in fructose manufacture, p. 51-72. In G. G.
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Clewell, D. B. 1972. Nature of Col E1 plasmid replication in
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Dekker, K., H. Yamagata, K. Sakaguchi, and S. Udaka. 1991. Xylose
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Dekker, K., H. Yamagata, K. Sakaguchi, and S. Udaka. 1991. Xylose
(glucose) isomerase gene from the thermophile Clostridium
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Devereux, J., P. Haeberli, and O. Smithies. 1984. A comprehensive set

of sequence analysis programs for the VAX. N uc. Acids Res. 12:387-
395.

Dishe, 2., and E. Borenfreund. 1951. A new spectrophotometric
method for the detection and determination of keto sugars and trioses.
J. Biol. Chem. 192:583-587.

Drocourt, D., S. Bejar, T. Calmels, J. P. Reynes, and G. Tiraby. 1988.
Nucleotide sequence of the xylose isomerase gene from Streptomyces
violaceoniger. Nuc. Acids Res. 16:9337.

Hastup, S. 1988. Analysis of the Bacillus subtilis xylose regulon. p. 79-
83. In A. T. Granesan, and J. A. Hoch (eds.), Genetics and biotechnology
of Bacilli. vol. 2. Academic Press, New York.

Ghangas, G. S., and D. B. Wilson. 1984. Isolation and characterization
of the Salmonella typhimurium LT2 xylose regulon. J. Bacteriol.
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Kreuzer, P., D. Gartner, R. Allmansberger, and W. Hillen.
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Lawlis, V. B., M. S. Dennis, E. Y. Chen, D. H. Smith, and D. J.
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47:15-21.

Lee, C., B. C. Saha, and J. G. Zeikus. 1990. Characterization of
Thermoanaerobacter glucose isomerase in relation to saccharidase

synthesis and development of single-step processes for sweetner
production. Appl. Environ Microbiol. 56:2895-2901.

Lee, C., L. Bhatnagar, B. C. Saha, Y.-E. Lee, M. Takagi, T. Imanaka, M.
Bagdasarian, and J. G. Zeikus. 1990. Cloning and expression of the
Clostridium thermosulfurogenes glucose isomerase gene in
Escherichia coli and Bacillus subtilis. Appl. Environ. Microbiol.
56:2638-2643.

Lee, C., M. Bagdasarian, M. Meng, and G. J. Zeikus. 1990. Catalytic
mechanism of xylose (glucose) isomerase from Clostridium
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21.

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210

Lee, C., and J.G. Zeikus. 1991. Purification and characterization of
thermostable glucose isomerase from Clostridium thermosulfurogenes
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Lee, Y.-E., M. K. Jain, C. Lee, S.E. Lowe and J. G. Zeikus. 1992.
Taxonomic distinction of saccharolytic thermoanaerobes: Description
of Thermoanaerobium gen. nov., xylanolyticum sp. nov. and
Thermoanaerobium saccharolyticum sp nov. Reclassification of
Thermoanaerobium brockii, Clostridium thermosulfurogenes and
Clostridium thermohydrosulfuricum E100-69 as Thermoanaerobacter
brockii comb. nov., Thermoanaerobacterium thermosulfrigenes comb.
nov. and Thermoanaerobacterium thermohydrosulfuricus comb. nov.,
and transfer of Clostridium thermohydrosulfuricum 39E to
Thermoanaerobacter ethanolicus. Manuscript submitted to the
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Marcel, T., D. Drocourt, and G. Tiraby. 1987. Cloning of the glucose
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Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with
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from Escherichia coli: characterization of the protein and the structural
gene. J. Biol. Chem. 259:6826-6832.

31.

32.

33.

35.

36.

37.

38.

211

Schink, B., and J. G. Zeikus. 1983. Clostridium thermosulfurogenes sp.
nov., a new thermophile that produces elemental sulphur from
thiosulphate. J. Gen. Microbiol. 129:1149-1158.

Shamanna, D. K., and K. E. Sanderson.1979. Genetics and regulation of
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Stormo, G. D., T. D. Schneider, and L. Gold. 1982. Characterization of
translational initiation sites in E. coli. Nucleic Acids Res. 10:2971-2996.

Wilhelm, M., and C. P. Hollenberg. 1984. Selective cloning of Bacillus
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Res. 16:1220.

Chapter VII

Characterization of the active site and thermostability

regions of endoxylanase from Thermoanaerobacter

212

AB STRACT

Deletion mutants were constructed from pZEP12 which contained the
intact Thermoanaerobacter endoxylanase gene (xynA). Deletion of 2 kb from
N-terminal end of xynA resulted in a mutant enzyme that retained activity
but lost thermostability. Deletion of 1.5 kb from the C-terminus did not alter
thermostability or activity. The deduced amino acid sequence of
Thermoanaerobacter B6A-RI endoxylanase xynA was aligned with five other
enzymes in family F B-glycanases by using the PILEUP program of the GCG
package. This multiple alignment of amino acids sequences revealed 6 highly
conserved motifs which included the consensus sequence ITELD in the
catalytic domain. Endoxylanase was inhibited by EDAC suggesting that Asp
and/ or Glu were involved in catalysis. Three aspartic acids, two glutamic
acids, and one histidine were conserved in all six enzymes aligned and they
were targeted for analysis by site-specific mutagenesis. Substitution of Asp-612
by Asn, Glu-508 by Gln, and His-539 by Asn had little effect on the enzyme
activity, whereas substitution of Asp-504 and Asp-569 by Asn and Gln-567 by
Gln completely destroyed endoxylanase activity and implicated their role in

general acid catalysis mechanism.

213

INTRODUCTION

B-1,4-xylan is a major component of hemicellulose and has a backbone of
B-1,4-linked D-xylopyranoside residues substituted with acetyl, arabinosyl and
uronyl side chains (3). After cellulose, xylan is the most abundant renewable
polysaccharide in nature, accounting for 20 to 30% of the dry weight of woody
tissue (39).

Complete breakdown of branched acetyl xylan requires the action of
several hydrolytic enzymes (3, 28, 41). The most important microbial
enzymes involved in xylan degradation are endo-1,4-B-D-xylanase (EC 3.2.1.8)
and B-D-xylosidase (EC 3.2.1.37). Endo-1,4-B-D-xylanase acts on chains of
xylans, arabinoxylans and 4-MeO-glucuronoxylans. B-xylosidase breaks down
short oligosaccharides from the nonreducing end to xylose, and has
substantial transferase activity (28).

These xylan degrading enzymes are produced by a wide variety of
microorganisms including aerobic and anaerobic mesophiles and
thermophiles (3, 38). The majority of the studies have been carried out in
aerobic, mesophilic fungi and bacteria (3, 28, 38, 41).

Recently B-glycanases were grouped into 9 different structural
paradigms (families A to families 1) according to hydrophobic cluster analysis
with the finding that most xylanases belong to either family F or family G
(40). Generally family F xylanases are larger in size (269 to 1,011 amino acid
residues) than family G xylanases which have a small molecular weight (182
to 234 amino acid residues). Family F includes Bacillus sp. strain C-125 xynA
(14), Butyrivibrio fibrisolvens xynA (18), Caldocellum saccharolyticum CelB,
xynA and ORF4 (19, 32), Cellulomonas fimi Cex (25), Clostridium

thermocellum xynZ (12), Cryptococcus albidus Xyn (5), Pseudomonas

214

215
fluorescens subsp. cellulosa xynA and xynB (13, 17), and Thermoascus

aurantiacus xyn (33).

Despite the interest in the xylanases for the bioconversion of renewable

plant cell materials and for their industrial applications, little is known about
their mechanism of catalysis. Xylanases and cellulases, like other
glycosidases, have been suggested to act by general acid catalysis involving 2
amino acid carboxy groups (1, 6, 7, 9, 16, 21, 23, 24, 26, 35, 42).
In the absence of X-ray crystallography data, some informations about the
amino acid residues involved in the active site can be obtained by using
labeled substrates and chemical modification. These approaches are limited
as it is not possible to identify the specific amino acid residues in the active
site. Identification of conserved amino acid residues through alignment of
amino acid sequences from similar enzymes can be a useful method to predict
essential amino acids for catalysis, and this prediction can be confirmed by
site-directed mutagenesis.

We have recently cloned and sequenced the endoxylanase A gene (xynA)
and showed it belongs to family F (Lee 8: Zeikus, manuscript prepared for
submission). The purpose of the present report is to identify the catalytic and
thermostability domains of endoxylanase A of Thermoanaerobacter and to

provide evidence of the specific Glu and Asp residues involved in catalysis.

MATERIALS AND METHODS

Strains, enzymes, and chemicals

E. coli strain SDM (hst17, mcrAB, recAI, supE44, Tet’, A (lac-proAB), [F'
traD36, pro AB+,lacI<1ZAM15]) was used as a host strain for site-directed
mutagenesis. -Restriction endonucleases and T4 DNA ligase used for
subcloning experiments were obtained from Bethesda Research Laboratories
(Gaithersburg, MD) and Boehringer Mannheim Biochemicals (Indianapolis,
IN). [a-3SS]dATP (12.5 mCi/ ml) was purchased from DuPont-New England
Nuclear (Bannockburn, IL). 1-(3-Dimethylaminopropenyl)-3-ethylcarbodi-
irnide hydrochloride (EDAC) was obtained from Aldrich (Milwaukee, WI).

All other chemicals were reagent grade.

General DNA procedures

Plasmid DNA preparation, electrophoresis and. DNA fragment isolation
were performed using standard procedures (30). The alkaline denaturation
method (4) was used for rapid extraction of plasmid DNA. Enzymes were
used as recommended by the manufacturers. Bacterial transformation was

performed by the Hanahan method as described by Perbal (27).

Subcloning experiments

Plasmid pZEP12 was the parent of all the plasmids generated throughout
this work (Figure 1). It carries the xynA gene on a 4.5 kb EcoRI-Pstl restriction
fragment inserted at the multicloning site of the pUC18 vector. Deletion
mutants were derived from pZEP12 by subcloning as described previously

Lee et al., manuscript prepared for submission).

216

2 l 7
A 4.5 kb EcoRI-PstI DNA fragment, containing the intact xynA gene, was

isolated from pZEP12 by partial digestion and inserted at the EcoRI-PstI sites
of the phagemid vector pUC119 (37). The resulting recombinant plasmid
pSDM12 was transformed into E. coli strain SDM and used for the isolation of

single-stranded DNA for site-directed mutagenesis.

Computer analysis

The amino acid sequences of six family F xylanases and cellulases were
aligned with the deduced amino acid sequence of Thermoanaerobacter xynA
using the Sequence Analysis Software Package of the Genetics Computer

Group (GCG), version 7 (University of Wisconsin) (10).

Chemical modification

The inhibitory action of EDAC on xylanase activity was studied by
varying EDAC concentrations in the reaction mixture from 10 to 100 mM.
The reaction mixture contained EDAC with enzyme (500 pg) with 50 mM
glycine methyl ester with 50 mM imidazole (pH 6.0) and was incubated at
45°C. After 1 h a 10 pl sample of the each reaction mixture was withdrawn
and added to 90 pl of 100 mM sodium acetate buffer, pH 5.5, to quench the
residual reagent. The remaining endoxylanase activity of the modified
enzymes were determined by the 3,5-dinitrosalicylic acid assay (Miller, 1959)
using oat spelt xylan (1%) as a substrate. To assess the ability of the soluble
xylan to protect xylanase from modification with EDAC, the enzyme (500 pg)
was incubated with 100 mM EDAC in the presence of soluble xylan (1%), and

the remaining activity measured.

218

Site-directed mutagenesis and nucleotide sequencing

Plasmid pSDM12 was used for site-directed mutagenesis. Based on the
sequence alignment, the mutagenic oligonucleotides were designed to be
complementary to the single-stranded template DNA and to contain
appropriate mismatches as indicated in Table 1. The oligonucleotide primers
were synthesized by the Michigan State University Macromolecular facility
using a automated DNA synthesizer (Applied BioSystems, Foster City, CA).
Synthesis of mutant genes and selection was performed by the method of
Vandeyar et al. (36), using a kit of “IV-GENTM In Vitro Mutagenesis kit (United
State Biochemical Co., Cleveland, OH). Mutant strains were cultured in the
presence of M13 helper phage M13KO7 (37) and single stranded DNA was
prepared from the culture supernatants as described previously (30).
Nucleotide sequences of the isolated single-stranded DNA were determined
by the dideoxy chain termination method (31), using a Sequenase version 2.0

kit (U. 5. Biochemical Co.).

219

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RESULTS

Subcloning and characterization of xynA thermostability domain

The physical map of the Thermoanaerobacter B6A-RI xynA gene and its
deletion mutants are shown in Fig. 1. Deletion of 2 kb from N-termial end of
xynA using deletion mutant resulted in a reduction of thermostability and
thermophilicity (Table 2). Although the deletion mutant was still active at
high temperature, the optimum temperature for activity was 65°C versus
75°C for wild type enzyme. Deletion of 1.5 kb from C-terminal end of xynA

did not affect thermostability or thermophilicity.

Comparison of the deduced amino acid sequences of xylanase A with the
family F B-1,4-glycanases.

I The complete nucleotide sequence of the endoxylanase gene (xynA) from
Thermoanaerobacter B6A-RI was determined previously (Lee 8: Zeikus,
manuscript prepared for submission). It has an open reading frame of 3471 bp
encoding 1157 amino acid residues. The deduced amino acid sequence was
also. compared to those of other proteins deposited in the data base. Most
proteins showing strong homology to xynA were xylanases and cellulases of
family F B-1,4-glycanases. This is also confirmed by hydrophobic cluster
analysis which grouped B-glycanases into 9 families based on the sequence
analysis of conserved sequences found in discrete domains (11).

The consensus sequence, ITELD, is found in the catalytic domain of the
family F glycanases, which currently comprises 3 glucanases and 11 xylanases
(2, 11, 15, 35). Alignment of the deduced amino acid sequences of

endoxylanase from Thermoanaerobacter B6A-RI with those of family F B-

220

221

 

 

 

 

 

 

 

 

 

11kb.]

I 1111]] III

I j
1 III :>

1 pZEP12 1

L, pZEPl I -----------------

__________________________ 1 pZHPI 1

 

Figure 1. The physical map of the DNA fragment containing the
Thermoanaerobacter B6A-RI xynA gene and the deletion mutants.
The black box indicates the location of consensus sequence in the

putative catalytic domain. Dashed line represents deleted fragment.

222

Table 2. Comparison of temperature optimum and thermostability of

endoxylanase with its deletion derivatives.

Mutant Size
plasmid deleted
pZEP12 none
pZEPI 1.5 kb
pZHPl 2.0 kb

Deleted Temp Thermostabilitya
region optimum

none 70°C 75°C

C-terminus 70°C 75°C

N-terminus 65°C 65°C

a The activity was not decreased at this temperature at least 1 h.

223
glycanases reveals that Thermoanaerobacter B6A-RI xylanase also contains

the conserved sequence, ITELD, in its putative catalytic domain (Fig. 2).
Thermoanaerobacter B6A-RI endoxylanase A has 68% similarity to the
Bacillus sp. strain C-125 endoxylanase and 43 to 66% similarity to other
family F B-glycanases. For more detailed analyses the deduced amino acid
squence of the Thermoanaerobacter B6A-RI endoxylanase xynA was aligned
with those of the other five enzymes in family F B-glycanases by using the
PILEUP program of the GCG package (University of Wisconsin) (Fig. 3). This
multiple alignment of amino acids sequences revealed 6 highly conserved
motifs shown in the box of Fig. 3 which includes the consensus sequence

ITELD.

Role of Glu and Asp in catalysis

In order to confirm that amino acid carboxy groups are essential for
endoxylanase activity, we investigated the inhibition effect of EDAC on the
endoxylanase activity (Table 3). Incubation of the enzyme in the presence of
100 mM of EDAC for 1 h resulted in total loss of activity and addition of xylan
before EDAC treatment prevented the loss of activity. This result indicated
that Aspartic acid and/ or Glutamic acid are involved in the active site of the
endoxylanase.

The predicted importance of aspartic and glutamic acids in catalysis of
the endoxylanase were tested by altering the conserved amino acids found in
the sequence alignment. Three aspartic acids, two glutamic acids, and one
histidine were conserved in all six enzymes aligned and these six amino acids
were targeted for site-specific mutagenesis. Table I shows the oligomers used
and the position of the mutations introduced. The conserved aspartic acid

residues at position 504, 569 and 612 were changed to asparagine. The

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227

Table 3. Effect of EDAC on recombinant endoxylanase A activity.“

EDAC conc. Residual activity

(mM) (%)
0 100
5 73
10 48
20 29
30 17

50 12.5

100 1.7
100 ( + 1% xylan) 92

a Purified enzymes (500 pg) in 50 mM imidazole buffer (pH 6.0) were
incubated with various EDAC concentration at 45°C for 1 h. The residual
endoxylanase activity was assayed as described in Materials 8: Methods
and expressed as percentage of specific activity found in the control

Without EDAC.

2 2 8
conserved glutamic acids at position 508 and 567 were changed to glutamine.

The conserved histidine at position 539 was changed to asparagine. The
mutations were confirmed by nucleotide sequencing.

Table 4 shows that substitution of Asp-504 and Asp-569 by Asn and Glu-
567 by Gln completely destroyed endoxylanase activity whereas the other

amino acid substitutions had little effect on enzyme activity.

229

Table 4. Specific activity of mutated endoxylanases

Enzyme Mutation Specific activity, U/ ml (%)a
Wild type None 3.01 :t 0.057 (100.0)
D504N Asp504 —> Asn 0.03 21:0.008 (1.0)
E508Q Glu503 —-> Gln 1.97 21:0.045 (65.4)
H539N His539 -—-> Asn 2.23 i0.055 (74.1)
E567Q Glu567 —-> Gln 0.065 i 0.011 (2.1)
D569N Asp569 --> Asn 0.056 :1: 0.003 (1.9)
D612N Asp6l7- --> Asn 3.68 10.364 (122.2)

a The enzyme activity present in crude extract was determined and percent

of specific activity relative to wild-type enzyme is given in parenthese.

DISCUSSION

To date only active site domains and substrate binding domains have
been characterized in glycanases including xylanases. Essentially nothing is
known about thermostability domains in thermophilic enzymes. The N-
terminal end of xynA appear to confer thermostability and thermophilicity
on endoxylanase A from Thermoanaerobacter B6A-RI. Further deletion from
the N-terminal end may result in complete loss of activity, demonstrating a
region responsible for activity and stability at high temperature.

Despite the structural differences in the substrates cellulose and xylan,
cellulases and xylanases share a high degree of homology to each other. The
highly conserved regions were suggested to be part of the catalytic and
substrate binding site. It is assumed that B-l,4-glycanases, like other enzymes
which hydrolyze glycosidic bonds, use a general acid-base catalytic
mechanism, promoted by aspartate and/ or glutamate residues. Stabilization
of the carbonium intermediate is acheived by a residue of the active site with
a negatively charged group (Asp, Glu) or by a histidine residue. As active site
residues are usually highly conserved during evolution, the catalytic domains
of cellulases and xylanases have been analyzed for conserved aspartates and
glutamates in an attempt to target catalytic residues (15).

Hydrophobic cluster analysis has been proven to be an appropriate
method for the comparison of cellulases and xylanases (15), which were
found to comprise 9 different families. Sequence alignment and hydrophobic
cluster analysis data indicated that Thermoanaerobacter B6A-RI endoxylanase
(xynA) belongs to family F B-glycanases. A search for Asp and Glu residues

occurring at the same position in all members of family F may identify the

230

2 3 I
putative catalytic amino acids. A conserved motif, ITELD, was found in all

family F 1.3—glycanases and was assumed to be involved in catalytic site.

In Cellulomonas fimi which is a member of family F, the active site
nucleophile has been identified as glutamic acid residue 274 by using a
tritium-labeled inactivator and sequencing the radiolabeled peptide (35). This
glutamic acid residue is completely conserved in the family F enzymes,
occurring in all of these enzymes. Using chemical modification studies,
evidence was provided for the importance of carboxy groups for catalysis in
the active site of the Schizophyllum commune xylanase (6). The importance
of Glu residues had been suggested by chemical modification of various
cellulases (2), including endo-B-I,4-glucanase from Schizophyllum commune
(34), and cellobiohydrolase I from Trichoderma reesei (9). Using site-directed
mutagenesis Glu-194 and Glu-169 were changed to the isosteric glutamine
form in endo-B-1,4-glucanase from Bacillus polymyxa and Bacillus subtilis
respectively, resulting in a dramatic loss of carboxymethyl cellulase activity
which could be restored by reverse mutation (1). The involvement of the
carboxy group is consistent with the view that cleavage of a B-1,4-glycosidic
linkage proceeds through a mechanism based on general acid catalysis.

To date only two small molecular weight xylanases, a xylanase from
Bacillus pumilus IPO (22, 29) and a xylanase from Trichoderma harzianum
(29), were crystallized and analyzed by X-ray crystallography. In the absence of
a known three dimensional structure, sequence comparisons among enzymes
with similar function have proven useful for defining minimum functional
size, and binding and catalytic domains. We used multiple sequence
alignment among family F B—glycanases to predict essential, functional
residues whose catalytic role was tested by site-directed mutagenesis. Six

conserved amino acids were found in the putative catalytic domains of these

2 3 2
enzymes, and within these regions two glutamic acids, three aspartic acids

and one histidine are conserved in all of the sequences aligned. Changing
Asp-504 to Asn, Glu-567 to Gln and Asp-569 to Asn resulted in a dramatic loss
of endoxylanase activity, suggesting that these carboxylic residues in the
enzyme could act by general acid catalysis as has been shown for other
hydrolytic enzymes such as lysozyme (8). The loss of activity which follows
the relatively conservative change of a charged residue to an isosteric, neutral
but polar residue, is expected if this Asp-504, Glu-567 or Asp-569 is responsible
for donating the proton during hydrolysis. Alternatively, loss of a charged
residue might alter protein folding enough to affect either the stability of the
enzyme or the ability of this enzyme to bind to the substrate and this may be
the case for Glu-508 and His-539 as their substitution resulted in partial loss of

activity.

10.

11.

LITERATURE CITED

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Beguin, P. 1990. Molecular biology of cellulose degradation. Ann.
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Biely, P. 1985. Microbial xylanolytic systems. Trends Biotechnol. 3:286-
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Boucher, F., R. Morosol, and S. Durand. 1988. Complete nucleotide
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Chauvaux, S., P. Beguin, and J.-P. Aubert. 1992. Site-directed
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Devereux, J., P. Haeberli, and O. Smithies. 1984. A comprehensive set
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Gilkes, N. R., B. Henrissat, D. G. Kilbum, R. C. Miller, Jr., and R. A. J.

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Grepinet, O., M.-C. Chebrou, and J.-P. Beguin. I988. Nucleotide

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Hall, J., G.P. Hazelwood, N.S. Hiskisson, A.J. Durrant, and H.J. Gilbert.
1989. Conserved serine-rich sequences in xylanase and cellulase from
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and unusual protein processing. Mol. Microbiol. 3:1211-1219.

Hamamoto, T., H. Honda, T. Kudo, and K. Horikoshi. I987.
Nucleotide sequence of the xylanase A gene of alkalophilic Bacillus
sp. strain C-125. Agric. Biol. Chem. 51:953-955.

Henrissat, B., M. Claeyssens, P. Tomme, L. Lemesle, and J.-P. Mornon.
1989. Cellulase families revealed by hydrophobic cluster analysis. Gene
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Hurst, P. L., P. A. Sullivan, and M. G. Shepherd. 1977. Chemical
modification of a cellulase from Aspergillus niger. Biochem. J. 167:549-
556.

Kellett, L. E., D. M. Poole, L. M. Ferreira, A. J. Durrant, G. P.
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contain identical cellulose-binding domains and are encoded by
adjacent genes. Biochem. J. 272:369-376.

Lin, L.-L., and J. A. Thomson. 1991. Cloning, sequencing and expression
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Luthi, E., D. R. Love, J. McAnulty, C. Wallace, P. A. Caughey, D. Saul,
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Miller, G. L. 1959. Use of dinitrosalicyclic acid reagent for
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Mitsuishi, Y., S. Nitisinprasert, M. Saloheimo, I. Biese, T. Reinikainen,
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23.

24.

26.

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29.

30.

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2 3 5
Moriyama, H., Y. Hata, H. Yamaguchi, M. Sato, A. Shinmyo, N.
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preliminary X-ray studies of Bacillus pumilus IPO xylanase. J. Mol.
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33.

35.

36.

37.

38.

39.

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41.

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2 3 6
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9:337-338.

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R. Aebersold. 1991. Glutamic acid 274 is the nucleophile in the active
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Woodward, J. 1984. Xylanases: functions, properties and applications.
TOp. Enzyme Ferment. Biotechnol. 8:9-30.

Yaguchi, M., C. Roy, C. F. Rollin, M. G. Paice, and L. Jurasek. 1983. A
fungal cellulase shows sequence homology with the active site of hen
egg-white lysozyme. Biochem. Biophys. Res. Commun. 116:408-411.

Chapter VIII

Evidence for xylanosomes on the cell surface of

Thermoanaerobacter B6A-RI

237

ABSTRACT

Negatively charged surface structures were visualized on cells of
Thermoanaerobacter strain B6A-RI grown on xylan or xylose, using
cationized ferritin and gold labelling with scanning and transmission electron
microscopy. These structures were absent on glucose grown cells that were
non-xylanolytic. The presence of these putative xylanosome structures
coincided with production of cell bound endoxylanases and the ability of the
cells to bind tightly to xylan. The xylanosomes appeared to be specific for
cellular adhesion to xylan, as cells would not bind to cellulose. These findings
extend the surface structures known to exist on the bacterial cell wall, and
provide the first example of a cell surface structure specific for cellular
adhesion to and degradation of xylan. These hydrolysome structures further
demonstrate the ability of microorganisms to effectively form a glycoprotein
complex to efficiently degrade insoluble substrates by facilitating both the
concerted action of hydrolytic enzymes and the transfer of degradation

products into the cell.

238

INTRODUCTION

Hemicelluloses are heteropolymers of various pentoses and hexoses
and contain mainly xylan which is a B-I,4-linked polymer of D-xylose with D-
glucuronic acid, L-arabinose and other sugar substituents. The complete
hydrolysis of this rather complex substrate to monosaccharides requires the
action of many hydrolytic enzymes such as endoxylanase and B-xylosidase (5,
21). Many xylanolytic microorganisms produce multiple xylanases (6, 27).
This multiplicity could be as a result of degradation of one protein, post-
translational modification of one enzyme, or the products of different genes
(27).

C. thermocellum is one of the most actively studied cellulolytic
microorganisms from which at least 15 genes encoding endoglucanases and
genes encoding endoxylanases were cloned (4, 9). The various components of
the cellulase system of C. thermocellum form an extracellular multienzyme
complex with an Mr of about 2.1 million. The complex, termed the
cellulosome, comprises 14-18 different polypeptides, has a strong affinity for
cellulose and is responsible for the strong activity toward cellulose (12, 15).
The cellulosome is present both in the culture supernatant and at the surface
of the cells, where it is found in mutimeric aggregates which form
protuberances mediating adhesion of the organism to the substrate (2, 3, 14).
Several of the endoglucanases purified from C. thermocellum are
glycoproteins (20).

The ability of microorganisms to adhere to the surface of insoluble
substrates rather than to excrete soluble enzymes might be critical for their

survival in the environment, especially in hot water, not only because of

239

240
intense competition for limited energy sources but also the diffusion of the

polymer hydrolysis products.

Little is known about the microbial transformation of hemicellulose in
thermoanaerobic habitats. A number of thermophilic, anaerobic,
saccharolytic bacteria, including Thermoanaerobacter ethanolicus,
Thermobacteroides acetoethylicus, Thermoanaerobium brockii, and
Clostridium thermocellum ferment xylan albeit at a very slow rate (25, 26).
Certain Thermoanaerobacter strains readily degrade xylan (16, 23).
Thermoanaerobacter strain B6A-RI (17) degrade xylan but not cellulose and is
a model organism to understand the mechanisms thermoanaerobes employ
to efficiently degrade hemicellulose. We have previously demonstrated
that Thermoanaerobacter B6A-RI possesses multiple endoxylanases that
induced by growth on xylan or xylose (Lee et al., manuscript prepared for
submission). In addition, this organism produced multiple endoxylanases
which were excreted when growth on limiting amount of xylan.

Because of the structural complexity of xylan, xylanolytic organisms
produce several different kinds of hydrolytic enzymes for xylan degradation
which might be organized in a discrete complex on the cell surface with a
function analogues to the cellulosome. Although thermoanaerobes have
been studied with respect to their growth on xylan, little is known about the
enzymes involved in the degradation of this insoluble substrate and currently
nothing is known about cell surface structures involved in adhesion and
degradation of xylan.

The purpose of the present paper is to provide evidence for the
presence of xylanosome structures on the cell surface of Thermoanaerobacter

which enables the cell to succesfully bind and degrade insoluble xylan.

MATERIALS AND METHODS

Chemicals and gases

All chemicals were reagent grade or better and were obtained from
Sigma Chemcal Co., St. Louis, Mo., or Mallinckrodt, Inc., Paris, Ky. All gases
used were at least 99.9% pure and were passed over copper-filled Vycor

furnaces (Sargent Welch Scientific Co., Skokie, 111.) to remove oxygen.

Organism and culture conditions

Thermoanaerobacter strain B6A-RI was grown on TYE medium at
60°C in anaerobic tubes containing 10 ml of medium wih either glucose,
xylose or xylan as the substrate as described previously (16, 17). The organism
was subcultured at least three times in medium containing the same substrate
prior to visualization of cell surface structures, to avoid any substrate carry

over.

Morphological studies
Bacterial morphology and cellular adhesion to xylan was investigated

using an Olympus phase-contrast microsc0pe model BHS.

Affinity cytochemistry

Thermoanaerobacter strain B6A-RI was grown on glucose, xylose or
xylan to midexponential phase (30 ml of culture were grown on each
substrate). Cells were treated with cationized ferritin as described previously
(3). The cells were washed twice with 0.9%(w/v) NaCl and resuspended in 5
ml of the same solution with 2 ml of 2.5%(v/v) glutaraldehyde in 0.1 M
NazHPO4-KH2PO4 (pH 7.2). After 20 mins at room temperature the cells were

241

242
washed three times with saline and then resuspended in 3 ml of saline. To

1.5 ml of this suspension was added 5 ml of the glutaraldehyde-phosphate
buffer, and the cells were stored at 4°C. To the remaining 1.5 ml of cell
suspension, was added 0.25 ml cationized ferritin (Sigma), and the solution
incubated at room temperature for 1 hour before being centrifuged. The cells
were washed twice with saline and then resuspended in glutaraldehyde-
phosphate buffer, and stored at 4°C overnight before being processed for

electron microsc0py.

Scanning electron microscopy .

Cells were processed in the absence and presence of cationized ferritin
as described above and collected on to a 13 mm diameter polyester membrane,
pore size 4 pm (Nucleopore, Pleasanton, CA) by suction and placed in a
solution of cold 2.5% glutaraldehyde in 0.1 M Na2HPO4-KH2PO4 (pH 7.2)
and then prepared for scanning electron microscopy as described previously

(11).

Transmission electron microscopy
Cells were fixed overnight to 2 days in cold 2.5% glutaraldehyde in
0.1 M Na2HPO4-KH2PO4 (pH 7.2). The cells were embedded in agar and, after

a buffer wash, post-fixed for 1 h at room temperature in 1% 0504 in the same
buffer as above. They were then dehydrated through an ethanol series,
treated with propylene oxide, and embedded in either Poly/Bed 812
(Polysciences Inc., Warrington, Pa.) or VCD/HXSA (Ladd Research Industries,
Inc., Burlington, Vt.) epoxy resin. Thin sections were cut with a diamond

knife mounted on ‘an Ultratome III (LKB Instruments, Inc., Rockville, Md),

243
stained with uranyl acetate and lead citrate, and examined with an electron

microscope (CM-10; Philips Electronic Instruments, Co., Mahwah, NJ).

RESULTS

Adhesion of cells to xylan

When cells of Thermoanaerobacter strain B6A-RI were mixed with
xylan, the cells attached to xylan and were sedimented leaving a clear
supernatant devoid cells. Using light microscopy, cells of
Thermoanaerobacter strain B6A-RI cultured on xylan were observed to
closely bind to the insoluble substrate (Fig. 1A). To determine the importance
of the growth substrate on cellular adhesion to xylan, cultures were grown on
xylose or glucose and incubated with xylan for up to 1 h. Samples were taken
at intervals and examined using light microsc0py. Cells grown on xylose or
xylan closely adhered to xylan (Fig. 1B), whereas cells grown on glucose did
not adhere to the substrate and tended to either associate together or remain
free in solution (Fig. 1C). To determine if the substrate played a role in
cellular adhesion of Thermoanaerobacter B6A-RI, cells were grown on xylose
and then incubated in the presence of another insoluble substrate, cellulose.
Examination of these cells using light microscopy, showed that the cells did
not bind to the cellulose, but rather remained free in the medium (data not

shown).

The presence of cell surface structures

As both the cellular location of the endoxylanases and adhesion of cells
to xylan varied depending on the growth conditions, cells were examined for
the presence of cell surface structures using cationized ferritin and gold
labelling to identify negatively charged structures with scanning and
transmission electron microscopy. In the absence of cationized ferritin,

surface structures were not evident on cells of Thermoanaerobacter strain

244

 

245

Figure 1. Light microscopy of cells of Thermoanaerobacter strain
B6A-RI grown on (A) xylan, (B) xylose and (C) glucose.

Cells were cultured on the different substrates and incubated
in the presence of xylan for one hour and then examined. Scale bar

represents 30 pm.

 

246

 

247
B6A-RI grown on glucose, xylose or xylan (Fig. 2). Glucose grown cultures

treated with cationized ferritin appeared similar to non treated cells, with the
cell surface remaining smooth in appearance (Fig. 2A 8: B). This differed
from cultures grown on xylose or xylan. Treatment of these cells of
Thermoanaerobacter strain B6A-RI with cationized ferritin revealed the
presence of numerous protruberances covering the cell surface (Fig. 2D 8: F).
To investigate these cell surface structures in more detail, transmission
electron microscopy was performed on cells grown on xylan. This study
revealed the presence of distinct particulate structures surrounding the cell
that appear as differentiation of the outer wall S—layer to which cationized
ferritin bound (Fig. 3). These structures varied in size, being either closely
associated with the cell, stretching out away from the cell or appearing as a

matrix linking the cells together.

 

248

Figure 2. Scanning electron microscopy of gold labelled cells of
Thermoanaerobacter strain B6A-RI grown on glucose (a, b), xylose
(c, d), and xylan (e, 0. Figures a, c, and e are of cells in the absence
of cationized ferritin, and figures b, d, and f, are cells treated with

cationized ferritin (d). Scale bar represents 20pm.

 

249

 

 

250

 

Figure 3. Transmission electron microscopy of cells of Thermoanaerobacter
strain B6A-RI grown on xylan and treated with gold and cationized ferritin.

Scale bar represents 0.2 pm.

DISCUSSION

This report provides the first ultrastructural study of the cell surface
components of a hemicellulolytic bacterium, and demonstrates the presence
of cell surface structures which appear to be involved in cellular adhesion to
xylan and coincide with the production of cell bound endoxylanases necessary
for substrate hydrolysis. This provides evidence for a xylanosome that is
induced by xylan and which maybe be analogous to cellulosome
demonstrated in cellulolytic anaerobes.

In Clostridium thermocellum a protein complex, termed the
cellulosome, containing enzymes necessary for the degradation of cellulose
has been demonstrated. This complex exists in cell surface bound and cell-
free forms, and has been shown to be responsible for cellular adherence to
cellulose and for the degradation of cellulose to cellobiose by the intact
organism (1, 14, 15). The cellulosome constitutes the majority of the
endoglucanase activity (~70%) and about one third of the total extracellular
protein, and possesses the major proteins so far reported for the entire
cellulolytic apparatus in this organism (12). Recently, xylanase activity has
also been found to be localized in the cellulosome (19).

The ultrastructure of cellulosomes located on the surfaces of C.
thermocellum have been studied by transmission electron microscopy using
specific labels (3), immunocytochemical techniques (I), and by scanning
electron microscopy using cationized ferritin (13).

Many anaerobic cellulolytic bacteria possess high molecular weight,
multisubunit cellulases which are often found associated with the cell surface
(28) or sedimentable membranous fragments (10). Bacteroides

succinogenes produces CMCase, B-glucosidase, xylanase and B-xylosidase

251

2 5 2
during growth on cellulose, and approximately 50% of each of the released

enzymes was associated with sedimentable subcellular membrane vesicles
which adhered to cellulose (7). In these cellulolytic anaerobes enzyme
complexes analogous to the cellulosome may be present.

In Thermoanaerobacter strain B6A-RI, the finding of predominantly
cell bound endoxylanase and other xylanolytic activity, a strong affinity of the
cells for the substrate and the presence of cell surface protuberances suggests
the presence of a xylanosome which functions similar to the cellulosome in
hydrolysis of insoluble polymer. Moreover the xylanosome appears to be
specific for cellular adhesion to xylan, as cells of Thermoanaerobacter strain
B6A-RI would not bind to cellulose. These findings mark a distinct difference
between Thermoanaerobacter strain B6A-RI and Thermoanaerobacter strain
B6A, as the former organism binds very tightly to xylan whereas there was a
lack of extensive attachment of the latter organism to hemicellulose
substrates (23).

There existed some similarities between the cell surface complex in
Thermoanaerobacter B6A-RI and the cellulosome in C. thermocellum. The
cell surface structures as visualized using SEM were very similar in size and
abundance when compared to cells of C. thermocellum LQRI but differed
from other strains of C. thermocellum (13). During the same study Lamed et
al. (13) demonstrated the presence of similar structures on cells of anumber of
bacteria, including Acetovibrio cellulolyticus, Bacteroides cellulovorans,
Clostridium cellulovorans and Clostridium cellobioparum. In all of the
mesophilic, anaerobic, cellulolytic bacteria, cell surface structures similar but
not identical to those found on Thermoanaerobacter B6A-RI were found,
thus, it would seem that the exact topology of these structures varies between

organisms and strains.

25 3
Transmission electron microscopy of the cationized ferritin labeled

cells of C. thermocellum YS showed labeling over the entire cell surface,
fibrous structures which sometimes connected adjacent cells, and distinct
evenly shaped protuberances (1). Cells of Thermoanaerobacter B6A-RI grown
on xylan had a similar ultrastructure, and the less defined and more diffuse
nature of the cell surface protruberances may be due to differences between
the two organisms, and unrelated to cellular function.

Most Eubacteria and Archaebacteria possess layered assemblies of
polymers and heteropolymers external to the plasma membrane. The upper
most layer is referred to as the S-layer and is composed of protein or
glycoprotein (22). The finding that endoxylanase from Thermoanaerobacter
B6A-RI was glycosylated and associated with the cell surface, and the presence
of a thin dark layer on the surface of cells of Thermoanaerobacter B6A-RI (as
visualized with transmission electron microscopy), suggests that the
xylanolytic complex may be part of the S-layer. This is further substantiated
by a recent study in which a novel linkage of O-linked carbohydrates was
found in the crystalline surface layer glycoprotein of Clostridium
thermohydrosulfuricum (18). A similar type of linkage was found in the
glycan chains of the cellulosome of C. thermocellum (8), further
substantiating the interrelationship between the S-layer and catalytic cell
surface complexes.

From this study we propose that the majority of the xylanolytic activity
is associated with the cell, and that Thermoanaerobacter strain B6A-RI binds
xylan on to the cell surface to facilitate the concerted action of xylanasesand to
enhance the transfer of the degradation products into the organism. The
genes encoding the major endoxylanase and B-xylosidase activities in

Thermoanaerobacter strain B6A-RI have been cloned (Lee 8: Zeikus,

254
manuscripts in preparation), and the endoxylanase gene contains a leader

sequence whereas the B-xylosidase gene does not, confirming the findings
from our previous study of endoxylanase activity being cell bound to the
surface or excreted and B-xylosidase being almost completely intracellular.
These findings extend the cell surface structures known to exist on the
bacterial cell, and provide the first example of a cell surface structure specific
for cellular adhesion to and degradation of xylan. Such structures further
demonstrate the ability of microorganisms to effectively form a hydrolytic

complex of enzymes to efficiently degrade insoluble substrates.

10.

11.

LITERATURE CITED

Bayer, E. A., and R. Lamed. 1986. Ultrastructure of the cell surface
cellulosome of Clostridium thermocellum and its interaction with
cellulose. J. Bacteriol.167: 828-836.

Bayer, E.A., R. Kenig, and R. Lamed. 1983. Adherence of Clostridium
thermocellum to cellulose. J. Bacteriol. 156:818-827.

Bayer, E. A., E. Setter, and R. Lamed. 1985. Organization and
distribution of the cellulosome in Clostridium thermocellum. J.
Bacteriol. 163:552-559.

Bégiun, P. 1990. Molecular biology of cellulose degradation. Ann.
Rev. Microbiol. 44:219-248.

Biely, P. 1985. Microbial xylanolytic systems. Trends Biotechnol. 3:286-
290.

Dekker, R. F. H., and G. N. Richards. 1976. Hemicellulases: their
occurrence, purification, properties, and mode of action. Adv.
Carbohydr. Chem. Biochem. 32:277—352.

Forsberg, C. W., T. J. Beveridge, and A. Hellstrom. 1981. Cellulase and
xylanase release from Bacteroides succinogenes and its importance in
the rumen environment. Appl. Environ. Microbiol. 42:886-896.

Gerwig, G.J., P. de Waard, J.P. Kamerling, J.F.G. Vliegenthart, E.
Morgenstern, R. Lamed, and EA. Bayer. 1989. Novel O-linked
carbohydrate chains in the cellulase complex (cellulosome) of
Clostridium thermocellum. J. Biol. Chem. 264:1027-1035.

Grepinet, O., M.-C Chebrou, and P. Beguin. 1988. Nucleotide sequence
and deletion analysis of the xylanase gene (xynZ) of Clostridium
thermocellum. J. Baceriol. 170:4582-4588

Groleau, D., and C. W. Forsberg. 1981. Cellulolytic activity of the
rumen bacterium Bacteroides succinogenes. Can. J. Microbiol. 27:517-
530.

Klomparens, K., S. L. Flegler, and G. R. Hooper. 1986. In
Procedures for Transmission and Scanning Electron Microscopy for
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12.

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14.

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Lamed, R., R. Kenig, and E. Setter. 1985. Major characteristics of the
cellulolytic system of Clostridium thermocellum coincide with those
of the purified cellulosome. Enz. Microb. Technol. 7:37-41.

Lamed, R., J. N aimark, E. Morgenstern, and EA. Bayer. 1987.
Specialized cell surface structures in cellulolytic bacteria. J. Bacteriol.
169:3792-3800.

Lamed, R., E. Setter, and E. A. Bayer. 1983a. Characterization of a
cellulose-binding, cellulase-containing complex in Clostridium
thermocellum. J. Bacteriol. 156:828-836.

Lamed, R., E. Setter, R. Kenig, and E. A. Bayer. 1983b. The cellulosome:
a discrete cell surface organelle of Clostridium thermocellum which
exhibits separate antigenic, cellulose-binding and various cellulolytic
activities. Biotechnol. Bioeng. Symp. 13:163-181.

Lee, C., B. C. Saha, and J. G. Zeikus. 1990. Characterization of
Thermoanaerobacter glucose isomerase in relation to saccharidase
synthesis and development of single-step processes for sweetner
production. Appl. Environ. Microbiol. 56:2895-2901.

Lee, Y.-E., M. K. Jain, C. Lee, S.E. Lowe and J. G. Zeikus. 1992.
Taxonomic distinction of saccharolytic thermoanaerobes:
Description of Thermoanaerobium gen. nov., xylanolyticum sp. nov.
and Thermoanaerobium saccharolyticum sp nov. Reclassification of
Thermoanaerobium brockii,Clostridium thermosulfurogenes and
Clostridium thermohydrosulfuricum E100-69 as Thermoanaerobacter
brockii comb. nov., Thermoanaerobacterium thermosulfrigenes comb.
nov. and Thermoanaerobacterium thermohydrosulfuricus comb.
nov., and transfer of Clostridium thermohydrosulfuricum 39E to
Thermoanaerobacter ethanolicus. Manuscript submitted to the
International Journal of Systematic Bacteriology.

Messner, P., R. Christian, J. Kolbe, G. Schulz, and U.B. Sleytr. 1992.
Analysis of a novel linkage unit of O-linked carbohydrates from the

crystalline surface layer glycoprotein of Clostridium
thermosulfuricum 5102-70. J. Bacteriol. 174:2236-2240.

Morag, B., E. A. Bayer, and R. Lamed. 1990. Relationship of
cellulosomal and noncellulosomal xylanases of Clostridium
thermocellum to cellulose-degrading enzymes. J. Bacteriol. 172:6098-
6105.

20.

21.

23.

24.

25.

26.

27.

28.

29.

30.

2 5 7
Ng, T. K. and J. G. Zeikus. 1981. Purification and characterization of an
endoxylanase (1,4-b-D-glucan glucanohydrolase) from Clostridium
thermocellum. Biochem. J. 199:341-350.

Reilly, P. J. 1981. Xylanases: structure and function, p.111-129. In A.
Hollaender (ed.), Trends in the Biology of Fermentations for Fuels
and Chemicals. Plenum Press, New York.

Sleytr, U.B., and P. Messner. 1983. Crystalline surface layers on
bacteria. Ann. Rev. Microbiol. 37:311-339.

Weimer, P. J. 1985. Thermophilic anaerobic fermentation of
hemicellulose and hemicellulose-derived aldose sugars by
Thermoanaerobacter strain B6A. Arch. Microbiol. 143:130-136.

Weimer,. P. J., L. W. Wagner, S. Knowlton, and T. K. Ng. 1984.
Thermophilic anaerobic bacteria which ferment hemicellulose:
characterization of organisms and identification of plasmids. Arch
Microbiol. 138:31-36.

Wiegel, J., L. H. Carreira, C. P. Mothershed, and J. Puls. 1983.
Production of ethanol from bi0ploymers by anaerobic, thermophilic,
and extreme thermophilic bacteria. ILThermoanaerobacter
ethanolicus JW200 and its mutants in batch cultures and resting

cell experiments. Biotechnol. Bioeng. Symp. 13:193-205.

Wiegel, J., C. P. Mothershed, and J. Puls. 1985. Differences in xylan
degradation by various noncellulolytic thermophilic anaerobes and
Clostridium thermocellum. Appl. Environ. Microbiol. 49:656-659.

Wong, K.K.Y., L.U.L. Tan, and J.N. Saddler. 1988. Multiplicicty of [34,4-
xylanase in microorganisms: Functions and applications. Microbiol.
Rev. 52:305-317.

Wood, T. M., C. A. Wilson, and C. S. Stewart. 1982. Preparation of the
cellulase from the cellulolytic anaerobic rumen bacterium
Ruminococcus albus and its release from the bacterial cell wall.
Biochem. J. 205:129-137.

Woodward, J. 1984. Xylanases: Function, properties and applications.
Topics Enz. Ferment. Biotechnol. 8:9-30.

Zeikus, J. G., A. Ben-Bassat, and P. W. Hegge. 1980. Microbiology of
methanogenesis in thermal, volcanic environments. J. Bacteriol.
143:432-440.

Chapter IX

Conclusions and recommendations for future research

258

Until recently only one thermoanaerobic strain, Thermoaerobacter
strain B6A, that actively grows on xylan has been reported. Our group has
isolated several xylanolytic strains from thermal spring ecosystems in
Yellowstone National Park. New isolates Thermoanaerobacter strain B6A-RI
and LX-II expressed high xylanase activities and grew readily on xylan.

In order to define the genetic relationship among xylanolytic
thermoanaerobes and to facilitate the establishment of species names, we
have compared DNA homologies of these strains by DNA-DNA
hybridization. This technique has been useful for taxonomic studies of other
species and a variation of this procedure that uses free-solution hybridization
and SI nuclease treatment of the hybrids was employed here. The results
indicate the existence of three groups among the xylanolytic thermoanaerobes
known to date: Group I includes one species Clostridium thermocellum since
this organism exhibits less than 14% homology to other species studied;
Group II is composed of Thermoanaerobacter strains B6A-RI, LX-II and
Clostridium .thermosulfurogenes 4B that showed more than 40% of
homology among themselves; Group III comprised of Clostridium
thermohydrosulfuricum 39B and E100-69, Thermoanaerobium brockii HTD4
and Thermoanaerobacter ethanolicus JW200 as they exhibited homologies
between each other's DNA ranging from 57% to 97%.

From these findings three different groups emerged and new
taxonomic assignments were proposed. C. thermocellum LQRI was placed in
group I retaining its original taxonomic assignment without change. C.
thermosulfurogenes strain 48 and the new isolates B6A-RI and LX-Il are
closely related and fell into group II for which a genus name

Thermoanaerobacterium gen. nov. was given. Group III was termed

259

260
Thermoanaerobacter and includes T. ethanolicus strain JW200, C.

thermohydrosulfuricum strain 39B and strain E100-69, and
Thermoanaerobium brockii strain HTD4. Thermoanaerobacter B6A-RI was
selected as a model organism for further studies, because of its high
xylanolytic activity and lack of cellulases.
During growth on xylan and xylose Thermoanaerobacter strain B6A-
RI produced endoxylanase, B-xylosidase, arabinofuranosidase and
acetylesterase and these activities appeared to be produced coordinately.
Under nonlimiting xylan conditions these enzyme activities were
predominantly cell associated, however, during growth on limiting
concentrations of xylan the majority of endoxylanase activity was
extracellular rather than cell associated. Endoxylanase, B-xylosidase and
arabinofuranosidase activities were induced by xylan, xylose and arabinose
respectively. Acetylesterase activity was constitutive and endoxylanase
activity was catabolite repressed by glucose. For purification of endoxylanases
an affinity chromatography method was developed by coupling of oat spelt
xylan to Sepharose CL-4B and the enzymes bound at >45°C and were eluted
using soluble xylan. Activity staining of SDS-PAGE indicated that
Thermoanaerobacter B6A-RI possesses multiple forms of endoxylanse. At
present it is not clear whether they represent the products of different genes,
multiple molecular forms of the same subunit polypeptides, or forms with
different degrees of post-translational modification such as glycosylation or
proteolysis. Evidence for these possibilities can be obtained using antibodies
prepared from the cloned endoxylanase, by deglycosylation studies or by
cloning more endoxylanase genes.
To isolate the genes involved in xylan degradation and to study their

genetic organization in Thermoanaerobacter B6A-RI, a genomic library was

2 6 1
constructed using a cosmid vector pHC79 and a cosmid clone coding for

endoxylanase and B—xylosidase activities was isolated. A 28 kilobase fragment
of Thermoanaerobacter genomic DNA insert was subjected to a series of
subclonings to identify the location of the genes. The DNA sequence of this
endoxylanase was determined using the dideoxy sequencing method. One
open reading frame (ORF) of 3471 bp which encodes a xylanase of 1157 amino
acid residues (Mr 130 kDa) was found. The distribution of endoxylanase
activity was approximately equal between the periplasm and cytoplasm in a
subclone containing the endoxylanase gene. This finding suggests that the
signal peptide is recognized and the protein is processed by Escherichia coli
cells. The N-terminus of the processed enzyme in E. coli was determined by
amino acid sequencing and a putative 33 amino acid signal peptide was found
from the deduced amino acid sequence. The cloned endoxylanase was
purified and its physico-chemical properties were determined. The pH
optimum was 5.5 and the optimum temperature was 70°C. The cloned
endoxylanase hydrolyzed xylan to mainly xylobiose and xylotriose, and
xylobiose was not cleaved.

The location of a gene encoding B-xylosidase activity was identified in a
cosmid clone and subsequently subcloned into pUC18. The complete
nucleotide sequence of the B-xylosidase gene and its flanking regions were
established. A 1500-bp open reading frame for B-xylosidase and another open
reading frame of unknown function were observed. The amino acid
sequence of the N-terminal region and the molecular weight (55 kDa) of the
B-xylosidase, deduced from the DNA sequence, agreed with the result
obtained for the purified enzyme. The endoxylanase gene of the same strain
was 12 kbp downstream of the 3' end of the B—xylosidase gene and they had

the same orientation. The cloned B-xylosidase was purified using heat

2 6 2
treatment, ion-exchange and gel-filtration chromatography. The pH optimum

was 5.5, and the optimum temperature was 65°C. The cloned B-xylosidase
cleaved both xylotriose and xylobiose to xylose and also had transferase
activity. No activity was found on xylan.

Xylose isomerase activity is responsible for the conversion of xylose to
xylulose prior to xylolysis. The gene encoding for xylose isomerase was
cloned and its nucleotide sequence determined. The amino acid sequence
deduced from the coding sequence of the gene exhibited considerable
homology to the sequences of other xylose isomerases studied to date
including C. thermosulfurogenes strain 4B, C. thermohydrosulfuricum strain
39E, E. coli and B. subtilis. The xylose isomerases from three
thermoanaerobes, Thermoanaerobacter strain B6A-RI, C. thermosulfurogenes
strain 4B and C. thermohydrosulfuricum strain 39E, shared a higher
homology among themselves than with E. coli or Bacillus subtilis. Codon
usage analysis of three genes from Thermoanaerobacter strain B6A-RI
showed a bias for A or T for the third base, and is reflected by the low G+C
mol% content of 36 found in this organism.

In order to identify the putative catalytic sites on the endoxylanase gene
more precisely, multiple sequence analysis was performed using sequence
data available through the Wisconsin-GCG Sequence analysis program. There
were very significant homologies between endoxylanase from
Thermoanaerobacter strain B6A-RI and the family F B-glycanases. This
grouping of B-glycanases in family F is based on the hydrophobic cluster
analysis program, which was used to confirm that endoxylanase from
Thermoanaerobacter strain B6A-RI should be placed in this family.

The catalytic domains in endoxylanase from Thermoanaerobacter B6A-

RI were investigated. The catalytic mechanism of endoxylanase has been

263
modeled after the acid-base catalytic mechanism proposed for lysozyme.

Chemical modification of the endoxylanase from Thermoanaerobacter strain
B6A-RI with group specific reagents indicated that either aspartate or
glutamate was involved in catalysis. Based on sequence alignment,
hydrophobic cluster analysis and chemical modification, 6 amino acids were
chosen and mutated by site—specific mutagenesis. Changing Glu-567 to Gln
and Asp-504 and Asp-569 to Asn resulted in a dramatic loss of xylanase
activity, suggesting that endoxylanase from B6A-RI, like other hydrolytic
enzymes, could act by general acid catalysis involving carboxylic residues.

Ultrstructural studies were performed to characterize cell topological
features involved in xylan adhesion and hydrolysis. It was observed that in
the exponential phase of growth Thermoanaerobacter B6A-RI cells grown on
xylan or xylose would bind tightly to particles of insoluble xylan, whereas the
cells grown on glucose remained unattached. Negatively charged surface
structures were visualized on cells of Thermoanaerobacter strain B6A-RI
grown on xylan, using cationized ferritin and gold labelling with scanning
and transmission electron microscopy. These structures were absent on cells
grown on glucose. The presence of these structures (i.e. xylanosome)
coincides with production of cell associated endoxylanases and the ability of
the cells to bind tightly to xylan.

It has been shown that cellulolytic enzymes of C. thermocellum were
organized into distinct multi-subunit complexes called cellulosomes. The
xylanosomes of Thermoanaerobacter strain B6A-RI may represent a putative
cell surface enzyme complex enabling rapid hydrolysis and absorption of the
xylan degrading products by the cell. Using antibodies prepared from the
purified cloned enzymes, endoxylanase, B-xylosidase and xylose isomerase,

immunogold labelling experiments will be performed to demonstrate their

264
cellular location and possible juxtapositio of different xylan degrading

enzymes.

The following studies should be continued to reveal the molecular
nature of Thermoanaerobacter xylanase system: (1) isolate and identify the
individual components which exist in the xylanosome; (2) establish the role
of the xylanosome components, and the basis for organization; (3) elucidate
the synergistic interactions between xylanolytic enzymes, and their potential
juxtapositions in the xylanosome; (4) isolate and characterize the putative
xylan-binding factor; (5) elucidate the mechanism for thermophilicity in the
endoxylanase; (6) chemically characterize the glycoconjugate of rndoxylanase;
(7) characterize the S-layer relationship to xylanosome components. Finally, it
would be worth continuing studies on establishing process uses for

thermophilic xylanases in the manufacturing of foods, feeds, paper and fibers.