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This is to certify that the

dissertation entitled

BIOCHEMICAL CHARACTERIZATION OF
AMYLOPULLULANASE FROM
CLOSTRIDIUM THERMOHYDROSULFURICUM 3913

presented by

Saroj Priyantha Mathupala

has been accepted towards fulfillment
of the requirements for

Ph.D. Biochemistry

degree in

 

 

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BIOCHEMICAL CHARACTERIZATION or
AMYLOPULLULANASE FROM
CLOSTRIDIUM THERMOHYDROSULFURICUM 39E
By

Sam} Priyantha Mathupala

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1992

m- 4545'

ABSTRACT

BIOCHEMICAL CHARACTERIZATION OF AMYLOPULLULANASE FROM
CLOSTRIDIUM THERMOHYDROSULFURICUM 39E

By
Saroj Priyantha Mathupala

A novel pullulanase, which hydrolyzes both a-1,6 bonds in pullulan and a-1,4
bonds in amylose, was characterized from Clostridium thermohydrosulfuricum 39E,
a thermophilic, anaerobic bacterium. Conditions were optimized for
overproduction and secretion of the enzyme by using a maltose limited
chemostat culture. The enzyme was purified by using affinity chromatography
on an inhibitor-linked matrix, and the biochemical properties determined.
Activity staining of PAGE gels, and inhibition kinetics using dual alternate
substrates showed that both a-1,4 and (at-1,6 glucosidic bond cleavage resided on
the same enzyme. This amyIOpullulanase had higher affinity for pullulan than
amylose. Both a-amylase and pullulanase activities were inhibited by B-
cyclodextrin, a known inhibitor for both pullulanases and a-amylases. Amylose,
glycogen, and amylopectin were hydrolyzed by the enzyme to maltose,
maltotriose, and maltotetraose. 13C N MR spectroscopy showed that the enzyme
was capable of hydrolyzing both a-1,6 and a-1,4 bonds in glycogen. The gene
encoding amylopullulanase was identified in a 6.1 kbp chromosomal DNA
fragment and Cloned into Escherichia coli and subcloned into Bacillus subtilis. The
cloned enzyme was processed to the periplasmic space of E. coli, while in B.
subtilis it was extracellular. The cloned enzyme from E. coli expressed both or-
amylase and pullulanase activities and maintained thermostability and

thermophilicity. The 4.4 kbp amylopullulanase (apu) gene was sequenced. Nested

deletion mutants and fusion proteins constructed from the gene allowed the
identification of a 2.9 kbp segment in the middle of the coding region, that
encoded a Mr 100,000 protein which maintained both a-amylase and
pullulanase activities and thermostability, indicating that a single active site was
probably involved in both a-1,6 and a-1,4 hydrolytic activities. Chemical
modification of the enzyme with group specific reagents enabled the putative
identification of either aspartate or glutamate, to be involved in catalysis. Site
directed mutagenesis determined that Asp597, Glu626, and Asp7o3 were
involved in catalysis, with each amino acid substitution resulting in loss of both

a-1,4 and a—1,6 glucosidic bond cleavage.

Copyright by
SARO] PRIY ANT HA MATHUPALA
1992

Dedicated to my Parents

in gratitude

ACKNOWLEDGEMENTS

I would like to acknowledge my gratitude to the many people who have
made the completion of this dissertation possible. First and foremost, I wish to
thank my advisor Dr. I. Gregory Zeikus for his guidance, encouragement, and
support, and for allowing me to gain experience while completing this thesis
research, in the broad areas of anaerobic microbiology, enzymology, and
molecular biology. I am greatly indebted to him for his broad-minded approach
in developing my thesis research program. I am also greatly indebted to all the
members of my guidance committee, Dr. R.P. Hausinger, Dr. P.K. Kindel, Dr. A.
Tulinsky, and Dr. ].E. Wilson, for their guidance and advice, specially during the
initial stages of my thesis research. Each of them contributed to my training

greatly.

I would like to acknowledge the support of my co-workers, the past and
present members of our lab, who taught me the techniques, and for their
encouragement, friendship, helpful discussions, and for making my stay in this
lab one of the best times in my life. I am particularly indebted to Dr. Sue Lowe,
Dr. Badal Saha, Dr. Raphael Lamed, Dr. Yong Lee, Mr. John Kemner, Dr. Michael
Bagdasarian, Mr. Keith Strevett, Dr. Chan Lee, Mr. Maris Laivenicks, Dr. Matur

Ramesh, Dr. Sergey Podkovirov, and Dr. Bassam Annous.

Finally, I wish to thank my father, sister, and brothers for their
encouragement and support ”from across the globe,” and I am specially grateful
to my wife Lathika, for her support and encouragement during all these years of

my ”graduate-student life.”

vi

TABLE OF CONTENTS

page
LIST OF TABLES .................................................................................................. ix
LIST OF FIGURES ................................................................................................ xi
ABBREVIATIONS ................................................................................................. xv
INTRODUCTION ................................................................................................. 1
OBJECT IVES .......................................................................................................... 5
CHAPTER 1. LITERATURE REVIEW .................................................... 7
Literature Review .............................................................. 8
List of References ............................................................... 30
CHAPTER II. IMPROVED PURIFICATION AND BIOCHEMICAL
CHARACTERIZATION OF EXTRACELLULAR
AMYLOPULLULAN ASE OF Clostridium
thermohydrosulfuricum 39E ........................................................................ 38
Abstract ............................................................................... 39
Introduction ........................................................................ 40
Materials and Methods ..................................................... 43
Results ................................................................................. 51
Discussion ........................................................................... 70
List of References ............................................................... 73

CHAPTER III. SUBSTRATE COMPETITION AND SPECIFICITY AT THE
ACTIVE SITE OF AMYLOPULLULANASE FROM

Clostridium thermohydrosulfuricum 39E ................................................... 77
Abstract ............................................................................... 78
Introduction ........................................................................ 78
Materials and Methods ..................................................... 78
Results and Discussion ..................................................... 79
References ........................................................................... 83

vii

CHAPTER IV.

viii

CLONING AND IDENTIFICATION OF

THERMOSTABILITY AND CATALYTIC REGIONS, AND
CHARACTERIZATION OF OL-l,4 AND a-1,6 BOND SPECIFICITY OF

AMYLOPULLULAN ASE FROM

Clostridium thermohydrosulfuricum 39E .................................................. 85
Abstract ............................................................................... 86
Introduction ........................................................................ 87
Materials and Methods ..................................................... 89
Results ................................................................................. 103
Discussion ........................................................................... 125
List of References ............................................................... 128

CHAPTER V. SEQUEN CIN G OF THE AMYLOPULLULAN ASE

GENE(apu) OF Clostridium thermohydrosulfuricum 39E,

AND IDENTIFICATION OF THE ACTIVE SITE BY SITE

DIRECTED MUTAGENESIS .................................................................. 132
Abstract ............................................................................... 133

Introduction ........................................................................ 134

Materials and Methods ..................................................... 136

Results ................................................................................. 146

Discussion ........................................................................... 165

List of References ............................................................... 169

CHAPTER VI. CONCLUSIONS AND PERSPECTIVES ........................ 174
APPENDIX ............................................................................................................. 179

LIST OF TABLES

Chapter I.‘
1. Substrate and bond specificity of various amylolytic
enzymes .................................................................................................... 10

2. General biochemical properties of a-amylase from bacteria and

fungi .......................................................................................................... 12
3. General biochemical properties of bacterial pullulanases ................ 22
Chapter II.

1. Effect of cultural conditions on a-amylase and pullulanase activities
of C. thermohydrosulfuricum 39E ............................................................ 52
2. Purification scheme of extracellular amylpullulanase from C.
thermohydrosulfuricum 39E ..................................................................... 53
3. Amino acid composition of amylopullulanase from C.

thermohydrosulfuricum 39E ..................................................................... 57

Chapter III.
1. Reaction products of amylopullulanase from C.
thermohydrosulfuricum 39E on low MW oligosaccharides ................. 8O
2. Reaction products of amylopullulanase from C.

thermohydrosulfuricum 39E on high MW polysaccharides ................ 80

Chapter IV.
1. E. coli and B. subtilis strains used in Cloning and subcloning

experiments ............................................................................................. 9O

ix

x
2. Activity and location of recombinant amylopullulanase isolated from

E. coli and B. subtilis ............................................................................... 109

3. Activity, location, induction, and thermostability of recombinant

constructs ...................................................................................................... 112

4. Purification of recombinant amylopullulanase from

E. coli SURE ................................................. 114
5. Purification of recombinant amylopullulanase from B. subtilis ...... 115
Chapter V.

1. Activity of oligonucleotide directed mutant constructs of

apu gene ................................................................................................... 163

LIST OF FIGURES

Chapter I.
1. Schematic structure of amylopectin and the action pattern of some
amylolytic enzymes ................................................................................. 9
2a.Stereo view of three dimensional structure of a-carbon chain of a-
amylase from Aspergillus oryzae ............................................................ 16
2b.Topological diagram of the secondary and super-secondary structures
of oz-amylase from A. oryzae .................................................................. 16

3. Proposed substrate binding and catalytic site for a-amylase of A.

oryzae ......................................................................................................... 18 ’
4. Conserved regions (I to IV) of some microbial amylases ................. 20
5. Action pattern of pullulanase degrading enzymes ............................ 26
Chapter II.
1a.SDS—PAGE of affinity purified amylopullulanase .............................. 56
lb.Native-PAGE of affinity purified amylopullulanase ......................... 56

1c.Isoelectric focusing gel electrophoresis of purified
amylopullulanase ................................................................................... 56

2. Comparison of N-terminal sequences of various amylases of

microbial origin ....................................................................................... 58
3. Analysis of glycoprotein components of amylopullulanase ............ 59
4. Effect of temperature and Ca2+ on stability 0f C~

thermohydrosulfuricum 39E amylopullulanase .................................... 62

xi

xn
Inhibition of pullulanase and a-amylase activity of C.
thennohydrosulfuricum amylopullulanase by B-cyclodextrin ............ 64
HPLC analysis of the product formation profile of amylopullulanase
on pullulan, glycogen, and amylose .................................................... 66
Thin layer chromatographic analysis of product hydrolysates upon
action by amylopullulanase from C. thermohydrosulfuricum 39E on
oligosaccharides ..................................................................................... 68
Chemical modification of aspartate and glutamate residues of

amylopullulanase from C. thermohydrosulfuricum 39E using 1-(3—

Dimethylaminopropyl)-3-ethyl carbodiimide .................................... 69
Chapter III.
1. KmaPP determination ............................................................................ 81
2. Kinetics of competitive inhibition with mixed substrates ................ 82
Chapter IV.
1. Plasmids used in cloning of amylopullulanase (apu) gene from C.
thermohydrosulfuricum 39E ..................................................................... 92
2. Southern hybridizationanalysis of C. thermohydrosulfuricum 39E
chromosomal DNA for cloning of the apu gene ................................. 104
3. Cloning strategy for the amylopullulanase gene from C.
thermohydrosulfuricum 39E in to E. coli SURE ...................................... 105
4. Physical map of the pUC18 clone (pAPZ 71) containing the
apu gene ................................................................................................... 106
5. Physical map of the pAPZ 74 subclone in B. subtilis NA-l ............. 108
6. Construction of fusion proteins containing the amylopululanase

gene from C. thermohydrosulfuricum 39E ............................................ 111

7. Agarose gel electrophoresis pattern of nested deletion mutants

constructed from the 3’ direction ....................................................... 116
8. Construction of nested deletion mutants of lacZ construct

of pAPZ 72 .............................................................................................. 117
9. SDS-PAGE analyis of recombinant amylopullulanase purified

from E. coli and B. subtilis ..................................................................... 118
10. 13 C NMR spectra of maltose and iso-maltose .................................. 121
11. 13 C NMR spectra of glycogen and the hydrolysate of glycogen... 123

Chapter V.

1. Nested deletion mutants constructs used for sequencing the

apu gene ................................................................................................... 140
2. Synthetic oligonucleotide primers used in oligonucleotide directed ’

mutagenesis of active site amino acids .............................................. 143
3. Strategy used for construction of site directed mutants ................. 145
4. Nucleotide sequence and the deduced amino acid sequence of apu

gene of C. thermohydrosulfuricum 39E ................................................. 148
5. Multiple sequnce alignment of deduced amino acid sequnce of apu

from C. thermohydrosulfirricum 39E with sequences of

(at-amylases ............................................................................................. 159
6. Multiple sequence alignment of the deduced amino acid sequence of

apu with sequences of a-1,6 hydrolyzing enzymes ........................ 161
7. Overall alignment of the deduced sequence of amylopullulanase of C.

xiii

thermohydrosulfuricum 39E with amylases from microbial and

fungal origin .......................................................................................... 164

xiv
Appendix
1. Scanning electron microscopy of gold coated cells of
C.thermohydrosulfuricum 39 grown on maltose ................................... 188
2. Seanning electron microscopy of gold coated cells of C.
thermohydrosulfuricum 39E grown on glucose and soluble

starch ........................................................................................................ 189

kbp
DEPC
DEAC

EDTA
EGTA
TLC
GLC
HPLC
N MR
pCMB
PEG
LB
PAGE
CAP
CAMP
OD
FPLC
SDS

ABBREVIATIONS

kilo base pair

diethylpyrocarbonate
1-(3-dimethylaminopropyl)-3-ethyl carbodiimide.HCl
molecular weight

ethylenediamine tetraacetic acid

ethylene g1ycol-bis-N,N,N’,N’-tetraacetiC acid
thin layer chromatography

gas-liquid chromatography

high performance liquid chromatography
nuclear magnetic resonance spectroscopy
p-chloro mercury benzoate

poly ethylene glycol

Luria-Bertani medium

polyacrylamide gel electrophoresis

catabolite gene activator protein

cyclic AMP

optical density

fast protein liquid chromatography

sodium dodecyl sulfate

XV

INTRODUCTION

Most natural products are biodegradable, due to the action of the
enzymes inherent in microorganisms present in the environment.
Microorganisms and their enzymes have been used by society due to their
ability to alter the chemical nature and composition of food material,
especially in fermentative processes. More recently, enzymes have been used
in more chemically-specific processes such as chiral synthesis of natural
products, for example in the production of optically pure pharmaceuticals.
Most industrial processes using enzymes have relied on simple hydrolases,
often carbohydrases or proteases, to hydrolyze macromolecules, sinCe
expensive and often unstable co—factors are not used in these enzymatic
conversions. Enzymes are of value in manufacturing because of their rapid
and efficient action at low concentrations and under milder conditions when
compared to chemical mediated processes, and due to their high degree of
substrate and product specificity and their low residual toxicity.

Thermophilic bacteria have tremendous potential in microbial and
enzymatic technology because of their ability to function and maintain
stability at higher temperatures, which enables the development of improved
or new biotechnological processes. In addition, the biochemical properties of
these enzymes are of great interest, since unique structural features might be
expected to account for their high degree of thermostability in comparison to
labile enzymes that function at mesophilic temperatures. Thermostable starch
hydrolyzing enzymes play an important role in the starch processing

industry. Several anaerobic bacteria have been investigated with respect to

2
their ability to produce extracellular saccharidases, which might be of applied

importance.

Most the thermostable starch-processing enzymes that are available for
industrial application, however, are a-amylases, glucoamylases, and
pullulanases that are produced by aerobic bacteria and fungi. These enzymes
catalyze the cleavage of tit-1,4 or (X-I,6 glucosidic linkages of starch.

We chose to study the pullulanase activity reported in C.
thermohydrosulfuricum 39E, that cleaved both a-1,4 and a-1,6 glucosidic
bonds, for two reasons. First, because of the potential insights that structural
and functional biochemical analysis of the enzyme would give with regard to
the mode of catalysis and nature of thermostability, and secondly because of
it's potential use as both a solubilizing and debranching enzyme working at
high temperatures during starch liquefaction or saccharification. ‘

We chose to characterize further the dual pullulanase and (at-amylase
activities of this enzyme using kinetic analysis and molecular biological
techniques. These studies enabled us to analyze this enzyme at the molecular
level and to identify specific characteristics of the enzyme, including
structural similarity to other amylases, factors responsible for structural
intergrity, and the putative structure of the catalytic center.

The thesis is divided into six chapters: a literature review, a chapter
on amylopullulanase production, purification and general biochemical
characterization, a chapter characterizing the substrate kinetics and dual
specificity of the enzyme, which has been published in Biochemical and
Biophysical Research Communications, a chapter describing the
amylopullulanase gene and the putative catalytic and thermal stability
domain of the gene product, a chapter identifying the active site and

mechanism of catalysis by site directed mutagenesis, a summary of the

3

research and future research directions, and an appendix that deals with
preliminary characterization of cell surface microstructures, with respect to
enzyme localization and cultural conditions.

Chapter I reviews the literature describing (at-amylases, pullulanases,
and of amylases harboring dual activities, with special emphasis on those
enzymes isolated from microbes. The biochemical properties of the
individual enzymes, cloning, sequencing, and expression of the genes
encoding these enzymes, and structural information, where available, are
also described in this chapter.

Chapter H, "Improved purification and biochemical characterization of
extracellular amylopullulanase from Clostridium thermohydrosulfuricum
39E," describes optimization of the levels of enzyme production by C.
thermohydrosulfuricum 39E, and release of the cell-bound enzyme into the
culture medium, simplifying subsequent purification procedures.
Purification of the enzyme from extracellular fractions of the culture, and the
biochemical properties of the enzyme are described.

Chapter III, "Substrate competition and specificity at the active site of
amylopullulanase from Clostridium thermohydrosulfuricum 39E," details
an inhibition kinetic study with the purified enzyme using dual alternate
substrates, which showed that both a-amylase and pullulanase activities
reside on the same enzyme, and suggested the presence of a single active
center. Product analyses are described that show the catalytic pattern of this
enzyme on branched and unbranched starch related polysaccharides and on
linear oligosaccharides, hydrolyzing both a-1,4 and a-1,6 linkages.

Chapter IV, "Cloning, identification of thermostability and catalytic
regions and characterization of (it-1,4 and a-1,6 bond specificity of

amylopullulanase from Clostridium thermohydrosulfuricum 39E" describes

4
the cloning and subcloning strategies used to identify the gene encoding for

amylopullulanase activity. By constructing nested deletion mutants of the
amylopullulanase gene, gene products were isolated that possessed both a-1,4
and a-1,6' activities and thermostability, but were of a smaller molecular
weight compared to the native enzyme. A structural study using NMR to
characterize the ability of the cloned enzyme to debranch polysaccharides is
described.

Chapter V, "Sequencing of the amylopullulanase (apu) gene of
Clostridium thermohydrosulfuricum 39E, and identification of the active site
by site directed mutagenesis" describes mutants constructed by creating nested
deletions and single point base mutants, to identify the catalytic site of the
enzyme. The experimental results provided supporting evidence of the dual
specificity of amyIOpullulanase being due to a single enzyme with one active
site, and identified within the gene, the regions that encoded for
thermostability and catalysis.

The final chapter summarizes the findings of this study and the

conclusions that can be made.

OBJECTIVES

This thesis research was undertaken to obtain a better

understanding of the biochemistry and molecular biology of the novel

pullulanase produced by C. thermohydrosulfuricum 39E. The enzyme is

novel because it has high thermostability and can degrade both (Jr—1,4 and a—1,6

glucosidic bonds, suggesting a new enzyme class.

To elucidate the biochemical features of the pullulanase from C.

thermohydrosulfuricum 39E, including characterization of the enzyme and

the gene encoding it, the following research objectives were undertaken.

(i)

(ii)

(iii)

(iV)

(v)

Enhancement/overexpression of this novel amylase in C.
thermohydrosulfuricum 39E by altering the physiological growth
conditions, to induce the release of the cell-bound enzyme into
the growth medium.

Development of an effective purification scheme for the
purification of pullulanase from C. thermohydrosulfuricum 39E.
Biochemical characterization of the purified enzyme from C.
thermohydrosulfuricum 39E, in terms of activity against (Jr-1,6 and
(It-1,4 linkages.

Determination of substrate specificity and product formation by the
enzyme, and Characterization of the active center by group specific
chemical modification and inhibition kinetics, to obtain a
preliminary understanding of the mode of action of the enzyme.
Cloning, sequencing and expression of the gene encoding for

amylase activity, into Escherichia coli and Bacillus subtilis.

(vi)

(vii)

6
Construction of fusion proteins, and deletion mutants of the gene,

to obtain a better understanding of the various encoded regions of
the primary sequence in enzyme function, and stability.

Site directed mutagenesis of the putative active site residues to
identify the residues involved in catalysis, and to confirm the dual

activity—single active site nature of the enzyme.

CHAPTER I.

LITERATURE REVIEW.

1. Starch composition

Starch is abundant in nature and is present at high concentrations in a
variety of plants such as maize, potato, rice and wheat. This polysaccharide is
made up of glucose molecules and composed of about 80% amylopectin and
20% amylose. The latter is a linear polysaccharide in which the glucose units
are exclusively bound by a-1,4-glycosidic linkages. Amylopectin on the other
hand, is a branched polysaccharide and contains a-1,6-glycosidic linkages in
addition to the (at-1,4 -glycosidic linkages. Every 20 to 25th glucose molecule in
amylopectin is linked via an a-1,6-bond. A variety of yeast, fungi and bacteria
are capable of degrading starch by the formation of extracellular enzymes
(Fogarty and Kelly, 1979; Ingle and Erickson, 1978; Priest, 1977). Such enzymes
include, a-amylase, B—amylase, glucoamylase, and a-glucosidase (Fig. 1, and

Table 1).

2. Pullulan composition

Pullulan is a linear a-glucan produced by Aureobasidium pullulans
(Bender and Wallenfels, 1961), and consists of maltotriose units linked by on-
1,6 glucosidic bonds. Pullulan cannot be degraded by a-amylases or B-
amylases. AIsoamylase, which can hydrolyze the (at-1,6 linkages in amylopectin,

cannot hydrolyze pullulan (Yokobayashi et al., 1970).
3. (Jr-Amylase (1,4-a-D-glucan glucanohydrolase EC 3.2.1.1)
(i) Biochemical properties

(Jr-Amylase, which catalyzes the hydrolysis of a-1,4-glucosidic linkages

in starch and related polysaccharides, is an endoacting amylase that liberates

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10

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oligosaccharides of various chain lengths. The released end products have an

a-configuration at the C1 position (reducing end) of the terminal glucose.

Some physical-Chemical properties of a-amylases of bacterial and
fungal origin are given in Table 2. Since starch cannot penetrate into cells,
most of the enzymes are extracellular, being released into the culture
medium, or associated with the outside of the cell. Membrane bound or-
amylases are found in the fungus Lipomyces starkeyi (Moulin and Galzy,
1979) and in Bacillus sp. (Srivastava et al., 1981).

pH optima of a-amylases ranges from 2.0 to 10.5, which reflects the
ecological niche and optimal growth conditions of these organisms, which
also varies from 3.0 to 10.5. Similarly, the temperature optima for activity of
the tat-amylases are reflected in the growth temperature of the
microorganisms in their natural environment. The lowest temperature
optima reported are 25°C to 30°C, while temperatures of 95°C to 100°C have
been reported to be Optimal for a-amylases from hyperthermophiles isolated
from deep sea vents and fumaroles (Brown et al., 1990; Schumann et al.,
1991). While temperature stability of most (at-amylases is Ca2+ dependent,
those from halophiles were dependent on NaCl for stability (Nachum and
Bartholomew, 1969), due to the high salt environment these organisms
occupy.

Molecular weights of or-amylases vary from 10,000 to 140,000 (Table 2),
while most microbial a-amylases have a molecular weight between 50,000 to
60,000. Carbohydrate moieties increase the molecular weight of some a-
amylases, and glycosylated (Jr-amylases have been reported from Aspergillus
oryzae (McKelvy and Lee, 1969), Bacillus stearothermophilus (Srivastava,
1984), and Bacillus subtilis (Matsuzaki, 1974) with the proportion of the

carbohydrate in these enzymes being about 10% (w/ w).

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a-Amylases are metalloenzymes that contain at least one activating and

stabilizing Ca2+ ion (Valee et al., 1959). Heavy metal ions, sulfhydryl group
reagents, and metal chelating reagents EDTA and EGTA inhibit a-amylases
(Table 2). ‘EDTA and EGTA are thought to destabilize a-amylase due to their
chelating effect on the Ca2+ ions. The amount of bound Ca2+ varies from 1 to
about 10 per mole of enzyme. Crystalline a-amylase from A. oryzae contains
10 bound Ca2+ ions, of which only one is tightly bound (Oikawa and Maeda,
1957). a-Amylase from B. subtilis, B stearothermophilus, or B.
amyloliquifaciens contain four Ca2+ ions (Yutani, 1975) which are necessary
for stability and conformation, although in other systems usually one Ca2+ is
sufficient to stabilize the enzyme. Recent X-ray crystallographic studies have
revealed that the essential Ca2+ is bound between the domains of a-amylase
(Fig. 2) (Vihinen and Mantsala, 1990; Boel et al., 1990). ‘
Almost all a-amylases contain and require Ca2+, and it is essential for
the folding of the enzyme in A. oryzae (Matsuura et al., 1984). It has been
reported that Ca2+ inhibits the activity of a-amylases from A. oryzae strain E1
212 (Kundu and Das, 1970), though this effect may be due to excess calcium,
which has been shown to bind within the catalytic cleft at concentrations

above 5 mM, and inhibit the enzyme (Boel et al., 1990).

(ii) Catalytic mechanism and structure

Three-dimensional structures are available for (at-amylase of A. oryzae
(Taka-amylase A; TAA) (Matsuura et al., 1984), and porcine pancreatic a-
amylase (Buisson et al., 1987). a-Amylases are multi-domain proteins

belonging to a structural subfamily of those enzymes that contain an (a/ [”8-

barrel, described first in triose phosphate isomerase (Banner et al., 1975).

Fig. 2A

Fig. 28

15

Stereo view of the three dimensional structure of a-carbon

chain of a-amylase from Aspergillus oryzae (Adapted from
Matsuura et al., 1984). Individual domains are denoted as
A,B, and C. Active site and substrate binding cleft is

indicated by arrow. A single Ca2+ atom is located between
domains A and B.

Topological diagram of the secondary and super-secondary

structures of (at-amylase from A. oryzae (Adapted from
Matsuura et al., 1984). Individual domains are denoted

A,B, and C. The (oz/[3)8 barrel is denoted by domain B. The
open circles represent (it-helices while the open squares
represent B-sheets.

2A

 

2B /

1 7
Several amino acid side chains in the loops that connect the eight B-sheets to

the succeeding a-helices constitute the extended substrate binding cleft
(Matsuura et al., 1984; Klein and Schulz, 1991; Farber and Petsko, 1990). A
characteristic long loop at the C-terminus of the third B-sheet is grafted on to
the (a/B)8-barrel (domain A) in a-amylases. This separate structural unit,
termed domain B, participates in substrate binding, and is linked to domain
A via an essential Ca2+ ion (Vihinen and Mantsala, 1990). Depending on the
enzyme, domain B has 48 to 133 amino acid residues (Matsuura et al., 1984;
Buisson et al., 1987; Klein and Schulz, 1991; MacGregor and Svensson, 1989;
Svensson et al., 1991). Domain C, succeeding the (a/ IDS-barrel domain, has an
immunoglobin type of fold (Matsuura et al., 1984; Buisson et al., 1987; Klein
and Schulz, 1991), and has not been assigned to a specific function.

Based on X-ray crystal structures of A. oryzae and porcine pancreatic a-
amylase, a subsite model has been proposed for substrate binding, where
seven consecutive glucose units of the substrate are bound by at least two
amino-acids on the substrate binding region of the enzyme (Matsuura et al.,
1984) (Fig.3). The essential Ca2+ is bound near the active center and appears
to stabilize the two domains (A and B) forming the active site/substrate
binding cleft. The catalytic mechanism of a-amylase has been modeled after
the general acid-base catalytic mechanism proposed for lysozyme (Vernon,
1967). Using X-ray crystallography Glu230 and Asp297 have been proposed to
be the catalytic residues in a-amylase from A. oryzae (Matsuura et al., 1984). In
porcine pancreatic oc-amylase, similar studies have been used to propose
Asp197 and Asp300 (corresponding to Asp206 and Asp297 of a-amylase from
A. oryzae) as the catalytic residues, while Glu233 (corresponding to Glu230 of
a-amylase from A. oryzae) was not suggested to be involved in catalysis

(Buisson et al, 1987). In a-amylase from A. oryzae, the substrate is bound by

Trp83

'r- 74
1.45pr "'

Asp297

 

so“

NI ' :
His 296 W" H\ Glu35
Arg344
Asp340

Tyr79

Fig. 3 Proposed substrate binding and catalytic site for a-amylase of A.
oryzae (Adapted from Matsuura et al., 1984).

Proposed catalytic residues; Aspzoa, Asp297 (Buisson et al., 1987)
Glu23o, Asp297 (Matsuura et al., 1984)

l 9
the residues located on the walls of the active site cleft (Matsuura et al., 1984).

The residues predicted to be involved in either catalysis or substrate binding
are highly conserved among the a-amylases from various mammalian, plant,
fungal, and bacterial sources, according to amino acid sequence data analysis

(Svensson, 1988).

(iii) Molecular biological studies

The structural genes encoding oc-amylase have been cloned from
various Bacillus spp. (Palva, 1982; Aiba et al., 1983; onet at al., 1984),
Aeromonas hydrophila (Gobius and Pemberton, 1988), Dictyoglomus
thermophila (Fukusumi et al., 1988), Streptomyces limosus (Long et al., 1987),
and Aspergillus oryzae (Tada et al., 1989). Two B. stearothermophilus strains
have been found to harbor plasmid borne a-amylase genes (Mielenz, 1983).
Host organisms most frequently used have been Escherichia coli and
Bacillus subtilis, and to a much lesser extent, B. brevis (Tsukagoshi et al.,
1985), B. stearothermophilus (Aiba et al., 1983), Brevibacterium
lactofermentum (Smith et al., 1986), Pseudomonas aeruginosa (Filloux et al.,
1985), Saccharomyces cerevisiae (Kunze et al., 1988), and several Streptomyces
strains (Thudt et al., 1985). Sequences of the a-amylase genes have been
determined from all the microbes mentioned above.

The sequenced d—amylase genes contain signal peptide coding regions
preceded by sequences similar to the E. coli and B. subtilis consenses promoter
sequences (Rosenberg and Court, 1979; McConnell et al., 1986). For the
sequenced a-amylases, the length of the signal peptide varies from 29 to 41
amino acids (Gray et al., 1986; Yamane et al., 1984). Signal peptides of cloned B.
stearothermophilus a-amylase are processed at the same site in E. coli and the

donor strain (Nakajima et al., 1985; Ihara et al., 1985).

20

I II
TAA 117 BVVANH 122 202 GLRIDTVKH 210
BStA 101 DVVFDH 106 230 GFRLDAVKH 238
BAA 98 DVVLNH 103 227 GFRIDAAKH 235
BLA 100 DVVINH 105 227 GFRLDAVKH 235
BME 109 DLVVNH 114 202 GFRLDAAKH 210
ES? 103 DVVMNH 108 233 GFRIDAVKH 241
BSU 107 DAVINH 112 181 GFRFDAAKR 190
SHY 88 DAVVNH 93 170 GFRIDAAKH 178
III . IV
TAA 229 GEVLD 233 292 FVENHD 297
BStA 263 GEYUS 267 326 FVDNHD 332
BAA 260 AEYUQ 264 323 FVENHD 328
BLA 260 AEYUQ 264 323 FVDNHD 328
BME 246 GEVVD 250 308 FLTNHD 313
85? 265 VEFUK 270 329 FVDNHD 334
BSU 217 GEILQ 221 274 UVESHD 279
SHY 199 QEVIY 203 257 FVDNWD 262

FIG. 4 Conserved regions (I to IV) of some microbial amylases.
TAA = Aspergillus oryzae

BSta = Bacillus stearothermophilus
BAA = B. amyloliquefaciens

BLA = B. licheniformis

BME = B. megaterium

BSP = Bacillus sp.

BSU = B. subtilis

SHY = Saccharomycopsys hygroswpicus

2 l
a-Amylase sequences contain three (Rogers, 1985) or four (Nakajima et

al., 1986) conserved regions (Fig 4), and these regions have been proposed to
be essential for the function of a-amylase because they are aligned and
spaced at similar intervals in all the a-amylases studied so far. These regions
form the active center, the substrate binding site, and the site for binding the
stabilizing calcium ion, according to comparison with the refined 3-
dimensional structure of (it-amylases from A. oryzae (Matsuura et al., 1984;

Boel et al., 1990) and porcine pancreas (Buisson et al., 1987).

4. Pullulanase (a-dextrin 6—glucanohydrolase EC 3.2.1.41)

- Pullulanase hydrolyzes (it-1,6 linkages of pullulan and other branched
oligosaccharides. The a-1,6 linkages are considered to mimic partially the a-
1,6 branch points of amylopectin, and pullulan has been widely employed as a
model substrate for starch debranching enzymes (Plant et al., 1986). A specific
pullulanase is an enzyme that can hydrolyze the a-1,6 linkages in pullulan,
forming maltotriose as the sole reaction product. Pullulanase has been
isolated from relatively few microorganisms (Table 3) compared to the
number of a-amylases identified from various microorganisms. Aerobacter
aerogenes, in which the enzyme was initially found (Bender and Wallenfels,
1961), is now classified as Klebsiella aerogenes (Enterobacter aerogenes).
Pullulanase is also produced by mesophilic organisms such as Bacillus
acidopullulyticus, B. cereus var. mycoides, Streptomyces mitis, and
Bacteroides thetaiotaomicron (Table 3). Recently, thermostable pullulanase
activities have been reported from several thermophilic microorganisms,
including, Clostridium thermosulfurogenes (Burchhardt et al., 1991),
Thermus aquaticus YT—1(Plant et al., 1986), T. finii (Koch et al., 1987), T.

ethanolicus (Koch et al., 1987), Thermobacteroides acetoethylicus (Koch ct al.,

22

 

 

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2 3
1987), Thermoactinomyces thalophilus (Odibo et al., 1988) and B.

stearothermophilus (Kuriki et al., 1988).

(i) Biochemical properties

pH optima of pullulanases vary from 4.9 to 8.5, while the temperature
optima range from 30°C to 85°C. The biochemical properties of these
pullulanases reflect the growth environment of the individual organisms
from which the enzyme was isolated. Molecular weights reported are from
58,000 to 450,000. pCMB, heavy metal ions, a-,B-,and y-cyclodextrins have

been found to inhibit pullulanase activity.

(ii) Molecular biology studies

The gene responsible for pullulanase activity have been cloned from K.
aerogenes (Takizawa and Murooka, 1985), K. pneumoniae (Michaelis et al.,
1985), B. thetaiotamicron (Smith and Salyers, 1989) and B. stearothermophilus
(Kuriki et al., 1988). Pullulanase from K. aerogenes and K. pneumoniae have
been the most studied, and the nucleotide sequences of the genes are
available. However, no X-ray crystallographic data with regard to the residues
involved in catalysis or substrtae binding, or the catalytic mechanism, are yet

available for any pullulanase.

Amylases with dual activity

Amylases that harbor dual activities are a recent addition to the
repertoire of starch-hydrolyzing enzymes, and have yet to be categorized by
the Enzyme Commission. Almost all the enzymes with dual activity
reported so far are from anaerobic thermophiles. Initial reports of an enzyme

containing dual amylase activities (on-amylase and pullulanase) were reported

2 4
from B. subtilis (Takasaki, 1987), where it was inferred that the two activities

are due to two individual enzymes forming a complex dimer of 450,000
molecular weight.

A chromosomal DNA segment encoding both a-amylase and
pullulanase activities was cloned from T. brockii into E. coli and B. subtilis
(Coleman et al., 1987), although no further studies were carried out to
determine whether the DNA insert encoded a single protein imparting the
dual activities.

An amylase-pullulanase enzyme of molecular weight 220,000,
produced by B. circulans F-2, which can hydrolyze soluble starch (on-amylase
activity) and pullulan (pullulanase activity) at equivalent rates, has been
characterized using inhibition kinetics to show that the enzyme has two
distinct and separate sites, each site having either a-amylase or pullulanase
activity (Sata et al., 1989). However, using the same approach, a similar
enzyme was isolated from Thermoanaerobium Tok6-Bl and has been
suggested to contain a single active site for both activities (Plant et al., 1987).

An amylase with dual activity, denoted a-amylase-pullulanase, has
been reported from C. thermohydrosulfuricum strain E101. This enzyme is
reported to be a dimer with a subunit molecular weight of 190,000, containing
a M;- 20,000 satellite protein that is necessary for structural and functional
stability (Melasniemi, 1988). This differed markedly from the subunit
structure and molecular weight of the amylase with dual activities reported
from C. thermohydrosulfuricum 39E (Saha et al., 1988; Mathupala et al., 1990),
where the enzyme is a monomer of mw 136,000. Based on sequence analysis,
it has been suggested that the C. thermohydrosulfuricum E101 (at-amylase-
pululanase may contain two distinct sites, one for each activity (Melasniemi

et al., 1990), and that the enzyme is encoded such that the initial segment of

2 5
the gene encodes a pullulanase, while the subsequent half encodes the a-

amylase activity.

Therefore, detailed biochemical characterization with regard to gene
organization, subunit structure, catalytic and substrate binding sites,
mechanism of action, is necessary to obtain a better understanding of these

enzymes with dual activities.

Other enzymes that hydrolyze starch

B-Amylase (EC 3.2.1.2) attacks the a-1,4 glycosidic linkages in
polysaccharides from the non-reducing ends and forms maltose as the major
product. This enzyme does not process debranching activity, and therefore is
not capable of hydrolyzing a-1,6-linkages in branched polysaccharides lik‘e
amylopectin or glycogen. Glucoamylase (EC 3.2.1.3) attacks (1-1,4 linkages in
polymers from the non-reducing end forming one glucose at a time. This
enzyme also has the capability of splitting the a-1,6-linkages, but at a slower
rate (Saha and Zeikus, 1989). The enzymatic action of a-glucosidase (EC
3.2.1.20) is similar to glucoamylase, but it attacks preferentially (it-1,4 linkages

in short chain polysaccharides (Kelly and Fogarty, 1983).

Other enzymes that hydrolyze pullulan

' Isoamylase is capable of hydrolyzing (at-1,6 linkages in branched

polysaccharides, but not in pullulan (Yokobayashi et al., 1970). Isoamylases

are usually secreted by fungi and are also classified as debranching enzymes.
Four different types of enzymes hydrolyze pullulan and produce

different end products (Fig. 5). Many glucoamylases hydrolyze pullulan by

liberating glucose units from the non-reducing end. Isopullulanase cleaves

the first (at-1,4. linkage following the a-1,6 bond, yielding isopanose (Sakano et

26

 

 

 

 

u “ on u o .
.6 N
H 01 I O!
u. um
l l GLUCOAMYLASE _ .
l - GLUCOSE
001m “3
o u n o
_ u u
H 0“ N
N N
1.6 LINKAGE
PULLlLANASE
:O-O-O
.. MALTOTROSE
l .
t mtmwsg
isoemose
l
ENZYME 0F
.., . PANOSE
T. vuusms

AND- esremomeauoemws

Fig. 5 Action pattern of pullulan degrading enzymes. The circles
represent glucose units, the horizontal lines (it-1,4 linkages, and

the vertical lines oc-l,6 linkages (Adapted from Vihinen and
Mantsala, 1989).

27
al., 1971). Neopullulanase of B. stearothermophilus cleaves the a-1,4

glucosidic bond preceding the (at-1,6 linkage liberating panose from pullulan,
but cannot hydrolyze starch or any other polysaccharide efficiently (Kuriki et
aL,1988l ~

The formation of debranching enzymes besides a-amylase, B-amylase
orlglucoamylase enables microorganisms to convert branched complex
polysaccharides completely and efficiently into small utilizable sugars. The
ability to produce such enzymes is distributed among aerobic and anaerobic
microorganisms. Compared to aerobes, little information, is available
concerning the physiology and enzymology of starch hydrolyzing anaerobic,

thermophilic, bacteria.

Thermostable enzymes

Thermophilic microorganisms, capable of growth at temperatures of
75°C and higher, were first isolated from hot springs at Yellowstone National
Park (Brock, 1986). Thermostable as well as thermophilic enzymes have been
isolated from many of these organisms, having temperature optima for
stability and activity comparable to the physiological or natural growth
temperature of the respective microorganisms from which they were isolated
from, and in some cases surpassing the temperature optimum found in
nature (Zeikus et al., 1977; Lamed and Zeikus, 1980; Saha et al., 1991; Zeikus et
al., 1991). Current evidence suggests that the upper limit of temperature for
growth of thermophilic bacteria is about 110°C (Stetter, 1982; Stetter et al.,
1987; Costantino et al., 1990; Brown et al., 1990). Three enzymes from the
hyperthermophilic archaebacterium Pyrococcus furiosus, have the highest
temperature optima reported so far of 105° to 115°C, for an a-glucosidase

(Costantino et al., 1990), an a-amylase with an optimum greater than 108°C

2 8
(Brown et al., 1990), and a serine protease with an optimum of 115°C (Eggen et

al., 1990).

Thermophilic enzymes are ideal for studying the structural basis of
protein stability. Since the initial discovery of these thermophilic enzymes,
many attempts have been made to identify and characterize any special
molecular properties that could impart such stability at higher temperatures.
These studies are important for basic research into protein structure and
folding (Dill, 1985; Schellman, 1987), and because of the possibility of using
these enzymes as practical catalysts under different experimental conditions
(Torchillin and Martinek, 1979; Mozhaev and Martinek, 1984; Shami et al.,
1989). Biotechnological applications of meSOphilic enzymes are often
hindered by their intrinsic lability toward environmental factors such as heat,
organic solvents, detergents, proteolytic enzymes, etc., which may be
overcome by using thermostable enzymes due to their greater stability
against these environmental parameters (Zeikus, 1979). However, detailed
structural studies carried out on thermostable enzymes, in comparison with
their mesophilic counterparts, has therefore failed to identify any unifying
properties accounting for thermostability. Instead, current evidence shows
that the intrinsic stability of thermophilic enzymes cannot be attributed to any
common determinant, but is due to the overall contribution of a variety of
stabilizing effects including hydrophobic interactions, ionic and hydrogen-
bonding, disulfide bonds, metal ion binding etc., similar to those effects that

have already been observed for mesophilic proteins (Mathews et al., 1974).

Clostridium thermohydrosulfuricum 39E
C. thermohydrosulfuricum 39E (ATCC 33223) is an obligate spore

forming anaerobe obtained from glucose and xylose enrichment cultures

2 9
from bacterial-algal mats associated with OctOpus hot spring in Yellowstone

National Park (Zeikus et al., 1980). The optimum temperature of growth of
this organism is 65°C. The organism is capable of catabolizing starch, hexoses
and pentOses to form ethanol as the only significant fermentation product.
Highly thermostable cell bound amylase activities have been reported from
this organism (Hyun and Zeikus, 1985a), and enzyme synthesis was inducible
and subject to catabolite repression (Hyun and Zeikus, 1985b). The enzyme
responsible for pullulanase activity has been purified and initial
characterizations carried out (Saha et al., 1988; Mathupala et al., 1990; this
study). Recent studies on C. thermohydrosulfuricum 39E using DNA-DNA
hybridization has showed that strain 39B has only 79% homology to C.
thermohydrosulfuricum neotype (Lee et al., manuscript submitted), while
97% homology is shown toward Thermoanaerobacter ethanolicus. Therefore,
a new name Thermoanaerobium ethanolicus has been proposed for this
organism (Lee et al., manuscript submitted). In this thesis, the organism will

be referred to as C. thermohydrosulfuricum 39E.

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CHAPTER 2

IMPROVED PURIFICATION AND BIOCHEMICAL CHARACTERIZATION
OF EXTRACELLULAR AMYLOPULLULANASE FROM Clostridium

themohydrosulfuricum 39E

38

ABSTRACT

Maltose-limited chemostat culture was used to investigate the
overexpression and excretion of amylopullulanase by C. thermohydrosulfuricum
39E. In maltose limited continuous culture, amylopullulanase was secreted and
produced at 10 fold higher levels than in batch culture. The extracellular
amylopullulanase was purified to homogeneity by using an inhibitor linked
affinity column matrix. The purified amylopullulanase had a specific activity of

480 U/ mg protein for pullulanase and 175 U/ mg protein for a-amylase. B-

Cyclodextrin inhibited both a-amylase and pullulanase activities, with a Ki of
0.065 mg/ ml. Amylopullulanase had a Mr of 140,000 on SDS-PAGE analysis and
a M,- of 133,000 using gel-filtration chromatography. The N-terminal sequence of
the enzyme was Glu—Thr-Asp-Thr-Ala-Pro—Ala. The purified enzyme displayed
Km values of 0.35 mg/ ml for pullulan and 1.00 mg/ ml for amylose. The enzyme
had a pI of 4.0, and displayed an optimum pH for stability and activity of 6.2 and
5.5 respectively. The enzyme was stable up to 85°C in the presence of Ca2+, and
had a half life of 40 min at 90°C (pH 6.2). Ca2+ was required for thermal
stability, but not for activity. Amylose, glycogen, and amylopectin were
degraded to maltose, maltotriose, and maltotetraose, whereas only maltotriose

was formed from pullulan.

39

INTRODUCTION

(at-Amylase (1,4-a-D-glucan glucanohydrolase EC 3.2.1.1) and pullulanase
(a-dextrin. 6-glucanohydrolase EC 3.2.1.41) are endo—amylases, which split
glucosidic linkages in the interior of the starch molecule in a random fashion. a-
Amylase hydrolyzes internal a-1,4 bonds, and can bypass a-1,6 linkages (or
branch points) in the starch molecule. Pullulanase is known as a debranching
enzyme, which hydrolyzes a-1,6 linkages (branch points) in starch molecules.
Pullulanase but not a-amylase can degrade the a-1,6 linkages of pullulan (poly
(it-1,6 maltotriose), a polysaccharide composed of a-1,6 linked maltotriose units,
into maltotriose (Bender and Wallenfels, 1961).

Both a-amylase and pullulanase are industrially important enzymes due
to their hydrolytic activity on starch, and derivatives of starch, to produce
conversion and glucose syrups (Allen and Dawson, 1975). For enzymatic
processing of starch, temperatures of 95°C to 105°C are required for periods
ranging from 5 min (105°C) to 2 h (95°C). The current technology uses two
enzymes, in a two stage system, a liquefaction step in which starch granules are
dispersed or gelatinized in an aqueous solution and then partially hydrolyzed by
a thermostable fungal a-amylase, and a saccharification step in which liquefied
starch is converted into glucose or low molecular weight saccharides by a
pullulanase in the presence of either glucoamylase or B-amylase. Pullulanase is
used in combination with saccharifying amylases such as glucoamylase, fungal
a-amylase or B-amylase for the production of various sugar syrups because it
improves saccharification and yield (Norman, 1982). In addition pullulanase has
gained significant attention as a tool for structural studies of carbohydrates

(Whelan, 1971).

40

41
Enzymes isolated from thermophilic bacteria, when compared to similar

enzymes of mesophilic microorganisms, have higher heat stability, while usually
possessing similar catalytic properties. Predictably, these thermostable enzymes
show catalytic activity only at high temperature (50°C to 100°C), and are almost
inactive at room temperature. The enhanced stability of these enzymes is
reflected in their greater stability against other extremes, such as extremes of pH,
solvent, salt concentrations, and in their resistance to the action of certain
proteases and strong denaturants such as urea and guanidium hydrochloride
(Brock, 1987).

Hyun and Zeikus (1985a) reported a highly thermostable and
thermoactive pullulanase activity from C. thermohydrosulfuricum 39E. They also
studied the regulation of the synthesis of the pullulanase in this organism (Hyun
and Zeikus, 1985b), with the finding that enzyme synthesis was inducible and
was subject to catabolite (glucose) repression. A cell bound pullulanase that
cleaved starch and pullulan was purified and partially characterized from this
organism (Saha et al., 1988).

Several studies have been conducted to determine the importance of
culture conditions on cellular localization of hydrolytic enzymes. In Klebsiella
aerogenes, pullulanase activity was detected in the culture medium in batch
culture with excess maltose, which acted as an inducer for the enzyme, while
under substrate limited chemostat culture, the enzyme activity remained firmly
cell-bound (Hope and Dean, 1974).

In Clostridia producing extracellular amylases, continuous culture has
been used to overproduce and increase the yield of amylases in the culture
medium (Antranikian et al., 1987a; Antranikian et al., 1987b; Madi et al., 1987). In

Clostridium thermohydrosulfuricum 39E, however, the amylolytic activities

42
remained mainly cell-associated during growth up to late exponential phase

under batch culture conditions (Hyun and Zeikus, 1985a,b).

The purpose of this study was three fold: first, to maximize conditions for
production and purification of amylopullulanase from C. thermohydrosulfuricum
39E, secondly, biochemical comparison of the extracellular pullulanase purified
here, with the cell-bound pullulanase (Saha et al., 1988); and thirdly, to provide
more detailed biochemical characterization of amylopullulanase with respect to
inhibitors, stability, amino acid composition and N-terminal sequence,
carbohydrate content, substrate and product hydrolysis rates and yields, and

amino acids involved in catalysis.

MATERIALS AND METHODS

Chemicals and gases

All chemicals used were reagent grade and obtained from either Sigma
Chemical Company (St. Louis, MO) or Aldrich Chemical Company (Milwaukee,
WI). Nitrogen was 99.9% pure and was made free of oxygen by passage over
heated (370°C) copper filings.

Chromatographic gel material were obtained from Pharmacia Limited
(Piscataway, NJ). The columns for standard chromatography were obtained from

Bio-Rad (Melville, NY).

Organism and cultural conditions

C. thermohydrosulfuricum 39E (ATCC 33223) was used as the source of
enzyme. The organism was grown at 60°C under anaerobic conditions on TYE
medium (Zeikus et a1, 1980), supplemented with 0.2% to 0.5% (w/v) soluble
starch, maltose or glucose as the substrate. Batch culture studies were performed
using pressure tubes containing 10 ml of medium, and samples were taken at

b

mid to late exponential phase of growth.

Enzyme production and continuous culture studies

For continuous culture of C. thermohydrosulfuricum 39E, a 300 ml
continuous culture vessel (Multigen, New Brunswick Scientific Co., Edison, NJ )
equipped with pH, temperature, and media flow controllers and a stirrer was
used, and the vessel was continuously gassed with nitrogen. Complex media
modified from Antranikian et al., (1987) contained 0.2% (w/v) maltose as the

carbon source; and the following components in % (w/v) : Tryptone, 0.25; Yeast

extract, 0.1; MgC12.6HzO, 0.016; KH2P04, 0.167; NazHPO4.7H20, 0.167;

43

44
CysteineHCl, 0.05; CaC12.2H20, 0.0008. Vitamin solution (Zeikus et al., 1980) and

trace mineral solution (Zeikus et al., 1980) were added at 1% (v/v). Cysteine.HCl,
maltose, and vitamin solution were filter sterilized together and added to the
autoclaved medium. The medium pH was varied from 5.5 to 7.5 using 2.0 M
KOH, and the temperature was maintained at 60°C. The culture medium was
monitored for extracellular a-amylase and pullulanase activity, by collecting the
outflow into vessels maintained at 4°C under anaerobic conditons, and then
assaying for enzyme activity.

For assay of enzyme activity, 1.0 ml of the cell culture was withdrawn
from the chemostat and centrifuged at 14,000 rpm for 1 min in an Eppendorf
microcentrifuge at room temperature. The supernatant was removed and
adjusted to 1.0 ml by using 1.0 M sodium acetate (pH 6.0) containing 0.1 M CaClz
stock buffer solution to give a final concentration of 50 mM sodium acetate, 5
mM CaClz, pH 6.0, and assayed for extracellular enzyme activity. The cell pellet
was resuspended in 100 ul of 50 mM sodium acetate buffer containing 5 mM

CaClz (pH 6.0), and assayed for cell bound enzyme activity.

Enzyme assays

For assay of pullulanase activity, 160 pl of 1.25% (w/v) pullulan in 50
mM acetate buffer, pH 6.0, containing 5 mM CaClz and 40 ul of enzyme solution
were mixed and incubated at 60°C for 30 min. The reaction was stopped by
adding 0.8 ml of dinitro salicylate (DNS) solution (Miller, 1959), and heated in a
boiling water bath for 15 min to develop the color reaction. The samples were
cooled on ice and centrifuged in an Eppendorf microcentrifuge (14,000 rpm x 10
min). The supernatant was recovered and measured at 640 nm, for

measurement of pullulanase activity. One unit of pullulanase activity is defined

45

as the amount of enzyme that produces 1 umol of reducing sugar (with glucose
as the standard) per min under the assay conditions.

For assay of (it-amylase activity, 160 pl of 1.25% (w/v) soluble starch in
50 mM acetate buffer, pH 6.0, containing 5 mM CaClz and 40 ul of enzyme
solution were mixed and incubated at 60°C for 30 min. The reaction was stopped
by adding 0.8 ml of DNS, and heated in a boiling water bath for 15 min to
develop the color reaction. The samples were cooled on ice and centrifuged in an
Eppendorf microcentrifuge (14,000 rpm x 10 min). The supernatant was
recovered and measured at 640 nm, for measurement of a-amylase activity. One
unit of a—amylase activity is defined as the amount of enzyme that produces 1
umol of reducing sugar (with glucose as the standard) per min under the assay
conditions.

For assay of glucogenic amylase activity, 160 ul of 1.25% (w/v) soluble
starch in 50 mM acetate buffer, pH 6.0, containing 5 mM CaClz and 40 uliters of
enzyme solution were mixed and incubated at 60°C for 30 min. The sample was
boiled for 10 minutes to destroy enzyme activity and centrifuged (14,000 rpm x
10 min) at 4°C. The supernatant was assayed for released glucose with a glucose
analyzer (Model 27, Yellow Spring Instrument Co., Yellow Springs, OH), or by
the hexokinase method (Sigma Glucose Diagnostic Kit 115-A). One unit of
glucogenic amylase activity is defined as the amount of enzyme that produces 1

mol of glucose per min under the assay conditions described.

Affinity column coupling

Sepharose CL-6B was activated with epoxy groups using bisepoxirane
(Sundberg and Porath, 1974; Janson and Ryden, 1989), or using epichlorohydrin
(Porath and Fornstedt, 1970; Janson and Ryden, 1989). Epoxy activation was

carried out as follows; 20 grams of Sepharose CL-4B was washed with water and

46
suction dried. The moist gel was resuspended in 20 ml of 1,4-butanediol

diglycidyl ether, and put under vacuum briefly to remove the air entrapped in
the gel matrix. 20 ml of 0.6 M NaOH containing 2 mg/ ml sodium borohydride
was added to the gel suspension while shaking on a rotary water bath (200 rpm)
and mixed for 8 h at room temperature. The reaction was stopped by washing
the gel with 2000 ml of water by suction filtration.

20 g of epoxy-activated Sepharose CL-48 was washed with 125 ml of 0.1 M
N aOH, and the moist gel transferred to a solution of 1.5 g of B-cyclodextrin in 60
ml of 0.1 M NaOH. Coupling of B-cyclodextrin proceeded for 16 h at 45°C in a
rotary shaker at 200 rpm. After completion of the coupling, the gel was washed
serially in 500 ml each of glucose (25 mg/ ml), 0.1 M borate buffer (pH 8.0)
containing 0.5 M N aCl, and 0.1 M acetate buffer (pH 4.0) containing 0.5 M NaCl.
Between each addition, the gel was washed with 500 ml of water. The final

product was stored at 4°C in water.

Purification of the extracellular enzyme:

(i) Concentration and ion-exchange chromatography: 1000 ml of the supernatant
from the continuous culture outflow was adjusted to 50 mM acetate and 5 mM
CaClz, pH 6.0 and was concentrated to 10 ml by ultrafiltration using an Amicon
YM-100 membrane (Amicon Co., Danvers, MA). The concentrate was passed
through a Q-Sepharose FF column (Pharmacia) (2.5 x 50 cm) pre-equilibrated
with 50 mM acetate buffer with 5 mM CaClz (pH 6.0) at a flow rate of 1 ml min'l.
The column was then washed with 50 mM NaCl in the same buffer until no
significant amount of protein eluted. The active enzyme was eluted with a NaCl
gradient from 0.05 M to 0.45 M in the same buffer.

(ii) Affinity purification: The active fractions from the Q-Sepharose FF column

were pooled and concentrated by ultrafiltration by using a YM 100 membrane

47

(Amicon). The concentrate was then applied to a B-cyclodextrin coupled
Sepharose 4B affinity column (10 ml bed volume), pre-equilibrated with 50 mM
acetate buffer with 5 mM CaC12 (pH 6.0). The column was washed with the same
buffer and then washed with buffer containing 0.5 M NaCl. The enzyme was
eluted with 1% (w/v) B-cyclodextrin at 4°C. The eluted peak, detected at 280 nm,
was collected and washed extensively using ultrafiltration to remove B-

cyclodextrin.

Characterization of the enzyme
Molecular weight and pl determination

Molecular weight was determined by gel-filtration chromatOgraphy on a
Superose-12 column (in acetate buffer containing 50 mM NaCl) calibrated with
known protein standards, and from SDS-polyacrylamide gel electrophoresis
(7.5%) by the method of Laemmli (1970) using a Bio-Rad Mini—Protean II gel
apparatus (Bio-Rad). For calibration of the gel filtration column, molecular
weight standards (MW-GF-200; Sigma) containing blue dextran (Mr >2,000,000),
B-amylase (Mr 200,000), alcohol dehydrogenase (Mr 150,000), bovine serum
albumin (Mr 66,000), bovine carbonic anhydrase (Mr 29,000), and horse heart
cytochrome C (Mr 12,400) were used. For SDS-PAGE electrophoresis, molecular
weight standards (high range) (Bio-Rad) containing myosin (Mr 200,000), E. coli
B-galactosidase (Mr 116,250), rabbit muscle phosphorylase b (Mr 97,400), bovine
serum albumin (Mr 66,200), and hen egg white ovalbumin (Mr 42,699) were
used. The isoelectric point was determined using a Servalyt Precote isoelectric
focusing gel (pH 3-10) (Serva Biochemicals Co., Westbury, NY), with an LKB

Multiphore II isoelectric focusing apparatus cooled to 10°C. Serva protein test

mixture 9 was used for calibration, and the gel was stained with Serva Blue W.

48
Amino acid composition and N-terminal analysis

Purified enzyme was desalted by using double distilled water with a
Centricon-30 (Amicon) ultrafiltration device. Amino acid composition was
determined by reversed phase HPLC using the PICO-TAG method (Waters Div.,
Millipore Co., Milford, MA), after modification of the enzyme with iodo-acetic
acid (for S—carboxymethylation) and with performic acid. N-terminal analysis of
the enzyme was carried out with a Beckman Model 890M sequencer at the
Macromolecular Facility, Department of Biochemistry, Michigan State

University.

Active site titration and chemical modification

Initial velocity studies were performed within the pH range of 3.5 to 9.9 at
0.5 pH intervals, using glycine, acetate, MES, and Tris buffers. KmaPP and
VmaxaPP were determined at each pH value. 1-(3-Dimethylaminopropyl)-3-
ethyl carbodiimide.HCl (DEAC) was used to modify aspartate and glutamate
residues (Carraway and Koshland, 1972). Diethylpyrocarbonate (DEP) was used
to modify histidine (Miles, 1977).

For asp or glu modification, ethanolamine or glycine methyl ester was
used as the nucleophilic reagent, and the reaction was performed in 50 mM
phosphate buffer, pH 6.0 at 25°C. DEAC concentrations of 0 to 100 mM in 25 mM
intervals were used, in the presence of 200 mM nucleophile. The reactions were
quenched in 1.0 M acetate buffer, pH 6.0 and he residual activity assayed.

Histidine residues were modified with DEP at 0 to 40 mM concentrations,
at 10 mM intervals in 50 mM acetate buffer (pH 6.0) at 25°C. The reactions were
monitored at 241.5 nm for 1 h, and quenched with 0.5 M imidazole buffer pH 6.0

before assay of residual activity.

49
Analysis of glycan component in amylopullulanase

Amylopullulanase was hydrolyzed with HCl and subsequently reduced
and acetylated (Chaplin and Kennedy, 1986; Knapp, 1986). The acetylated
hydrolysate was analyzed by capillary GC (Restec Rtx 2330; 90% biscyanopropyl-
10% phenylcyanopropyl polysiloxane) in a temperature gradient of 140°C to
200°C (2°C/ min), with nitrogen used as carrier gas at 0.5 ml/ min. Acetylated
monosaccharide standards (alditol acetates) were used for calibration and

identification of carbohydrate components.

HPLC analysis of product formation by action of enyme on polysaccharides
For determination of rate of product formation by enzyme action on
polysaccharides, 1.0% (w/v) solutions of pullulan, soluble starch, amylose,
amylopectin, mammalian glycogen, and oyster glycogen were incubated with
affinity-purified enzyme (0.05 U/ ml). 100 pl samples were withdrawn and boiled
for 10 min to destroy the enzymatic activity, and centrifuged. The supernatant
was analyzed by HPLC using an Aminex HPX-42A saccharide analysis column
(Bio-Rad Laboratories) and a refractive index detector (Model 410 differential
refractometer, Waters, Danvers, MA). Analysis was carried out at 85°C, using

water as the eluant.

Thin Layer Chromatographic analysis of product formation of enzyme action
on oligosaccharides

2 ul samples of the reaction mixture were applied to Whatman HP-K
high performance silica gel plates (4.5 m particle size; 10 x 10 cm). The plates
were developed with a n-butanolzethanolzwater (3:222 v/v) eluant mixture at

room temperature. The resolved sugars were detected by using a 1:1 mixture of

50
0.2% (w/v) orcinol in methanol and 20% (v/v) sulfuric acid in methanol (1:1,

v/ v) and the plates heated at 100°C for color development.

Activity staining by native PAGE

“ The affinity-purified native enzyme was electrophoresed at a
concentration of 1 ug/ well as for SDS-PAGE, on a Bio-Rad Mini Protean II
electrophoresis apparatus, except for the absence of SDS in the buffer systems
and in the gel. Prior to the polymerization of the gels, pullulan or soluble starch
were added to a final concentration of 1% (w/v). After electrophoresis, the gels
were washed with acetate buffer (pH 6.0), and incubated at 60°C for 5 to 10 min.
For activity staining of pullulan embedded gels, the gel was processed as for
determination of glycoprotein in SDS-PAGE gels (Pharmacia), and developed
using Schiff‘s reagent. For activity staining of soluble starch embedded gels, a
solution of 0.15% iodine:1.5% KI was added as an overlay. Pullulanase activity
could be detected as a clear band against the dark red background of the pullulan
embedded gel, while a—amylase activity could be detected as a clear band against

the dark blue background.

RESULTS

Cellular location and production of amylopullulanase

In order to determine the effect of culture conditions on the cellular
location and level .of amylopullulanase produced, chemostat cultures of C.
thermohydrosulfuricum 39E were compared with batch cultures of the organism
with the aim of producing higher levels of predominantly extracellular activity
that would simplify purification of the enzyme.

For comparison of enzyme activity and localization under different
growth conditions, cell bound and extracellular pullulanase and oc-amylase
activities were analyzed during growth on maltose and soluble starch (Table 1).
Under batch culture conditions low levels of amylase and pullulanase were
produced during growth of C. thermohydrosulfuricum 39E on starch, while higher
levels were detected during growth on maltose. With maltose, higher levels of
both a-amylase and pullulanase activities occured in late exponential phase, with
activities being predominantly extracellular. Under maltose limited continuous
culture conditions (0.2% w/v), a 12 fold increase in extracellular
amylopullulanase activity occurred when compared to cells grown under batch

culture conditions (0.2% w/v maltose).

Purification and comparison of excreted versus cell bound amylopullulanase
The scheme used for the purification of extracellular amylopullulanase is
given in Table 2. The purification of the cell-free enzyme from the culture
supernatant was simplified due to the higher specific activity of the starting
crude extract containing the pullulanase activity, and due to the approximately
12 fold higher yield of enzyme in the culture medium under maltose limited

chemostat culture. The final affinity chromatographic step resulted in purified

51

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54
enzyme with a specific activity of 480 U/ mg for pullulanase activity, and 175

U/ mg for oc-amylase activity, using soluble starch as the substrate. The purified
excreted enzyme gave a Mr of 140,000 on SDS-PAGE gels, and had a pI of 4.0 on
Serva Precote isoelectric focusing gels (pH 3-10) (Fig. 1). Native gel
electrophoresis on soluble starch or pullulan embedded gels showed that the
same protein band contained both a-amylase and pullulanase activities. The
molecular weight, temperature and pH optima for activity for the excreted
amylopullulanase were identical with that reported for cell bound enzyme (Saha

et al., 1988).

Amino acid composition

The amino acid composition of the purified enzyme is given in Table 3.
Analysis of amino acid composition showed that the enzyme is composed of 30%
hydrophobic amino acids, while the remainder consists of polar and acidic amino
acids, and 10% of basic amino acid residues. Comparison of the N-terminal
sequence of the initial seven amino acid residues in thermostable and
mesophilic pullulanses and (it-amylases of microbial origin are given in Fig. 2.
There were marked differences in the amino acid sequences of the N-terminal
end of these enzymes, with a-amylase and glucoamylase from Aspergillus oryzae

being the most similar.

Carbohydrate analysis

GLC analysis showed mannose and N-acetyl galactosamine as the major
carbohydrate components in amylopullulanase (Fig. 3). Glucose, rhamnose and
fucose were present in minor amounts. Based on GLC analysis,
amylopullulanase had a carbohydrate content of 9-10% (w/w). Recently,

galactose, rhamnose, mannose and glucose have been found to be the

Fig. 1A.

Fig. 1B.

Fig. 1C.

55

SDS-PAGE (7.5% cross-linked) of affinity purified
amylopullulanase.

lane 1 = high molecular weight standards

lane 2 = purified amylopullulanase

N ative-PAGE (7.5% cross-linked) of affinity purified
amylopullulanase.

lane 1,3 = Purified amylopullulanase after Coomassie
Blue staining

lane 2 = Staining for a-amylase activity, after overlay
of starch embedded gels with iodine.

lane 4 = Staining for pullulanase activity, after overlay
of pullulan embedded gels with Schiff’s Reagent.

Isoelectric focusing gel electrophoresis of purified
amylopullulanase (pH range 3—10).

lane 1 = Purified amylopullulanase

lane 2 = Isoelectric focusing protein standards
(Protein Test Mix 9 ; Serva).

56

3..

 

.Htx. ._ .

 

57

Table 3. Amino acid composition of amylopullulanase from
C. thermohydrosulfuricum 39E.

 

 

Amino Acid Molar Ratio (%)
Asp, Asn 15.1
Glu, Gln 10.3
Ser 8.6
Gly 10.7
His 1.4
Arg 3.6
Thr 7.7
Ala 7.2
Pro 5.9
Tyr 4.5
Val 7.5
Met 1.1
He 0.9
Leu 5.8
Phe 3.6
Lys 5.8
Trp not determined
Cys not detected

 

Total 100

 

58

398 6111 - Thr - Asp - Thr - Ala - Pro - Ala
Kaepula Asn - Lys - His - Ile - Arg - Asp - Tyr

Kpnpula Arg - Val - Tyr - Asn - Thr - Ser - Tyr

TAA Ala - Thr - Pro - Ala - Asp - Trp - Leu
Asagai Ala - Thr - Leu - Asp - Ser - Trp - Leu
Angglu Met - Ser - Phe - Arg - Ser - Leu - Leu
Figure 2. Comparison of N-terminal sequences of various amylases

of microbial origin. Abbreviations are: 39E, Clostridium
thermohydrosulfuricum 39E amylopullulanase; Kaepula,
Klebsiella aerogenes pullulanase; Kpnpula, Klebsiella

pneumoniae pullulanase; TAA, Aspergillus oryzae (it-amylase;
Asagai, Aspergillus oryzae glucoamylase; Angglu, Aspergillus
niger glucoamylase

59

 

 

§
:38: 5
i

  

Fig. 3. GLC Analysis of carbohydrate composition of amylopullulanase.

A = Monosaccharide standards
B = Monosaccharides released after acid hydrolysis of
amylopullulanase

Rha = Rhamnose; Fuc = Fucose; Xyl = Xylulose; Ara = Arabinose;
Gch = Glucosamine; GlcN Ac = N-acetyl glucosamine; GalNAc =
N-acetyl galactosamine; Gal = Galactose; Glc = Glucose; Man =
Mannose; GalA = Galactosamine; Gch = Glucosamine.

60
components of glycoproteins isolated from the outermost cell surface layer (S-

layer) of C. thermohydrosulfuricum (Messner et al., 1992).

Enzyme stability

The Ca2+ requirement for high thermal stability of amylopullulanase is
shown in Fig. 4. The purified pullulanase was thermostable up to 90°C, with a
half life of 40 min at that temperature. N 0 loss of activity was detected at 85°C for
a period up to 60 min in the presence of 5 mM CaClz. However, when the
enzyme was initially incubated at 60°C for 10 min with 25 mM EGTA or 25 mM
EDTA (pH 7.0) and then extensively dialyzed in the presence of the chelating
agent, the resultant enzyme solution rapidly lost activity at 85°C, with a half life
of <5 min, indicating the importance of Ca2+ in maintaining thermostability of
the enzyme. At 25°C, the enzyme was stable at pH 3.5 to 9.5 in the presence of 5
mM Ca2+. However, at 60°C, over the same period of time, the range narrowed

to pH 3.5 to 7.0.

Inhibition and substrate-product specificity

In order to identify any similarity between the catalytic site of C.
thermohydrosulfuricum 39E amylopullulanase and other characterized a-amylases
and pullulanases, the effect of B-cyclodextrin on activity was determined. Kinetic
analysis showed the ability of B-cyclodextrin to inhibit both pullulanase and a-
amylase activities completely, with a Ki of 0.065 mg/ ml (Fig. 5). This showed the
similarity of the active site of this enzyme to the active site of pullulanases and of
a-amylases, which are inhibited by B-cyclodextrin.

In order to determine the activity of the enzyme on various
polysaccharides, product formation on linear chain polysaccharides (i.e.,

amylose and pullulan) was compared to glycogen (Fig. 6). For glycogen, there

Figure 4.

61

Effect of temperature and Ca2+ on stability of C.
thermohydrosulfuricum 39E amylopullulanase in the
absence of starch. Determination of enzyme stability in the
absence of metal ions, was achieved by incubating samples
of the enzyme in the presence of 25 mM EGTA or EDTA
(pH 7.0) at 60°C, and desalted by ultrafiltration initially with
the chelating agent, and finally with distilled water. The
desalted enzyme (at a concentration of 0.05 mg / ml), was
used to determine the stability of the enzyme in the presence
and absence of Ca2+.

62

'ool‘l‘l'l'l‘l‘lfl‘rlfasoc

80

70-—~ .

or
O
l

a:
O
I

A
O
I

90°C

residual activity (%)

30—

20—

85°C, 25mMEDT 95.0
1 l l l l l I l

 

 

 

0 5 IO IS 2025 3055 4045 505560
minutes

Figure 5A.

Figure 5B.

63

Inhibition of pullulanase activity of C.
thermohydrosulfuricum amylopullulanase by [3-
cyclodextrin. Final pullulan concentrations of 0.4 (I),

0.8 (D ), 2.0 (O), and 4.0(.) mg/ ml were used, [3'
cyclodextrin concentrations were 0.05, 0.1, 0.15, and 0.2,

0.25 mg / ml. All assays were performed in 50 mM acetate
buffer, pH 6.0, containing 5 mM CaC12 at 60°C and samples

were taken at 15 minute intervals.

Inhibition of a-amylase activity of C.
thennohydrosulfuricum amylopullulanase by B-
cyclodextrin. Final amylose concentrations of 0.25 (I), 0.5
(D), 1.0 (O), and 2.0 (0) mg/ ml were used, B-cyclodextn'n
concentrations were 0.05, 0.1, 0.15, 0.2, 0.25 mg/ ml.

ilv (micro mol-i min ml)

mln ml)

tlv (mlcro mol-i

0.40 -

0.35 ~

0.30 't

0.25 -‘

0.20 '-

0.15-‘

0.10‘

0.05 *

 

0.00
-0.10

0.7 "‘

i

I

-0.05

64

Y

I

0.00

1

0.05

‘v’

I

0.10

['1 (mg/ml)

*1

I
0.15

v

I
0.20

fir

I
0.25

V

I
0.30

 

0.2
-O.10

 

V

I
'0.05

V

I
0.00

Y

I

0.05

1
0.10

I

0.15

I

0.20

V

65

Figure. 6. HPLC analysis of the product formation profile of
amylopullulanase on pullulan (A), glycogen (B), and
amylose (C). The products formed include maltose (V),
maltotriose (A), maltotetraose (D).

 

 

 

 

 

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67
was a significant lag period of approximately 24 h, before rapid and efficient

hydrolysis of the remaining polysaccharides occurred. This was not observed
with pullulan or amylose. Glycogen and amylose were hydrolyzed to maltose,
maltotriose and maltotetraose, whereas pullulan hydrolysis yielded only
maltotriose.

Thin layer chromatographic analysis identified what hydrolysis products
were obtained upon action of amylopullulanase on low molecular weight
oligosaccharides containing (at-1,4 linkages (Fig. 7). Maltoheptaose was
hydrolyzed to maltotriose and glucose, with a small amount of maltose.
Maltohexaose was cleaved to maltotriose. Maltose and maltotriose were the
products after hydrolysis of maltopentaose and maltotetraose was cleaved to

maltotriose and glucose. There was no enzyme activity against maltotriose.

Active site titration and chemical modification

In order to determine the amino acids involved in activity, an active site
titration of amylopullulanase, and chemical modification with group specific
reagents, were performed. The active site titration curve showed inflection points
at pH values of 3.9 and 6.1, which are approximate pKa values for aspartate or
glutamate residues, and for histidine, respectively. However, chemical
modification of histidines in amylopullulanase using DEP, which was monitored
spectrophotometrically, did not cause a loss in activity (data not shown), while
aspartate/ glutamate modification with DEAC resulted in loss of activity (Fig. 8).
Kinetic analysis of the rate of loss of activity with respect to the concentration of
DEAC showed the involvement of at least a single acidic amino acid residue in

catalysis.

0009

Figure 7.

68

 

Thin layer chromatographic analysis of product
hydrolysates upon action by amylopullulanase from C.
themohydrosulfuricum 39E on oligosaccharides.

1.0 ml aliquotes of 1.0% (w/ v) maltotriose (lane 2),
maltotetraose (lane 3), maltopentaose (lane 4), maltohexaose
(lane 5), and maltoheptaose (lane 6) were incubated with the
affinity purified enzyme (0.05 U) at 60°C for 72 hours. lanes
1,9 = standards (a=glucose; b=maltose; c=maltotriose;
d=maltotetraose)

69

 

 

 

 

 

 

 

 

 

A
2.00 ~——-
<3 . .
. . . , .
t-I A .
’0‘ ' I ° .
3*
v . . o
a - .
.2 ' . °
‘5 1 90— '
a .
... A .
a l'
a
E " I '
m k
0 ..
..‘:.
g) " A
"' 1.80-
1L I I J 1 ' l
o 15 30 45 60 75 90
min.
8
IE 1.4:
E 1.0—
5 -
o (3'er
i no ‘
8’ "' .
" log [[DEAC] (mM)]

Figure 0 Chemical modification of aspartate and glutamate residues
of amylopullulanase from C. thermohydrosulfuricum 39E
using 1-(3odimethylaminopropyl)-3oethyl carbodiimide.

A : Plot of Log [residual activity (%)] vs time at different
concentrations of DEAC; 0 (V) , 25 (O), 50 (O), 75 (D), 100 &)
mM.

B : Plot of rate of loss of activity vs log of DEAC
concentration (Eyzaguirre, 1987)
log Robs = n log [DEAC] + log k1

DISCUSSION

This is the first report on the use of maltose-limited chemostat conditions
for overexpression and secretion of amylopullulanase activity in
thermoanaerobes. Prior studies on continuous culture using substrate limitation
had centered on the use of starch for amylase overexpression (Antranikian et
al.,1987; Madi et al.,1987; Koch et al.,1987), where optimal conditions were found
to be 0.4% (w/v) starch, with the dilution rate being 0.05 h’l. With C.
thermohydrosulfuricum 39E grown under maltose limitation, it was possible to
obtain optimal overexpression of enzyme in 0.2% (w/v) maltose, at a dilution
rate of 0.03 h'l. The findings in this study of release of‘ cell-bound
amylopullulanase into the culture medium during maltose limited continuous
culture contrasted with the effect of the same conditions on the gram negative
organism Klebsiella aerogenes, where extracellular pullulanase became cell-bound
(Hope and Dean, 1984).

The recovery of enzyme activity from C. thermohydrosulfuricum 39E was
improved by using cell-free supernatant from maltose limited continuous
culture, since no residual starch related material was present in the starting
crude extract, which would tend to lower the efficiency of purification during
the column chromatography steps.

In all the purification steps, where the chromatographic fractions were
monitored with pullulan (for pullulanase activity) and with soluble starch (for 0:-
amylase activity), both activities responded almost identically to all parameters
tested, suggesting that both activities are the function of a single, novel

thermostable saccharidase.

7O

71
The purified secreted amylopullulanase had a similar molecular weight,

p1, and activity as the purified cell-bound enzyme from C. thermohydrosulfuricum -
39E (Saha et (21,1988).

The purified enzyme was able to hydrolyze a-1,6 branched and (at-1,4
unbranched polysaccharides. It lacked the ability to hydrolyze starch into
glucose, a characteristic of glucoamylase; although it was capable of producing
maltose, maltotriose and maltotetraose from starch, an activity typical of a-
amylase, and it produced maltotriose from pullulan, a characteristic of
pullulanase.

Similar enzymes with dual activties have been isolated from several
anaerobic thermophilic bacteria including C. thermohydrosulfuricum E101
(Melasniemi, 1988) where the enzyme is a dimer with 190,000 subunit molecular
weight. The enzyme was reported to contain 11-12% (w/w) carbohydrate
content, containing rhamnose, mannose, galactose, and glucose. A monomeric
pullulanase with dual activties isolated from C. thermosulfurogenes (Spreinat and
Antranikian, 1990) had a molecular weight of 102,000, with a higher affinity for
amylose than for pullulan. Amylase-pullulanase of Bacillus circulans F-2 (Sata et
al., 1989) is a monomer of 220,000 molecular weight and had similar affinity
toward both amylose and pullulan.

Inhibition of both a-amylase and pullulanse activities showed the
similarity of the substrate binding sites in amylopullulanase to those of known
pullulanase and (it-amylase (Marshall, 1973).

i The results from chemical modification studies showed that aspartates or
glutamates are involved in catalysis and the possibility exists for the second
titration point at pKa 6.1 to be due to another aspartate or glutamate in a
hydrophobic environment in the catalytic center, similar to the pKa value

observed for Glu:B of lysozyme in it's catalytic center (Walsh, 1979).

72
Although kinetic analysis of modification of amylopullulanase by DEAC

showed the involvement of only a single acidic amino acid in catalysis,
involvement of more than one acidic residue or histidine, in catalysis or substrate
binding cannot be ruled out, since the thermostable amylopullulanase may be
folded in to a rigid structure at 25°C, used during the chemical modification
studies. This may effectively make the residues in catalysis or substrate binding
inaccessible to the modifying reagents.

Further studies are required to identify whether both pullulanase and (X.-
amylase dual activities reside in the same enzyme, and to identify whether both

activities are located at the same active center.

LIST OF REFERENCES

Allen, W.G., and Dawson, H.G. 1975. Technology and uses of debranching
enzymes. Food Technol. 29 : 71-76

Antranikian, G., Zablowski, P., and Gottschalk, G. 1987a. Conditions for the
overproduction and excretion of thermostable (it-amylase and pullulanase from
Clostridium thermohydrosulfuricum DSM 567. 27 : 75-81

Antranikian, G., Herzberg, C., and Gottschalk, G. 1987b. Production of
thermostable a-amylase, pullulanase, and a-glucosidase in continuous culture by
a new Clostridium isolate. 53: 1668-1673

Bender, H. , and Wallenfels, K. 1961. Untersuchungen an pullulanase. 11.

Specifisher abbau durch ein bakterielles enzyme. Biochem. Z. 334 : 79-97

Brock, TD. 1986. in Thermophiles: General, Molecular and Applied
Microbiology (Brock, T.D. Ed.) John Wiley and Sons, New York, NY. pp. 1-16

Carraway, K.L., and Koshland, DE. 1972. Carbidiimide modification of proteins
in Methods Enzymology. 25: 616-623

Chaplin, M.F., and Kennedy, J.F. 1986. in Carbohydrate Analysis: a practical
approach. IRL Press, Washington, DC. pp. 68-70.

Hope, G.C., and Dean, A.C.R. 1974. Pullulanase synthesis in Klebsiella
(Aerobacter) aerogenes strains growing in continuous culture. Biochem. J. 144 :
403-411

Hyun, H.H. and Zeikus, J.G. 1985a. General biochemical characterization of

thermostable pullulanase and glucoamylase from Clostridium
thermohydrosulfuricum . Appl. Environ. Microbiol. 49 : 1168-1173

73

74
Hyun, H.H. and Zeikus, J.G. 1985b. Regulation and genetic enhancement of
glucoamylase and pullulanase production in Clostridium thermohydrosulfuricum .
J. Bacteriol.164: 1146-1152

Janson, J.-C. and Ryden, L. 1989. in Protein Purification: Principles, High
Resolution methods, and Applications (Ed. Janson, J.-C. and Ryden, L) VCH
Publishers, New York, NY.

Knapp, DR. 1979. in Handbook of Analytical Derivatization Reactions. John
Wiley and Sons, New York, NY. pp. 545-546.

Koch, R., Zablowski, P., and Antranikian, G. 1987. Highly active and
thermostable amylases and pullulanases from various anaerobic thermophiles.

Appl. Microbiol. Biotechnol. 27 : 192-198

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

Madi, E., Antranikian, G., Ohmiya, K., Gottschalk, G. 1987. Thermostable
amylolytic enzymes from a new Clostridium isolate. Appl. Environ. Microbiol.
53 : 1661-1667

Marshall, L]. 1973. Inhibition of pullulanase by schardinger dextrins. FEBS Lett.
37 : 269-273

Melasniemi, H. 1988. Purification and some properties of the extracellular 0L-
amylase-pullulanase produced by Clostridium thermohydrosulfuricum. 250 : 813-
818.

75 .
Messner, P., Christian, R., Kolbe, J., Schulz, G., and Sleytr, U.B. 1992. Analysis

of a novel linkage unit of O-linked carbohydrates from the crystalline surface
layer glycoprotein of Clostridium thermohydrosulfuricum S102-70. J. Bacteriol. 174 :
2236-2240.

Miles, B.W. 1977. Modification of histidyl residues in proteins by
diethylpyrocarbonate. in Methods. Enzymology 47 : 431-442

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

Norman, B.E. 1982. A novel debranching enzyme for application in the glucose
syrup industry. Starch, 10 : 340-346 '

Porath, J., and Fornstedt, N. 1970. Group fractionation of plasma prateins on
dipolar ion exchangers. J. Chromatography, 51 : 479-489

Saha, B.C., Mathupala, S.P., and Zeikus, J.G. 1988. Purification and
characterization of a highly thermostable novel pullulanase from Clostridium
thermohydrosulfuricum . Biochem. J. 252 : 343-348

Sata, H., Umeda, M., Kim, C.-H., Taniguchi, H., and Maruyama, Y. 1989.
Amylase-pullulanase enzyme produced by B. circulans F-2. 991 : 388-394.

Spreinat, A. and Antranikian, G. 1990. Purification and properties of a
thermostable pullulanase from Clostridium thermosulfurogenes EM1 which
hydrolyses both (at-1,6 and a-1,4-glycosidic linkages. 33 : 511-518.

Sundberg, L., and Porath, J. 1974. Preparation of adsorbants for biospecific
affinity chromatography. J. Chromatography. 40 : 87-98

Walsh, C. 1979. in Enzymatic Reaction Mechanisms. W.H. Freeman and Co., San
Francisco, CA

76
Whelan, W.J. 1971. Enzymic exploration of the structures of starch and
glycogen. Biochem. J. 122 : 609-622

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

CHAPTER 3
SUBSTRATE COMPETITION AND SPECIFICITY AT THE ACTIVE
SITE OF AMYLOPULLULANASE FROM Clostridium

thermohydrosulfuricum.

(BIOCHEM. BIOPHYS. RES. COMMUN. (1990)l66 [1], pp.126-l32)

77

78

SUBSTRATE mMPETITION AND SPECIFICITY AT THE ACTIVE SITE OF
AMYIDPULLULANASE FROM CLOSTRIDIUM THERMOHYDROSUU" URIC UM

Samj Mathupala‘. Badal c. sm’. and .1. Gregory Racism-3

Department of Biochemistry‘ and Department of Microbiology and Public Health’.
Michigan State University. East Lansing. MI 48824
Michigan Biotechnology Instinlte’. Lansing. MI 48909

Received October 19. 1989

 

A highly thermostable pullulanase purified from Clasuidiwn dremrohydrosulfim‘cwn strain 39B
displayed dual activity with respect to glycosidic bond cleavage. The enzyme cleaved a-1.6
bonds in pullulan. while it showed a-l.4 activity against malto-oligosaccharides. Kinetic
analysis of the purified enzyme in a system which contained both pullulan and amylase as. the
two competing substrates were used to distinguish the dual specificity of the enzyme from the
single substrate specificity known for pullulanases and a-amylases. o 1990 mo...“ m... Inc.

 

Clostfldium (hemlohydrosulfilricwn strain 39B produces a highly thermostable pullulanase
activitywhengrownonstarcha). Thisenzymewashomogeoeouslypurifiedandpartially
characterized in terms of MW. pl, pH stability, and thermal stability (2).

Pullulanase (pullulan 6-glucanohydrolase, EC 3.2.1.41) is a debranching enzyme that
specifically cleaves 0.4.6 links in starch. amlepectin. pullulan and related oligosaccharides
(3). while ct-amylases (1.+arD-glucan glucanohydrolase. EC 3.2.1.1) hydrolyse the a-l.4
linkages (4). Pullulanases do not show activity against linear (a-l,4-linked) oligosaccharides.
and a-amylases show no activity against pullulan.

The novel pullulanase of C. themahydrosulfiuicum strain 39B described in this
communication showed a-l.4 as well as a-1,6 cleavage activity against amylose and pullulan.
respectively. Detailed ldnetic studies with the homogeneously purified enzyme were performed
with pullulan. amylose, and linear low MW oligosaccharides in order to biochemically evaluate
theactivesiteandtopmposeanameforthisnewlyrecogniaedenzymeactivity.

METHODS

m. AllchemicalsusedwereobtainedfrcmeitherSigmaOremicalCompany (St. Louis,
MO) or Aldrich Chemical Company (Milwaukee. WI).

W Clomidiwn Wmosulfiuicum min 3915 (ATCC
33223)wasusedasthesomce0fpullulanase. Theorganismwasgrownat60°Cunder

79

anaerobic conditions in TYE medium (5) with 1% (w/v) soluble starch as the substrate.
Isolation and purification of the enzyme was performed as described previously (2).

W. 160 microliters of pullulan solution (1.25% w/v in 50 mM NaOAc buffer. pH
6.0. containing 5 mM CaCl,) and 40 microliters of enzyme solution were mixed and incubated
at 60°C for 30 min. The reaction was slapped by adding 0.8 ml of DNS solution (6) and
heated in a boiling water bath for 15 min. The samples were cooled in ice and the
absorbance of the reaction solution was measured at 640 nm. One unit of pullulanase activity
is defined as the amount of enzyme which produced 1 mieromole of reducing sugar (with
glucose as standard) per minute under the above assay conditions.

' ’nhi i ' ki ‘ 'v enzyme recognizes both
pullulan and amylase as substrates. It cleaves essentially the a-1.6 bonds in pullulan, while
in amylase, the a-l .4 bonds are cleaved. In this experiment bath substrates were present as
mixed alternative substrates (as outlined by Segel [7]). and was used to investigate the
following possibilities:

I. Ifboth activitiesaredue toanenzymecomplex ofapullulanaseand an (It-amylase.
orifbothactivitiesaccurredwithinthesameenzyme. butattwoindividual activecenters non-
interacting with each other. then the initial velocity obtained for product formation with both
substrates present should be approximately the sum of the individual initial velocities when

either of the substrates is present.

 

 

 

v = v, + vA
V... [A] V.. [P]
v = +
Km,+ [A] Km.+ [P]

where [A] - amylase concentration
[P] = pullulan concentration
V. maximum velocity for each substrate
v, initial velocity
Km. apparent Michaelis constant for each substrate

2. Ifbothactivitiesareduetoasingleenzymehavingasingleactivecenter.ortwa
active sites negatively interacting with each other, then the initial velocity obtained with both
substrates present will be less then the sum of the individual velocities.

v < v, + VA
VA. [A] V'.[P]

V'-'-' +

Kma (l+[P]/Km,)+{A] Kmr(1+[Al/Kmr)+lPl

 

RESULTS AND DISCUSSION

Incubation of the enzyme with low MW oligosaccharides, (maltotetraose to
maltoheptaose) resulted in the substrates being degraded into units of maltotriose and residual
sugars (i.e., glucose or maltose). depending on the parent aligosaccharide subjected to
enzymatic action (Table 1), demonstrating that the enzyme had no activity against maltotriose.
Maltotetraosewasapoarsubstratefortheenzymeandmaltosewasnatdetectedasits
hydrolysis product. These results reflect an important deviation from known a-amylase activity
and pullulanase activity, as maltotriose, as well as other oligosaccharides. would have been

80

IABLE 1. Reaction products of amylopullulanase from C. rhermohydrosulfun‘cwn strain 39B
on low MW oligasaccharides'

 

 

 

Substrate End Products
G, G3 G, G. G, G.
Maltotriose - -- ++ -- - -
Maltotetraoseb ++ - ++ ++ - --
MaltOpentaose - H- ++ -- .. --
. Maltohexaose - -- ++ -- - --
Maltoheptaose -H- - H -- - --

 

Major products observed are shown by the positive sign.

‘Salutions of 1% (va) mltotriose. maltotetraose. maltopentaose. maltohexaose. and
maltoheptaose were incubated at 60°C with purified enzyme (0.05 U/ml). Products were
analyzed after 72 hours by HPTLC (Whannan HP-K). Plates were developed with n-
BuOH:EtOH:H,O (3:2:2. v/v) at 25°C and the products detected with a mixture of 0.2% (w/v)
orcinol in MeOH and 20% (vlv) 11,80. in MeOH (1:1. v/v).

hMaltoteuaoseisaverypoarsubsuatefordleenzymeandthepraducts wereobservedonly
after long-term reaction.

finally degraded into glucose and maltose by saccharifying a-amylase. and no activity would
have been shown towards any of these cr-l,4-linked oligosaccharides by pullulanase.

The enzyme from C. themohydrosulfirricum strain 39E showed broad range substrate
specificity with regard to high MW polysaccharides. The final reaction producrs obtained were
maltose. maltotriose, and maltotetraose (Table 2). The only product obtained upon incubation
of the enzyme with pullulan was maltotriose, demonstrating that the enzyme shows typical
pullulanase activity.

The present data indicate that the pure enzyme has both "(z-amylase" and ”pullulanase-
like” activity. and its own unique mode of action. In general, these data show that the mode
of action of the enzyme from C. themrohydrosulfirricwn strain 39E is different from that

1mg. Reaction products of amylopullulanase from C. drenuolrydrosulfiuicwn strain 39B

 

 

 

 

on high MW polysaccharides'

Substrate W
G! G! G! on G‘ G,
Pullulan - - 100 -- - --
Amylase - 31 47 16 - -
Amylopectin - 36 36 28 - ~-
Soluble starch - 39 39 22 - -
Mammalian glycogen - 22 47 31 - --
Oyster glycogen - 17 50 33 - -

 

‘Solutians of 1% (w/v) Pullulan. amylose, amylopectin, mammalian glycogen. oyster glycogen
and soluble starch (pH 6.0) were incubated at 60°C with ptu'ified enzyme (0.05 Ulml).
SampleswerewithdrawnafwrZIGhrsandheatedat100°Cfor15minfarenzyme
inactivation. Thereactianproductswereanalyzedbymfarsugars (2).

81

 

   

Pullulan

   

mg ml"
( )Iosx E '5 "'
v

u min"

Amylase

  

 

 

 

rflrrrierrrrrrJ

-l.0 -.O -.6 —.4 -.2 O .2 .4 .6 .8 LO l.2 IA LG LG 2.0 2.2 2.4 2.6

 

S(mg ml")

F‘g. l.Km'°pdetermination. mum (I) and low MW amylase (u) at the concentrations
indicatedwereincubatedwithpurif'redenzymeatWC.

previously reported for pullulanase and a—amylase, in terms of bond cleavage specificity as
well as product formation.

The Km'" for pullulan (average MW 50.000) and low MW amylase (MW 4.100) wae
obtained at substrate concentrations between 0.4 mg/ml to 2.4 mg/ml at 0.4 mglml increments
(Fig. 1). The dependence of the rate of pullulan and amylase hydrolysis on the substrate
concentration followed Michaelis-Menteu kinetics. The apparent Km for pullulan (average
MW 50.000) and amylase (MW 4.100) as determined from the limes-Woolf plot were 0.35
mg/ml and 1.00 mg/ml. respectively (Fig. l). The apparent It. for pullulan was 16,000
min".

Kinetic experiments on competitive inhibition with mixed alternative substrates were
per'formedatpullulancoucentrationsofo.4t02.4tnglmlat0.8mg/mlincrements.while
amylaseconaenuadonswerevafiedfiomo.6to3.0mglmlat0.6mglmlinaements. Initial
vebddeswaedewrminedfaeachcambinafimofpuuulmandamyloseuthedifi'aem
concentrations. Iheinidalvelocitywasplouedagainstthetotalsubsuateconcenuatianina
SNvasusSpkx(whemSisthewmlsubsuateconceuuadon).andisshowninFig.2. For
clarity.anlytwosetsofdatawithamyloseat0.4mg/mland2.4mglmlwithvaryingpullulan
cancentrationsareshown. 'I'heinitialvelacitiesobtainedcloselyfallowedthetheareticalplot
forcase2(asdescribedinMethods),wheretheobservedvelocitywasleesthanthecumulative
valueoftheindividualinitialvelocidesobtainedfarthetwosubsuates. Thisdemanstrates
competition between the two substrates used. indicating that the enzyme possesses an active

82

 

2.0

Ert l03

 

0.8

(M)

0 rain" 9 °
0.4 _

0.2 -

 

 

 

7 o 2.0 3.0 4.0 5.0 ca 7.0
[S] (mg ml")

F‘g. 2Kinetics of competitive inhibition with mixed substrates. The solid lines A and C
indicate the theoretical plots for competitive inhibition at amylase concentrations of 0.6
and 2.4 mglml. respectively. Lines 8 and D are the theoretical plots for absence of
inhibition at the same respecdve amylase concentrations. Pullulan was used at
canwntrations of 0.4. 0.8. 1.2. 1.6. 2.0. 2.4 mg/mL For clarity. only two sets of data
points were used in the above plot. (I) and (A) are the practical data points obtained
at 0.6 and 2.4 mg/ml amylase concentrations. All reaction mixtures contained 5% (vlv)

dimethyl sulfoxide for solubility of amylase.
[S] = [A] + [P]. where S is the total substrate concentration. A and P are the

concentrations of amylase and pullulan. respectively.

site for cleavage of both a-l,6 and a-l,4-linlted substrates. This apparent competition between
two substrates has been used by several authors as proof of a given enzyme having a single
active center with two different modes of activity (8-12). However, as the same result can
occur under certain conditions for an enzyme with two active centers (8). the results obtained
for the enzyme from C. rhemrohydrosulfiu'icrun strain 395 can only be used as positive proof
for both activities belonging to the same enzyme.

The fust description of pullulanase activity was an enzyme from Klebsiella pneumonia:
(13). which specifically hydrolyses a-1,6 glycosidic linkages of pullulan to yield maltotriose.
Other pullulan degrading enzymes (e.g.. neopullulanase) which specifically cleave a-1.4
glycosidic bonds in pullulan to yield panose (l4) and isopanose (15) have been reported.

83

This is the first detailed kinetic and substrate hydrolysis studies reported on novel
pullulanases that cleave a—l.6 and a-l,4 bonds. Pullulanases possessing both at-l.6 and tit-1.4
activity have been isolated from Themoanaerobt‘wn Tolt 6-Bl (16. 17). T. brocla'r' (18).
00st themrosulfitrogenes (l9), and C. thermohydrosulfwicwn strain 8101-69 (20. 21).
The enzyme from T. brocldr‘ was reported to hydrolyze starch into various sized oligomers.
while the Themroanaerqbiwn strain Tok 6-Bl enzyme acted upon starch. amylopectin and
amylase to yield predominantly maltose and maltotriose. Maltotetraose was completely
hydrolysed to maltose by Themroanaerobiwn Tolr 6-Bl enzyme (16). and the enzyme was
found to hydrolyse low MW oligosaccharides at two glucose residues away from a terminal.
and release maltose as the product (17). The enzyme isolated from C. themrahydrosumrn’cum
strainBlOl-69wasintwofarmofMWoverSOOflOQandthepossibilityofthepresenceof
a third farm was suggested (21). Each farm was capable of hydrolysing both pullulan and
amylase but oligasaccharide hydrolysis studies were not reported. In contrast. the pullulanase
described in this paper was of monomeric form with a MW of 136.500. -

In comparison to the enzyme from Thennoanaerobr’wn Tolt 6-Bl. ”c. thermo-
hydrosulfiuicum strain 39B pullulanase produced maltose, maltotriose. and maltotetraose upon
reaction with high MW polysaccharides. Also, it hydrolysed low MW oligosaccharides three
glucose units away from a terminal. released maltotriose as the product. and did not form
maltose from maltotetraose. These results represent significant differences between the
pullulanases from Themroanaerobt‘um Tolt 6-81 and C. rhemrokydrosulfim‘cum strain 395.

Kinetic studies of the homogeneous enzyme from C. themrohydrosulfitrt’cum strain 39B
on low MW amylose and pullulan as competing substrates, gave further proof that both
activities resided on the same enzyme and putatively within the same active site. Therefore.
the enzyme described here as amylopullulanase higher affinity and activity toward pullulan
than amylose; and. it shows unique characteristics with regard to substrate utilization as well
as product formation. In light of these results. it is now necessary to distinguish pullulanases
which possess only at-l,6 cleavage activity from amylopullulanases that contain both a—l,6 and
a-l,4 cleavage activity. Thus. we propose the enzyme commission use the term
amylopullulanase to describe those enzymes which act on starch and cleave both a-l.6 bonds
in pullulan and a-l,4 bonds in amylase.

W We drank Dr. Susan Lowe for her help in preparing the manuscript. useful
discussion. advice and encouragement.

REFERENCES

Hyun. H. H., and Zeikus. J. G. (1985) Appl. Environ. Microbiol. 49. 1168-1173.
Saha. B. C., Mathupala. S. P., and Zeikus. J. G. (1988) Biochem. J. 242. 343-348.
Abdullah. M., Catley, B. J., Lee. E. Y. C.. Robyt. J., Wallenfels, K., and Whelan, W.
J. (1966) Cereal Chem. 43. 111-118.

9’2"!"

84

Thoma, .l. A., Spradlin. J. B., and Dygert. S. (1971) In The Enzymes (P. D. Boyer. Ed.).
Vol. V. pp. 115-189. Academic Press. New York.

Ng, T. K., Ben-Bassat. A., and Zeikus. I. G. (1981) Appl. Environ. Microbiol. 41. 1337-
1343.

Miller. G. L. (1959) Anal. Biochem. 31. 426-428.

Segel.1. H. (1975) In Enzyme Kinetics. pp. 113-118. John Wiley and Sons. New York.
Hiromi, K., Hamazu. Z. Takahashi, K., andOno. S. (1966) J. Biochem. 59. 411-418.
Sakano, Y., Hiraiwa. 8.. Fukushima. J., and Kobayashi, T. (1982) Agric. Biol. Chem.
46. 1121-1129.

Guranowski. A., and Schneider. Z. (1977) Biochim. BiOphys. Acta 482. 145-158.
Walker. D. B., and Axelrod. B. (1978) Arch. Biochem. Biophys. 187. 102-107.

Plant. A. R., Clemens. R. M., Morgan. Ii. W., and Daniel. R. M. (1987) Biochem. J.
246. 537-541.

Bender. H., and Wallenfels, K. (1961) Biochem. Z. 334. 79-97.

Kuriki, T., Okada. 8.. and Imanaka, T. (1988) 1. Bacteriol. 170. 1554-1559.

Sakano, Y., Matsuda. N., and Kobayashi, T. (1971) Agric. Biol. Chem. 35. 971-973.
Plant. A. R., Clemens. R. M., Daniel. R. M., and Morgan. H. W. (1987) Appl.
MicrobioL Biotechnol. 26. 427—433.

Plant. A. R., Clemens. R. M., Morgan. H. W., and Daniel. R. M. (1987) Biochem. J.
246. 537-541.

Coleman, R. D., Yang. S. 8.. and McAllister. M. P. (1987) .1. Bacteriol. 169. 4302-
4307. "
Madi, F... Antranikian, G., Ohmiya, K., and Gottschalk, G. (1987) Appl. Environ.
MicrobioL 53. 1661-1667.

Melasniemi, H. (1987) Biochem. J. 246. 193-197.

Melasniemi, H. (1988) Biochem. J. 250. 813-818.

CHAPTER 4

CLONING, IDENTIFICATION OF THERMOSTABILITY AND CATALYTIC
REGIONS; AND, CHARACTERIZATION OF a-1,4 AND (14,6 BOND
SPECIFICITY OF AMYLOPULLULANASE FROM Clostridium

thermohydrosulfuricum 39E.

85

ABSTRACT

A gene (apu), encoding amylopullulanase, which exhibits both a-1,4
cleaving amylase and tat-1,6 cleaving pullulanase activities, was cloned from
the thermophilic anaerobe Clostridium thermohydrosulfuricum strain 39B
and expressed in both Escherichia coli and Bacillus subtilis. In both host
strains the cloned enzyme was expressed constitutively and was located
intracellularly or within the periplasm of E. coli cells, and secreted by B.
subtilis. The recombinant amylopullulanase in E. coli displayed a molecular
weight of 160,000, a half-life of 40 min at 90°C, and hydrolyzed glycogen,
soluble starch, amylopectin, and amylose to maltose, maltbtriose, and
maltotetraose. The 6.] kbp DNA insert containing the apu gene was restricted
to 2.9 kbp resulting in a Mr 100,000 expressed protein, which did not show
any quantitative loss of activity or thermostability. Nested deletion mutants
from both 3’ and 5’ ends of the 2.9 kbp DNA fragment were prepared that lost
thermostability but retained activity. The Mr 100,000 protein was used to
prove by 13C NMR analysis the ability of the enzyme to cleave a-1,6 bonds
and at-l,4 bonds in glycogen.

86

INTRODUCTION

(at—Amylases are endoglucanases which randomly hydrolyze the a-1,4
linkages found in amylose, amylopectin, glycogen, and related
polysaccharides. Pullulanase, also known as a debranching enzyme, is capable
of attacking specifically the (at-1,6 linkages found in the linear polysaccharide
pullulan, and in other branched polysaccharides such as glycogen and
amylopectin.

Amylopullulanase of Clostridium thermohydrosulfuricum 39E (Saha
et al., 1988; Mathupala et al., 1990), a-amylase-pullulanase of C.
thermohydrosulfuricum E101-69 (Melasniemi, 1988), pullulanase of
Thermoanaerobium Tok6-Bl (Plant at al., 1987), debranching enzyme of T.
brockii (Coleman et al., 1987), and amylase-pullulanase of B. circulans F-2
(Sata et al., 1989) are thermophilic enzymes exhibiting a—amylase activity
against a-l,4 bonds in amylose and related polysaccharides as well as
pullulanase activity against a—1,6 bonds in pullulan. These proteins may
represent a new class of enzymes capable of cleaving both (IL-1,4 and (at-1,6 bond
in glycogen and starch, but this remains to be proven.

C. thermohydrosulfuricum 39E (ATCC 33223), a thermophilic,
obligately anaerobic bacterium isolated from OctOpus Spring in Yellowstone
National Park (Zeikus et al., 1980) can ferment a wide variety of starch related
polysaccharides, including amylose, amylopectin, glycogen, and pullulan
(Hyun and Zeikus, 1985). We have identified and characterized a specific
amylase from this organism (Saha et al., 1988; Mathupala et al., 1990),
tentatively named amylopullulanase, because of it's hydrolytic activity
against all the above mentioned polysaccharides. Our previous study had

centered on the biochemical characterization of this amylopullulanase to

87

8 8
demonstrate that the dual activities are due to the action of a single enzyme

(Mathupala et al., 1990).

The purpose of this study was to further substantiate these findings by
cloning, expressing, and characterizing the apu gene, followed by biochemical
characterization of the recombinant enzymes to demonstrate that the a—
amylase and pullulanase activities are encoded by a single gene, and to test
the ability of the enzyme to cleave both (It-1,6 and a-1,4 linkages in glycogen,
to identify thermostability regions of the gene and, to demonstrate that the

active site of this enzyme is encoded by a single region within the gene.

MATERIALS AND METHODS

Reagents
All chemicals were of molecular biology or analytical grade and were
obtained from Aldrich Chemical Co. (Milwaukee, WI), or Sigma Chemical Co
(St. Louis, MO).

Bacterial strains, plasmids, and transformation

C. thermohydrosulfuricum 39E (ATCC 33223) was grown anaerobically
at 60°C in TYE medium (Zeikus et al., 1980) containing 0.5% (w/v) glucose,
and was used for chromosomal DNA isolation. The bacterial strains used as
the recipient hosts for the recombinant plasmids are listed in Table 1. Plasmid
pUC 18 (Yanisch-Perron et al., 1985), was used as the initial cloning vector.
pUC 19 (Yanisch-Perron et al., 1985) and pUB 110 (Gryczan et al., 1978) were
used in subsequent subcloning experiments (Fig. 1). Plasmid pCPC 902,
containing a chromosomal DNA insert encoding for the debranching
enzyme of T. brockii (Coleman et al., 1987) was obtained from the American
Type Culture Collection (ATCC 53114) and was used in Southern
hybridization experiments to isolate and identify the chromosomal DNA
fragment of C. thermohydrosulfuricum 39E expressing amylopullulanase
activity. Escherichia coli strains used in DNA manipulations were made
competent by the Hanahan method as described by Perbal (1988) while
recombinant vectors were introduced into E. coli strains by heat-shock
treatment (Hanahan, 1983). Bacillus subtilis was transformed by PEG
mediated protoplast transformation (Chang and Cohen, 1979; Puyet et al.,
1987).

89

90

TABLE 1. E. coli and B. subtilis strains used in cloning and subcloning

experiments.

 

Strain Genotype Source or Reference

 

F'mch m" supE44 hsd520(rb' Bethesda Research Labs.
mb') recA13 ara14 proA2 lach
galKZ rpsL20(Sm5xy151-leu mtll

E. coli H8101

F'¢80dlacZAM15 A(lacZYA-
argF)U169 deoR recA1 endAl
hst17(rI<'mI<*) supE441'thi-1
gyrA96 relAl

E. coli DHSa Bethesda Research Labs.

supE hsdDS thi A(lac-proAB) F

Amersham International.
(traA36 proAB‘lachlacZAMIS)

E. coli TG-l

e14-(mcrA) A(mchB-hstMR-
mrr)171 endAI supE44thi-1
gyrA96 relAI lac recB rec] sbcC

umuC:Tn5(kan1) uvrC [F' proAB
lachZAMIS Tn10(tet')]

E. coli SURE Strategene Co.

arg-IS hst hde Amprr'

B. subtilis NA-I N akajima et al., 1985

 

Figure 1.

91

Plasmids used in cloning of amylopullulanase (apu)

gene from C. thermohydrosulfuricum 39E. Plasmid pUC 18
was used as the initial cloning vector (A), and pUB 110 was
used for subcloning into B. subtilis NA-I (B). Phagemid
vector pUC 119 (C) was used for sequencing.

92

 

 
        
    

259nm
252
M2
sanctum“: \' [M];
mm!
Q“ R
“(QM Phe
4mm

pucua I pucus
. (32 kb)

9 3
Growth media and preparation of cell extracts

E. coli and B. subtilis were grown at 37°C in modified LB broth
containing tryptone (10g/ liter), yeast extract (Sg/ liter), and NaCl (Sg/ liter)
without pH adjustment. The medium was supplemented with ampicillin or
kanamycin at a final concentration of 50 ug/ ml, for appropriate E. coli or B.
subtilis strains carrying a vector or a recombinant plasmid. For agar plates,
1.5% (w/v) Bacto-Agar (Difco Laboratories, Detroit, MI) was added. For soft
agar overlay, 0.7% (w/v) agar was used. For preparation of cell extracts,
cultures were grown to late exponential phase and harvested by
centrifugation at 8,000 x g for 10 min and washed twice with 50 mM acetate
buffer (pH 6.0) containing 5 mM CaClz. The periplasmic enzyme fraction was
isolated by osmotic-shock treatment of the cells (Willis, 1984; Neu and
Heppel, 1965). The intracellular enzyme fraction was isolated either by passing
the cells through a French-pressure cell at 20,000 lb/in2 (American Instrument
Co., Inc., Silver Spring, MD) for large scale enzyme isolation, or for small
volumes, cell samples (1.0 ml) were sonicated using a microsonicator for 5 s
(Kontes, Vineland, NJ). The disrupted cells were centrifuged and the

supernatant used as the source of amylopullulanase.

Enzyme assays

For determination of pullulanase activity, 160 pl of 1.25% (w/v)
pullulan (for pullulanase activity) or 1.25% (w/v) soluble starch (for on—
amylase activity) in 50 mM acetate buffer, pH 6.0, containing 5 mM CaClz and
40 uliters of enzyme solution were mixed and incubated at 60°C for 30 min.
The reaction was stopped by adding 0.8 ml of dinitro salicylate (DNS) solution
(Miller, 1959), and heated in a boiling water bath for 15 min to develop the

color reaction. The samples were cooled on ice and the absorbance of the

9 4
reaction solution measured at 640 nm. One unit of pullulanase or oc-amylase

activity is defined as the amount of enzyme which produces 1 umol of
reducing sugar (with glucose as the standard) per min under the assay
conditions.

For determination of a-amylase activity, 160 pl of 1.25% (w/v) soluble
starch in 50 mM acetate buffer, pH 6.0, containing 5 mM CaC12 and 40 ul of
enzyme solution were mixed and incubated at 60°C for 30 min. The reaction
was stopped by adding 0.8 m1 of DNS solution, and heated in a boiling water
bath for 15 min to develop the color reaction. The samples were cooled in ice
and the absorbance of the reaction solution measured at 640 nm. One unit of
a-amylase activity is defined as the amount of enzyme that produces 1 umol
of reducing sugar (with glucose as the standard) per min under the assay

conditions described.

DNA manipulation

Enzymes and kits for DNA manipulations were obtained from
Bethesda Research Laboratories (Gaithersburg, MD), United State Biochemical
Co. (Cleveland, OH), or Boehringer Mannheim Biochemicals (Indianapolis,
IN). Chromosomal DNA from C. thermohydrosulfuricum 39E was isolated
and purified by a modification of the procedure by Doi (1983) as described for
B. subtilis, where the cell lysate was treated with proteinase K (0.1 mg/ ml,
37°C, 1 h) prior to deproteinizing with buffer equilibrated phenol. Plasmid
DNA was routinely prepared by the alkaline lysis method (Sambrook et al.,
1989), while large scale plasmid DNA isolation was performed by the method
of Clewell and Helinsky (1969) followed by ultracentrifugation in a CsCl
density gradient as described previously (Sambrook et al., 1989). Enzymatic

manipulations of DNA, and separation of DNA fragments by agarose gel

9 5
electrophoresis were carried out as described by Sambrook et al., (1989) or

according to the manufacturer's instructions. A 2.2 kbp gene fragment
isolated from plasmid pCPC 902 after restriction with PstI, and by
electroelution using an electroelution apparatus, the Elutrap (Schleicher and
Schuell Inc., Keene, NH) was labelled with a-32P dATP (400 Ci/mmol, New
England Nuclear, Wilmington, DE), by nick translation (Nick Translation
Reagent Kit, Bethesda Research Laboratories, Gaithersburg, MD). This probe
was used in Southern hybridization experiments to identify the

amylopullulanase (apu) gene.

Southern hybridization

Chromosomal DNA from C. thermohydrosulfuricum 39E was
completely restricted by single or double digestion with SstI, BamHI, EcoRI,
and XbaI. The digested DNA was electrophoresed through 0.8% (w/v) agarose
gels and visualized by staining with ethidium bromide. For hybridization
with the 2.2 kbp radiolabelled probe, the DNA was transferred onto Zeta-
Probe membranes (Bio-Rad, Richmond, CA) by the method of Southern, as
described by Sambrook et al., (1989).

Cloning of the amylopullulanase (apu)gene into E. coli

Chromosomal DNA of C. thermohydrosulfuricum 39E was double
digested with EcoRI and XbaI. 5 to 8 kbp DNA fragments were isolated from
an agarose gel (0.8% w/v) by electroelution. The plasmid vector pUC 18 was
linearized by double digestion with EcoRI and XbaI. The 5 to 8 kbp
chromosomal DNA fragments were ligated with linearized pUC 18 at a molar
ratio of 1:3. The ligated DNA was transformed into competent E. coli SURE
cells by the Hanahan method (1983).

9 6 .
Identification of amylopullulanase positive transformants

E. coli SURE transformants harboring recombinant pUC 18 plasmids
were selected by plating the transformation sample on LB agar plates
containing 5-bromo-4-chloro-3-indolyl-B-D-thiogalactopyranoside (X-gal) and
isopropyl-B-D-thiogalactopyranoside (IPTG) as described by Rodriguez and
Tait (1983). For detection of E. coli SURE transformants expressing
amylopullulanase activity, individual transformant colonies were replica
plated onto LB agar plates containing 0.5%(w/v) soluble starch or 0.5%(w/v)
pullulan coupled to Reactive Red dye (Yang and Coleman, 1987). Upon
growth of colonies, the plates were transferred to 60°C for 24 h. The starch
containing plates were overlayed with 0.15% iodine/1.5% potassium iodide
solution to visualize colonies containing (it-amylase activity.

Positive clones showed a colorless halo around the colonies, against
the dark blue background of the starch-iodine complex. Pullulanase positive
clones gave a pale diffusion ring around the colonies against the dark red

background of Reactive Red-pullulan plates.

Subcloning of amylopullulanase (apu) gene into B. subtilis

Recombinant plasmids expressing amylopullulanase actvity in E. coli
SURE were double digested with EcoRI and XbaI. The EcoRI-Xbal
chromosomal DNA insert was recovered by agarose gel electrophoresis
followed by electroelution, and ligated into pUB 110 double digested and
linearized with EcoRI and XbaI at a molar ratio of 1:3. For PEG mediated
transformation of B. subtilis cells, protoplasts were prepared from cells grown
to exponential phase in SMMP medium (Chang and Cohen, 1979) using
lysozyme. The ligation mixture was used in PEG mediated transformation of

B. subtilis NA-l protoplasts (Chang and Cohen, 1979). The transformants

9 7
were plated onto cell wall regeneration media (Puyet et al., 1987) and the

resultant colonies were replica plated onto LB agar containing kanamycin. oz-
amylase and pullulanase positive colonies were identified as indicated above

for E. coli transformants.

Construction of lac 2 fusion proteins

Recombinant plasmid DNA isolated from transformants expressing
amylopullulanase activity, was partially digested with HindIII. The DNA
fragments were isolated by electrophoresis and subsequent electroelution. The
plasmid vector pUC 18 was linearized with HindIII and dephophorylated
with calf intestine alkaline phosphatase. The partially digested DNA
fragments were ligated with linearized and dephosphorylated pUC 18 at a
molar ratio of 1:3. The ligated DNA was used to transform E. coli H3101, TG-
1, and DHSa. Transformants were tested for a-amylase and pullulanase

activities, as described previously.

Construction of nested deletion mutants

Recombinant plasmid DNA was double digested with AatII and NdeI
restriction sites available on the pUC 18 vector, creating an exonuclease III
sensitive restriction site (Ndel) towards the DNA insert, which was used to
construct nested deletion mutants of the DNA insert from the 3’ direction. To
construct nested deletion mutants from the 5’ direction of the DNA insert,
double digestion was carried out with SSH and BamHI restriction sites
available on the multicloning site of the pUC 18 vector, creating an
exonuclease III sensitive site (BamHI). Nested deletion mutants were
constructed with exonuclease III and nuclease SI (Ausubel et al., 1988) at a

reaction concentration of 150 units of exonuclease 111 per pmol of susceptible

9 8
3’ ends, at 30°C. Each deletion mutant was transformed into E. coli DHSa, and

tested for activity and thermostability. E. coli DHSa harboring the deletion
mutants were grown at 37°C in 5.0 ml of LB media containing ampicillin (50
ug/ml). Cells were harvested from 1.0 ml of culture by centrifugation in a
microcentrifuge (14,000 rpm x 1 min) and the cell pellet sonicated in 150 pl of
50 mM acetate buffer (pH 6.0) containing 5 mM CaClz. To test for
thermostability, the cell lysate was centrifuged (14,000 rpm x 5 min) and the
supernatant heat treated at 85°C for 5 to 30 min and centrifuged (14,000 rpm x
5 min). The supernatant was recovered and tested for (at-amylase and
pullulanase activity. To test for activity in deletion mutants which lost

thermostability, the cell lysate was assayed directly without heat treatment.

Purification of recombinant amylopullulanase

(i) Preparation of cell extract: Unless otherwise noted, all operations were
performed at 25°C. E. coli DHSa carrying pAPZ72 were grown at 37°C for 12 h
(mid—exponential phase) in LB medium (1 L) containing ampicillin (50
ug/ ml), and harvested by centrifugation (8,000 x g, 10 min). The cells were
resuspended in 1/ 25 of the original volume in 50 mM acetate buffer (pH 6.0),
containing 5 mM CaClz, and then disrupted by passing through a French
Pressure cell (American Instrument Co., Silver Springs, MD) operating at
20,000 lb /in2.

Recombinant amy10pullulanase from B. subtilis NA-l, harboring
pAPZ 74 was recovered from the culture supernatant, by repeated dilution
(with 50 mM acetate buffer, pH 6.0, containing 5 mM CaClz) and
reconstitution, using an Amicon ultrafiltration cell, with a 100,000 molecular

weight cut off membrane (YM-IOO) (Amicon Co., Danvers, MA).

9 9
The periplasmic recombinant enzyme fraction of E. coli SURE (from

pAPZ 71 ), was recovered by subjecting the cells to osmotic shock for release of
periplasmic proteins (Neu and Heppel, 1965; Willis, 1984). The cells were then
centrifuged and the supernatant recovered as the periplasmic enzyme
fraction, and processed in a manner similar to enzyme recovery from B.
subtilis. The cell pellet was suspended in 50 mM acetate buffer (pH 6.0)
containing 5 mM C302, and lysed for recovery of the intracellular enzyme.
(ii) Heat treatment: After centrifugation of the cell lysate (38,000 x g, 30 min),
the supernatant was heat treated at 85°C for 15 min and chilled on ice for 10
min. The resultant suspension was centrifuged (38,000 x g, 30 min) to remove
the denatured proteins, and the supernatant containing the thermostable
protein fraction, was concentrated and desalted by repeated ultrafiltration
through an Amicon YM-IOO membrane equipped cell (Amicon), using 50
mM acetate buffer, pH 6.0, containing 5 mM CaClz.

(iii) Anion exchange chromatography: The concentrate (5.0 ml) was applied to
a Q-Sepharose column (30 cm x 2.5 cm) equilibrated with 50 mM acetate
buffer at pH 6.0 containing 5 mM CaClz, and washed with the same buffer
containing 0.05 M NaCl and then eluted with a 500 ml linear gradient of 0.05
M NaCl to 0.45 M NaCl at a flow rate of 1.0 ml/min.

(iv) Gel filtration chromatography: The amylopullulanase fractions collected
from anion exchange chromatography were pooled and concentrated in
Centricon-30 filter cartridges (Amicon) to 200 ul. 50 ul aliquotes were then
applied to a Superose-12 column (Pharmacia, Piscataway, NJ) column
equilibrated with 50 mM acetate buffer (pH 6.0) containing 50 mM NaCl and 5
mM CaClz.

(v) Affinity column chromatography: Amylopullulanase fractions obtained

from the Superose-12 column were applied directly to a B-cyclodextrin-

100
coupled Sepharose CL-4B affinity column (10 ml bed volume) (prepared as

described by Mathupala and Zeikus, manuscript submitted), at 25°C and
washed sequentially with 50 mM acetate buffer (pH 6.0) containing 0.5 M
NaCl, and then with buffer containing 1%(w/v) maltose. Purified
amylopullulanase was eluted from the column using 1%(w/v) B—cyclodextrin
in 50 mM acetate buffer (pH 6.0). The eluate, detected at 280 nm, was
concentrated and reconstituted in acetate buffer (pH 6.0) containing 5 mM

CaClz, by using ultrafiltration to remove B-cyclodextrin.

Protein determination and gel electrophoresis

Protein was determined by using either the dye binding assay
(Bradford, 1976) with the Bio-Rad protein assay kit (Bio-Rad), or by using
bicinchoninic acid with the BCA Assay Kit (Pierce Co., Rockford, IL), using
bovine serum albumin as the standard. SDS-PAGE was performed according
to the method of Laemmli (1970) using 7.5% polyacrylamide gels in a Mini-
Protean II apparatus (Bio-Rad), and protein bands were visualized by staining
with Coomassie Brilliant Blue R-250. The molecular weights of the
recombinant proteins were determined by using high range molecular weight
standards (Bio-Rad) containing myosin (200,000), B-galactosidase (116,250),
phosphorylase b (97,400), bovine serum albumin (66,200), and ovalbumin
(42,700).

Amino acid analysis and N-terminal sequence analysis

Purified recombinant enzymes were desalted using double distilled
water with a Centricon-30 ultrafiltration device (Amicon). Amino acid
composition was determined by reversed phase HPLC using the PICO-TAG
method (Waters Div., Millipore Co., Milford, MA) in a Beckman 7300 HPLC

101

analyzer. The N-terminal amino acid sequence of the recombinant enzyme
was identified by using a protein sequencer Model 477A (Applied Biosystems,
Foster City, CA) at the Macromolecular Facility, Department of Biochemistry,

Michigan State University.

13C NMR analysis

Natural abundance 13C NMR spectra were obtained on maltose and
iso-maltose, which were used as standards to identify the spectral positions
for (at-1,4 and a-1,6 linkages respectively, and on glycogen, prior to, and
following hydrolysis by amylopullulanase. For preparation of the product
hydrolysate, 60 units of recombinant enzyme were incubated for 48 h at 60°C
with 25 ml of mammalian glycogen (10 mg/ ml) in acetate buffer, pH 6.0.
Completion of hydrolysis was determined using TLC, and the product
hydrolysate from glycogen was desalted and deproteinized by anion exchange
(Amberlite IR-118H) (5 ml bed volume) and cation exchange (Amberlite IRA-
4000H) (5 ml bed volume) mini-column chromatography. All samples were
lyophilized twice in the presence of D20 prior to NMR analysis. The proton
decoupled 13C nuclear magnetic resonance spectra (NMR) were recorded at
75.429 MHz on a Varian VXR-300 spectrometer with 5 mm (diameter) NMR
tubes. The solvent deuterium resonance was used as a field-frequency lock
and chemical shifts are expressed relative to tetramethylsilane. Maltose,
isomaltose, and glycogen were analyzed at a concentration of 100 mg/ ml,
while the product hydrolysate was analyzed at a concentration of 250 mg/ ml.
Glycogen was analyzed for 5000 transients, while maltose, isomaltose, and the
product hydrolysate were analyzed for 1500 transients. NMR spectra of

maltose and iso-maltose standards were assigned carbon numbers using

published data (Colson et al., 1974; Nunez et al., 1977). C7 position carbon of

102
maltose and isomaltose, were assigned signals at 100.1 ppm (for (at-1,4

glucosidic bond) and 98.5 ppm (for a-1,6 glucosidic bond) respectively. C1
position carbon of maltose and isomaltose, was assigned signals at 96.3 ppm

and 96.6 ppm respectively.

Thin Layer Chromatography

2 ul aliquots of the samples prepared for NMR analysis were applied
to Whatman HP-K High Performance Silica gel plates (4.5 uM partcle size; 10
x 10 cm). The plates were developed with a n-butanolzethanolzwater (3:2:2
v/v) eluant mixture at room temperature. The resolved sugars were detected
by using a 1:1 mixture of 0.2% (w/v) orcinol in methanol and 20% (v/v)
sulfuric acid in methanol (1:1, v/v) and heating the plates at 100°C for color

development.

RESULTS

Cloning of amylopullulanase (apu) gene into E. coli and B. subtilis

In order to identify the apu gene of C. thermohydrosulfuricum 39E,
Southern analysis was performed on chromosomal DNA digested with
EcoRI, XbaI, and SSH, or double digested with EcoRI/ XbaI and SstI/ XbaI. The
results are shown in Fig. 2. An approximately 6 kbp chromosomal DNA
fragment, resulting from the EcoRI/Xbal double digestion, which was the
smallest DNA fragment that hybridized to the radiolabelled 2.2 kbp DNA
probe upon Southern analysis, was selected for cloning experiments to isolate
the apu gene, in E. coli SURE cells using pUC18 vector. The cloning strategy
is summarized in Fig. 3. Approximately 600 E. coli SURE transformants were
screened for amylopullulanase activity on the starch-iodine and Reactive
Red-pullulan plates. A transformant colony which produced a clear halo on
both pullulan and starch plates was identified. Plasmid DNA was isolated
from the positive transformant clone, and subjected to restriction enzyme
analysis. The recombinant plasmid designated pAPZ 71, contained a DNA
insert of 6.1 kbp. The physical map of pAPZ 71 is shown in Fig. 4.

In order to express the amylopullulanase activity in B. subtilis, the 6.1
kbp DNA insert was recovered from pAPZ 71, and ligated to plasmid pUBllO.
Transformation experiments were performed initially, using CaClz mediated
transformation of B. subtilis (Kuriki et al., 1988). Although transformants
could be selected on kanamycin/ LB agar plates by colony hybridization using
the 2.2 kbp radiolabelled probe DNA, none of the transformants showed
amy10pullulanase activity. Restriction enzyme analysis of the recombinant
pUBllO plasmid DNA indicated spontaneous partial deletions of the 6.1 kbp

chromosomal DNA insert upon transformation into B. subtilis. However,

103

104

1234567 23456

 

Figure 2.

 

59
12.216
— I I. I98 I0. mo

—6.|08 7.126 8.]44
-5.090

9. l62

 

— 4.072
— 3.054

- l.0|8

— 506. Sl'l

Southern hybridization analysis of C.
thennohydrosulfuricum 39E chromosomal DNA for
cloning of the apu gene. A 2.2 kbp DNA containing a
fragment of the debranching enzyme gene of

T. brockii was used as the probe. lanes 1,7 = 1 kb DNA size
markers; lane 2 = EcoRI digest; lane 3 = XbaI digest;

lane 4 = EcoRI/ XbaI double digest; lane 5 = SstI/ XbaI
double digest; lane 6 = SstI digest

105

 

 

 

 

 

 

 

_ E
O O
{2‘ 8
39B chromosomal DNA |-—-C
. Ap' loc'OPZ'
double digest with EcoRl/Xbal
- pUC l8
electroelute 4 to 8 kb DNA fragments - double digest with EcoRl/Xb:
H24!" '81“ dial flat ]
.‘LNIINW 5'“. ’“m . V
l——— N ____j hgate with T4 DNA ligase

pCPC 902 (Coleman and McAllstor)

Q

'ansfer into E. 9911 SURE (lac ZAM 15)

 

 

 

 

digeSt with Pitt 1 select transformants (white colonies) on
X-gal I Amp plates
isolate 2.2 kb Pst 1 fragment
replica plate
label with a32P-dATP
colony hybridization
autoradiography
select
identify amy10pullulanase positive colonies :‘
r

 

pAPZ?! (8.9 kb plasmid)
6.1 kb insert

Figure 3. Cloning strategy for the amylopullulanase (apu) gene
from C. thermohydrosulfuricum 39E into E. coli SURE

106

 

 

 

 

s E
.c .5
:i_::
E& 75 7'5: =3 7; E
..3. n ma 8.: s :3
mespucm
1k lpAPZ7I
5.3:
32%
I <
l l T l I l l l l I

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 kb

Figure 4. Physical map of the pUC 18 clone (pAPZ 71) containing the
apu gene.
The hatched area represents C. thermohydrosulfilricum DNA,

and the open region represents the plasmid DNA. mcs =
multicloning site

1 O 7
after PEG mediated transformation of B. subtilis protoplasts, expression of et-

amylase and pullulanase activity was observed for the recombinant B. subtilis
subclones. A plasmid recovered from a colony expressing amylopullulanase

activity was denoted pAPZ 74 (Fig. 5).

Expression and location of recombinant enzyme in E. coli and B. subtilis

Recombinant E. coli SURE containing pAPZ 71 expressed
amylopullulanase constitutively. The 6.1 kbp insert, when subcloned into
pUC 19 vector in the opposite orientation, expressed amylopullulanase in
quantitatively similar amounts to that in pUC 18 (data not shown). Since the
DNA insert is in opposite orientation to the lacZ promoter in pUC 19, and the
quantitative expression of the recombinant enzyme was not affected, it can be
inferred that the apu gene contains an indigenous promoter sequence
recognized by E. coli, allowing for efficient transcription. In E. coli SURE
transformants harboring pAPZ 71, approximately a third of the recombinant
enzyme was localized in the periplasmic space, while the remainder was
located intracellularly. The occurrence of amylopullulanase in the
periplasmic space indicates the presence of a putative signal sequence on the
apu gene, identified and processed by E. coli, although at low efficiency.

The molecular weight of the expressed recombinant enzyme from
pAPZ 71 as determined by SDS-PAGE analysis, was 160,000, which was 20 kDa
higher than the molecular weight observed for the native enzyme from C.
thermohydrosulfuricum. Therefore, a minimum coding region of 4.4 kbp,
within the 6.1 kbp DNA insert is necessary for the expression of the enzyme.
The expressed enzyme maintained amylopullulanase activity, and

thermostability at 90°C with a half-life of 40 min.

Psfl
Hindlll

EeoRl
Banll

1.0

Figure 5.

Hindlll

2.0

108

 

 

 

pUBIIO ‘f
I 1. l 1 pAPZ74

3.0 4.0 5.0 6.0 7.0 8.0 9.0

Physical map of the pAPZ 74 subclone in Bacillus
subtilis NA-l. pUB 110 was used as the subcloning

vector. The DNA insert is depicted by the hatched
area.

109
B. subtilis NA-l harboring pAPZ 74 expressed amylopullulanase

constitutively and secreted the enzyme into the growth medium. Growth of
the host into the late stationary phase was necessary to obtain optimal yields
of the recombinant enzyme. Secretion of amy10pullulanase by B. subtilis
suggests the recognition of the putative secretion signal sequence of apu by
the host. The quantitative amounts of recombinant enzyme expressed by

individual subclones are given in Table 2.

Table 2. Activitya and location of recombinant amylopullulanase
isolated from E. coli and B. subtilis .

 

 

 

Plasmid construct and host Activity
(U/ ml)
supernatant periplasmic intracellular
pAPZ71 in E. coli SURE <0.01 0.17 0.8
pAPZ74 in B. subtilis NA-l 4.2 -- 0.23

 

a pullulanase activity is shown

The native and recombinant amylopullulanase in E. coli and B. subtilis
displayed similar pH stability (pH 6.0) and activity (pH 5.5) optima (data not
shown). Ca2+ (up to 5 mM) was necessary for the stability of both
recombinant enzymes, which was similar to that observed for the native

enzyme. Product analysis by thin-layer chromatography for polysaccharides

1 1 O
and oligosaccharides were similar for both native and cloned enzymes, with

maltose, maltotriose, and maltotetraose being the products formed upon
hydrolysis of glycogen, soluble starch, amylopectin, and amylose. Glucose was
not observed in any of the polysaccharide product hydrolysates (data not
shown).

When pAPZ 71 plasmid DNA was subcloned into E. coli strains H3101,
DHSa, and TG-l, spontaneous deletions occurred within the recombinant
plasmid, resulting in loss of activity. Restriction enzyme analysis of the
deletion mutants identified an approximately 2.5 kbp EcoRI-Pstl region
within pAPZ 71, which was prone to deletion upon introduction into E. coli
H8101, DH50t, or TG-l.

In order to stabilize the apu gene within these hosts for further DNA
manipulations, construction of fusion proteins with the B-galactosidase N-
terminal encoded by the lacZ region in pUC18 vector was investigated.
Several subcloning experiments were performed where pAPZ 71 DNA was
partially digested with HindIII, and ligated into pUC18. Two subclones
harboring DNA inserts of 5.3 kbp and 4.5 kbp and expressing
amylopullulanase activity were isolated (Fig. 6). Recombinant plasmids pAPZ
72 and pAPZ 73, with 5.3kbp and 4.5 kbp DNA inserts respectively, were
expressed constitutively in hosts E. coli SURE, HB 101, and DHSa. ,However,
in E. coli TG-1, which overexpresses lac I protein, the presence of IPT G (1 mM)
in the growth medium was necessary for expression of both pAPZ 72 and
pAPZ 73 amylopullulanase fusion proteins, suggesting that in pAPZ 72 and
pAPZ 73, transcription is under the control of the lacZ promoter of the pUC
18 vector (Table 3). In E. coli harboring either pAPZ72 or pAPZ73,
amylopullulanase activity was located intracellularly. These data enabled

identification of the orientation of the apu gene within the recombinant

111

3 ’6
C C
o- 0'-
I I

 

 

 

 

 

5.:

t8
pUC 18 I _
J pAPZ/2
_ 11
m m
3913

'2
J-pAPZ73

 

 

 

 

 

- EeoRl
Sstl
‘ BamHl

 

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 kb

Figure 6. Construction of fusion proteins containing the
amylopullulanase gene from C. thermohydrosulfuricum
39E.

The DNA insert is depicted by the area of shading, and the
Open region represents the plasmid DNA. pAPZ72, and
pAPZ73 are plasmids used to transform E. coli HB 101,
DHSa and DHSOLF'. mcs = multicloning site. Note, the lac Z

promoter region and multicloning site are not
drawn to scale

Table 3.

112

Activity", location, induction and thermostability of recombinant
constructs.

 

 

 

Plasmid E. coli Activity IPTG t1 / 2
construct strain (U / ml) (mM) (min)
85°C
Intracellular Periplasmic Extracellular
pAPZ 71 SURE 0.8 0.17 <0.01 0 >30
pAPZ 72 H3101 4.6 <0.01 <0.01 0 >30
pAPZ 72 TG-l 0.15 <0.01 <0.01 0 -
pAPZ 72 TG-l 2.9 <0.01 <0.01 1.0 >30
pAPZ 73 H3101 0.3 <0.01 <0.01 0 <5
pAPZ 73 TG-l <0.01 <0.01 <0.01 0 -
pAPZ 73 TG-l 0.12 <0.01 <0.01 1.0 <5

 

*Pullulanase activity is shown.

1 l 3
plasmids, and the location of the putative promoter region of apu gene and

the putative secretion signal sequence-encoded by the gene, within the EcoRI-

HindIII region of the 6.1 kbp chromosomal DNA insert.

Purification and characterization of recombinant amylopullulanase
Recombinant amylopullulanase was purified from E. coli and B.
subtilis and characterized with respect to physical and biochemical properties.
A summary of the purification procedure is shown in Tables 4 and 5.
Amylopullulanase was purified 16-fold from E. coli and 14-fold from B.

subtilis.

Analysis of catalytic and thermostability regions

In order to further characterize the 6.1 kbp DNA insert containg the
apu gene, specifically to identify the gene region encoding peptide regions
necessary for thermostability and catalysis, nested deletion mutants were
constructed from 5’ and 3’ directions of the 5.3 kbp DNA insert of pAPZ72.
Nested deletion mutants obtained from pAPZ 72 were tested for their
expression of amylopullulanase activity, as well as for thermostability. Under
the conditions used, deletion of approximately 217 bp could be obtained per
min (Fig. 7). It was possible to restrict the 5.3 kbp gene insert to a size of 2.9
kbp without any quantitative loss of activity or thermostability. The mutant
plasmid containing the 2.9 kbp residual insert was denoted pAPZ 75 (Fig. 8).
The enzymes purified from individual subclones are shown in Fig. 9. The
thermostability of individual subclones are given in Table 3. These results
allowed location of the apu gene segment within the 6.1 kbp DNA insert
which encoded the thermostable domain of amylopullulanase involved in

catalysis. Further deletions in to the gene, enabled the identification of a 2.0

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Figure 7.

116

Agarose gel electrophoresis pattern of nested
deletion mutants constructed from the 3' direction.
The reaction time is indicated above the individual
lanes in minutes. lambda/ HindIII DNA size
markers are indicated on either side of the agarose
gels.

BamHl
Hindlll
thdlll

 

 

 

 

 

 

 

 

 

33 _ a
E E‘ E?" 2 E, 33?: Activity Thermostabilitu(min)
pUC 119 I
TR : 4’ >30
lacZ A! E =
——Z 3 + <5
—— + <5
—— " ‘5
———— ' <5
— <5
—-1 + >30
—— + >30
————~ + >30
— + <5
— "' <5

 

 

 

 

Construction of nested deletion mutants of lacZ
construct of pAPZ 72. For deletion from the 5'
direction, double digestion with Sstl and BamHI was
used. For deletion from 3' direction, double digestion
with AatII and Nde I was used. Amylopullulanase
activity and thermostability was determined as
described in the Materials and Methods.
Thermostability was tested at 85°C.

Fig. 9.

 

SDS-PAGE (7.5% cross-linked) analysis of recombinant
amylopullulanase purified from E. coli and B. subtilis.

lane 1,6 = high molecular weight standards

lane 2 = native amylopullulanase (Mr 140,000)

lane 3 = recombinant amylopullulanase (Mr 160,000)
isolated from the periplasmic space of E. coli SURE
harboring pAPZ71.

lane 4 = recombinant amylopullulanse (Mr 100,000) isolated

from E. coli DHSa harboring pAPZ 75. The presence
of a minor protein band below the Mr 100,000
protein is due to in vivo protease digestion of the
recombinant enzyme.
lane 5 = recombinant amylopullulanase secreted by B.
subtilis harboring pAPZ 74.

1 l 9
kbp region within the HindIII-Ball sites of the gene, which imparted

amylopullulanase activity, but not thermostability.

13C N MR analysis

In order to determine the ability of amylopullulanase to hydrolyze 0t-
1,6 bonds in branched polysaccharides, 13C NMR analysis of product
hydrolysates upon action by the Mr 100,000 recombinant thermostable
enzyme encoded by the 2.9 kbp insert in pAPZ75, was performed. Prior studies
(Mathupala et al., 1990) had shown the ability of the native enzyme from C.
thermohydrosulfuricum 39E to hydrolyze branched polysaccharides, i.e.
mammalian and oyster glycogen, and amylopectin. To test for catalytic action
by amylopullulanase against a-1,6 glucosidic bonds, mammalian glycogen
was chosen, due to it's higher solubility in DzO. 13C NMR of maltose and
isomaltose are included as standards (Fig. 10).

13C NMR was used to clearly identify (at—1,6 bond and a-1,4 bond
cleavage in glycogen (Fig. 11). The C7 of a-1,4 and 0t-1,6 bonds in glycogen

were assigned signals at 101.0 ppm and 99.8 ppm respectively. The signal for

C1 position was absent in glycogen since reducing ends are present in minute
quantities in the unhydrolyzed polymer. Upon hydrolysis by recombinant
amylopullulanase, the reaction products gave signals at 100.4 ppm (C7 of tat-1,4
bond) and 96.4 ppm (C1 of reducing carbon)(Fig. 11). A signal corresponding to

C70f tit-1,6 bond was not present in the spectrum (98 ppm), indicating efficient

hydrolysis of the branch points of glycogen by the recombinant
amylopullulanase.

The results from N MR analysis, where the signals corresponding to the
C7 of (11,6 bonds were removed upon enzymatic action, indicates the ability of

amylopullulanase to hydrolyze tit-1,6 bonds efficiently, and identifies the 2.9

Figure 10.

120

13C N MR spectra of maltose (A), and iso-maltose (3)
standards. The samples were scanned for 1500
transients. The individual carbon numbers

assigned to the NMR signals are given in bold face.

7 = non reducing C1 position of the a-1,4 or a-1,6
glucosidic bond; 1 = C1 reducing carbon position

C7 of a-1,4 bond ~ 100 ppm

C7 of a-I,6 bond ~ 98 ppm

 

 

 

 

 

 

 

 

 

 

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Figure 11.

122

13C NMR spectra of glycogen (C), and hydrolysate of glycogen
(D). The glycogen standard was scanned for 10,000 transients,

while the product hydrolysate of glycogen were scanned for 1500
transients. 25.0 ml of a glycogen solution (10 mg/ ml in D20) in

SOmM acetate buffer containing SmM CaCl2 was hydrolyzed with

80 Units of purified amylopullulanase from pAPZ 75 for 48 h

at 60°C. Completion of the hydrolysis was inferred by thin-layer
chromatography of reaction products, where no retentate was
observed for the starting substrate glycogen.

123

 

 

 

 

 

 

 

 

124
kbp region within the apu gene as the essential gene motif that encodes the

thermostable catalytic domains responsible for the a-1,4 and a-1,6 activities.
The presence of a-1,4 bonds in the NMR spectrum of the product hydrolysate
is due to the formation of maltose, maltotriose, and maltotetraose upon
action by amylopullulanase on glycogen, and the sharper peaks indicate the
total hydrolysis of glycogen to maltose, maltotriose, and maltotetraose, as

corroborated by TLC.

DISCUSSION

To our knowledge, this represents the first reported study on analysis
of activity and thermostability of an amylase by construction of nested
deletion mutants into the gene encoding for the enzyme, in order to test the
effects of progressive deletion of the gene from both 5’ and 3’ directions, and
hence the progressive truncation of the enzyme from both the N- and C-
terminal ends. Using this method, we were able to localize a 2.9 kbp region
within the 6.1 kbp DNA insert, which translated into a mature peptide of
approximately 100,000 molecular weight, that was thermostable and
maintained dual activities.

N-terminal analysis of the enzyme purified from pAPZ 71 was
identical to that of the native enzyme. The disparity in molecular weight
between the native enzyme (Mr 140,000) and the recombinant enzyme in E.
call (Mr 160,000) indicates post-translational processing of the C- terminal
region of the mature peptide in C. thermohydrosulfuricum 39E, or the
possibility of specific protease action against the native enzyme under the
culture conditions used for isolation of the native enzyme. Although the
molecular weight of the mature peptide of amylopullulanase denotes a
minimum coding region of 4.4 kbp, the result from nested deletion mutants
indicated that the major part of thermostability and activity of the enzyme
was provided by a peptide region corresponding to approximately 2.9 kbp.
Within this 2.9 kbp region, the DNA motif encoding for the dual activities
could be further narrowed to 2.0 kbp. Thus, this starch-degrading enzyme is
not a fusion of an a-amylase and a pullulanase as proposed for amylase-

pullulanase from B. circulans F-2 (Sata et al., 1989) or structured according to a

125

126
casette model, as described for a-amylase-pullulanase from C.

thermohydrosulfuricum E101 (Melasniemi et al., 1990; Sata et al., 1989).

Thermostability of the native and recombinant enzymes were similar,
suggesting that the glycan moiety reported for the native enzyme (Saha et al.,
1988) is not a contributing factor towards the thermostability of
amylopullulanase. Molecular weights of 220,000 (Sata et al., 1989), 165,000
(Melasniemi et al., 1989), 102,000 (Spreinat and Antranikian, 1990), and
105,000 (Coleman et al., 1987) have been reported for thermophilic enzymes
exhibiting dual activities isolated from different thermophilic
microorganisms. It will be of interest to identify whether these enzymes too
could be truncated from N- and C- termini to a smaller molecular weight
enzyme product without loss of thermostability or dual activities.

Construction of fusion proteins of amylopullulanase to the lac Z
promoter in pUC vectors enabled the overexpression of amylopullulanase
activity in E. coli, in addition to stabilizing the gene within the E. coli hosts.
The yield of secreted recombinant amylopullulanase from B. subtilis was
low, due to the secretion of highly thermostable protease into the culture
medium by the host, indicated by the substantial degradation of the
recombinant amylopullulanase detected by SDS-PAGE analysis.

NMR analysis indicating that tat-1,6 bonds in glycogen are efficiently
hydrolyzed by the M1- 100,000 enzyme expressed by the 2.9 kbp truncated gene
region, verified the ability of amylopullulanase to cleave 0t-1,6 linkages in
branched polysaccharides, in contrast to the debranching enzyme of T. brockii,
which could only cleave a-1,4 bonds in starch, although it was capable of
hydrolyzing (It-1,6 bonds in pullulan (Coleman et al., 1987).

Further studies (Mathupala and Zeikus, manuscript in preparation) in

sequencing the 2.9 kbp gene region have enabled the identification of four

127

conserved peptide regions common to a-amylases which are involved in
catalysis and substrate binding. This 2.9 kb region may code for two major
domains, similar to the domain structure of (it-amylase of Aspergillus
oryzae, for which the active site and substrate binding subsites are located in
the inter-domain region (Matsuura et al., 1988; Boel et al., 1987).

Since the catalytic activity was maintained in nested deletion mutants
that lost thermostability, it can be inferred that the loss of thermostability is
not due to loss of peptide regions essential for calcium binding, which in 0:-
amylase are located at the catalytic and substrate binding center (Matsuura et
al., 1984). The loss of thermostability is possible due to general destabilization
of the catalytic domain of amylopullulanase, where part of ' the peptide
contributing to it’s tertiary structure is deleted.

Findings in this study suggest the possible use of amylopullulanase in
the starch industry, to replace the current technology involving a-amylase
and pullulanase in the stepwise process for the production of conversion
syrups. Similar studies for determination of the gene regions encoding the
domains necessary for thermostability and activity within glucoamylase, an
industrially important amylase for conversion of oligosaccharides to glucose,
may enable the construction of a multifunctional fusion protein using the
thermostable catalytic domains from both glucoamylase and
amylopullulanase, for single step conversion of raw starch to glucose, for use

in bioprocessing.

LIST OF REFERENCES

Ausubel, P.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A.
Smith, and K. Struhl. 1988. In Current Protocols in Molecular Biology,
Vol.1 and 11, John Wiley and Sons, New York.

Boel, E., Brady, L., Brzozwski, A.M., Derewenda, Z., Dodson, S.G., Jensen,
V.J., Petersen, 8.3., Swift, H., Thim, L., and Woldike, H.F. 1990. Calcium
binding in tat-amylases : An X-ray diffrction study at 2.1 A resolution of two
enzymes from Aspergillus. Biochemistry. 29 : 6244-6249.

Chang, S., and SN. Cohen. 1979. High frequency transformation of
Bacillus subtilis protoplasts by plasmid DNA. Mol. Gen. Genet. 168:111-
115.

Clewell, D.B., and DR Helinsky. 1969. Supercoiled circular DNA-protein
complex in Escherichia coli: Purification and induced conversion to an
open circular DNA form. Proc. Natl. Acad. Sci. USA. 62:1159-1166.

Coleman, R.D., S.S. Yang, and M.P. McAllister. 1987. Cloning of the
debranching-enzyme gene from Thermoanaerobium brockii into
Escherichia coli and Bacillus subtilis. J. Bacteriol. 169:4302-4307.

Colson, P., H.J. Jennings, and I.C.P. Smith. 1974. Composition, sequence,
and conformation of polymers and oligomers of glucose as revealed by
carbon-13 Nuclear Magnetic Resonance. J. Am. Chem. Soc. 96:8081-8087.

Doi, R.I-1. 1983. p. 162-163. In Recombinant DNA Techniques: An
Introduction. R.L. Rodriguez, and RC. Tait (ed.), Benjamin-Cummings,
CA.

Gryczan, T.J., S. Contente, and D. Dubnau. 1978. Characterization of
Staphylococcus aureus plasmids introduced by transformation into
Bacillus subtilis. J. Bacteriol. 134 : 318-329.

128

1 2 9
Hanahan, D. 1983. Studies on transformation of Escherichia coli with

plasmids. J. Mol. Biol. 166 : 557- 580.

Hyun, H.H. and Zeikus, J.G. (1985) Regulation and genetic enhancement of
glucoamylse and pullulanase production in Clostridium
thermohydrosulfuricum. J. Bacteriol. 164 : 1146—1152.

Kuriki, T., Okada, S., Imanaka, T. 1988. New type of pullulanase from
Bacillus stearothermophilus and molecular cloning and expressiion of the
gene in Bacillus subtilis. J. Bacteriol. 170 :

Mathupala, S.P., B.C. Saha, and J.G. Zeikus. 1990. Substrate competition
and specificity at the active site of amylopullulanase from Clostridium
thermohydrosulfuricum. Biochem. Biophys. Res. Commun.166 : 126-132.

Matsuura, Y., M. Kusunoki, W. Harada, and M. Kakudo. 1984. Structure
and possible catalytic residues of Taka-Amylase A. J. Biochem. 95:697-702.

Melasniemi, H. 1988. Purification and some properties of the extracellular
a-amylase-pullulanase produced by Clostridium thermohydrosulfuricum.
Biochem. J. 250:813-818.

Melasniemi, H. and Paloheimo, M. 1989. Cloning and expression of the
Clostridium thermohydrosulfuricum oc-amylase-pullulanase gene in
Escherichia coli. J. Gen. Microbiol. 135 : 1755-1762.

Melasniemi, H., M. Paloheimo, and L. Hemio. 1990. Nucleotide sequence
of the a-amylase-pullulanase gene from Clostridium
thermohydrosulfuricum. J. Gen. Microbiol. 136 : 447-454.

Miller, G.L. 1959. Use of dinitrosalicylic acid reagent for determination of

reducing sugar. Anal. Chem. 31 : 426-428.

1 3 0
Nakajima, R., Imanaka, T., and Aiba, S. 1985. Nucleotide sequence of

Bacillus stearothermophilus (It-amylase gene. J. Bacteriol. 163 : 401-406.

Neu, H.C., and LA. Heppel. 1965. The release of enzymes from
Escherichia coli by osmotic shock and during the formation of
spheroplasts. J. Biol. Chem. 240 : 3685-3692.

Nunez, H.A., T.E. Walker, R. Fuentes, J. O'Connor, A. Serianni, and
R. Barker. 1977. Carbon-13 as a tool for the study of carbohydrate
structures, conformations and interactions. J. Supramol. Struct. 6: 535-550.

Perbal, B. 1988. in A Practical Guide to Moecular Cloning, 2nd ed. John
Wiley and Sons, New York.

Plant, A.R., R.M. Clemens, R.M. Daniel, and H.W. Morgan. 1987.
Purification and preliminary characterization of an extracellular
pullulanase from Thermoanaerobium Tok6—31. Appl. Microbiol.
Biotechnol. 26 : 427-433.

Puyet, A., H. Sandoval, P. LOpez, A. Aguilar, J.F. Martin, and M. Espinosa.
1987. A simple medium for rapid regeneration of Bacillus subtilis
protoplasts trans formed with plasmid DNA. FEMS Microbiol. Lett. 40 : 1-
5

Rodriguez, R.L. , and Tait, R.C. 1983. in Recombinant DNA techniques:
An introduction. The Benjamin/ Cummins Publishing Co., Inc. , London.

Saha, B.C., S.P. Mathupala, and J.G. Zeikus. 1988. Purification and
characterization of a highly thermostable pullulanase from Clostridium
thermohydrosulfuricum. Biochem. J. 252 : 343-348.

Sambrook, J., E.F. Fritch, and T. Maniatis. 1989. in Molecular cloning: a
laboratory manual. Cold Spring Harbor Laboratory, N. Y.

l3 1
Sata, H., Umeda, M., Kim, C.-H., Taniguchi, H., and Maruyama, Y. (1989)

Amylase-pullulanase enzyme produced by Bacillus circulans F-2. Biochim.

Biophys. Acta. 991 : 388-394

Spreinat, A., and Antranikian, G. 1990. Purification and properties of a
thermostable pullulanase from Clostridium thermohydrosulfurogenes
EM1 which hydrolyses both a-1,6 and a-1,4 glycosidic linkages. Appl.
Microbiol. Biotechnol. 33 : 511-518.

Willis, R.C., Morris, R.C., Cirakoglu, C., Schellenberg, G.D., Gerber, NH,
and Furlong, CE. 1984. Preparation of the periplasmic binding proteins
from Salmonella typhimurium and Escherichia coli. Arch. Biochem.

Biophys. 161: 64-75.

Yanisch-Perron, C., and J. Vieira, and J. Messing. 1985. Improved M13
phage cloning vectors and host strains: nucleotide sequences of the
M13mp18 and pUC19 vectors. Gene 33 : 103-119.

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

CHAPTER 5
SEQUENCING OF THE AMYLOPULLULANASE (apu ) GENE OF

Clostridium thermohydrosulfuricum 39E, AND IDENTIFICATION OF THE
ACTIVE SITE BY SITE DIRECTED MUTAGENESIS.

132

ABSTRACT
The complete nucleotide sequence of the gene encoding the dual active
amylopullulanase of C. thermohydrosulfuricum 39E was determined. The
structural gene (apu) contained a single open reading frame 4443 bp in length,
which corresponds to 1481 amino acids, with an estimated molecular weight
of 162,780 for the deduced amino acid sequence. The 5' region preceding the
structural gene contained sequences similar to the E. coli consensus promoter
sequences, and a sequence similar to the consensus sequence for binding of
the CAMP-CAP complex of E. coli. The N-terminal of the deduced primary
sequence contained a putative 31 amino acid signal sequence, which may be
involved in cell-surface localization of the mature peptide in C.
thermohydrosulfuricum 39E. Analysis of the deduced sequence of apu gene
with sequences of (at-amylases and tat-1,6 debranching enzymes enabled the
identification of four conserved regions putatively involved in substrate
binding and in catalysis. The conserved regions were localized within a 2.9
kbp gene fragment, which encoded a Mr 100,000 protein product that
maintained the dual activities and thermostability of the native enzyme.
Oligonucleotide directed mutants constructed within the 2.9 kbp gene
segment enabled the identification of three acidic amino acid residues,
located within the second, third and fourth conserved regions, that were
involved in tat-1,4 and tit-1,6 glucosidic bOnd cleavage. Asp597, G1u526, and
Asp7o3 were individually modified to their respective amide form, or the
alternate acid form, and in all cases both a-amylase and pullulanase activities
were lost, suggesting the involvement of three residues in catalysis, and the
presence of a single catalytic site within the enzyme and substantiated

amylopullulanase as a new enzyme class.

133

INTRODUCTION

Enzymes harboring pullulanase and a-amylase activity have recently
been reported from various bacteria (Sakano et al.,1982; Coleman et al., 1987;
Plant et al., 1987; Takasaki, 1987; Melasniemi, 1988; Saha et al., 1988; Sata et al.,
1989; Spreinat and Antranikian, 1990). The dual activity of an amylase from
B. subtilis is reported to be due to two individual enzymes forming a complex
dimer of 450,000 (Takasaki, 1987). An amylase-pullulanase enzyme of Mr
220,000 produced by B. circulans F-2, which has a-amylase and pullulanase
activities at equivalent rates, has been shown to contain two active sites for
the individual activities (Sata et al., 1989). a-amylase-pullulanase from C.
thermohydrosulfuricum E101 has been reported to be structured similar to a
cassette model, 'where one half of the enzyme codes for tit-amylase activity
while the other half encodes pullulanase activity, based on sequence analysis
studies (Melasniemi et al., 1990).

The amylopullulanase of C. thermohydrosulfuricum 39E has been
purified to homogeneity, and is a monomer (Mr 140,000), with a catalytic
optimum of 90°C (Saha et al., 1988). Ca2+ is required for the stability of the
enzyme, which has a half-life of 40 minutes at 90°C (pH 6.0).
Amylopullulanase is an enzyme exhibiting both a-amylase and pullulanase
activities, giving rise to a-1,4 and tit-1,6 glucosidic bond cleavage in starch and
related polysaccharides (Mathupala et al., 1990). The gene encoding
amylopullulanase, designated apu, has been cloned and expressed in
Escherichia coli and Bacillus subtilis (Mathupala and Zeikus, manuscript
submitted for publication). In E. coli, the enzyme was located in the
intracellular and periplasmic spaces, while in B. subtilis the recombinant

enzyme was secreted to the culture supernatant. The expressed recombinant

134

1 3 5
protein in E .coli had a M;- of 160,000. The identity of the recombinant protein

was verified by N-terminal amino acid sequencing, which was identical with
the native enzyme. Nested deletion mutants constructed from the
chromosomal DNA insert containing the apu gene enabled restriction of the
putative coding region to a size of 2.9 kbp, with concomitant decrease of the
molecular weight of the expressed gene product from 160,000 to 100,000,
without loss of dual activities or thermostability.

The purpose of this study was to sequence the apu gene and compare
the deduced amino acid sequence with known amino acid sequences of oz-
amylases and debranching enzymes, in order to identify the conserved
regions and the catalytic residues. In order to test the hypothesis'that a single
active site imparts dual activity, site directed mutagenesis of the amino acids
putatively identified as being involved in catalysis was performed and the

effect on amylopullulanase activity determined.

MATERIALS AND METHODS.

Reagents

All chemicals were of molecular biology or analytical grade and were
obtained from Aldrich Chemical Co. (Milwaukee, WI), or Sigma Chemical
Co. (St. Louis, MO).

Bacterial strains and plasmids

E. coli strain DHSaF' [F' ¢80dlacZAM15 A(lacZYA-argF)U169 deoR
recAl endAl hst17(rK" mK+) supE44 l' thi-1 gyrA96 relA1] was obtained
from Bethesda Research Laboratories (Gaithersburg, MD). E. coli TG-I [supE
hsdD5 thi A(lac-proAB) F' (traA36 proAB+ lach lacZAM15)]_was obtained from
Amersham Co. (Arlington Heights, IL). Phagemid vector pUC 119 and helper
phage M13KO7 were obtained from Dr. T. Freidman of Michigan State
University. Plasmids pAPZ 71 (containing the complete apu gene) in E. coli
SURE, and pAPZ 72 (containing a fusion construct of apu gene to the lac Z
promoter in pUC 18) in E. coli DH50t, are the parental plasmids used in this

work (Mathupala and Zeikus, manuscript submitted for publication).

Enzymes
Restriction enzymes were obtained from Bethesda Research
Laboratories, United States Bochemical Co. (Cleveland, OH), or Boehringer

Mannheim Biochemicals (Indianapolis, IN).
Oligonucleotides

Oligonucleotides were synthesized in an Applied Biosystems model

380A DNA synthesizer at the Macromolecular Facility, Department of

136

l 3 7
Biochemistry, Michigan State University. Purification of the oligonucleotides

used in site directed mutagenesis was performed using thin layer
chromatography (Sure Pure oligonucleotide purification kit; United States
Biochemicals, Cleveland, OH) and subsequently 5' phosphorylated using T4
polynucleotide kinase, for construction of oligonucleotide directed mutants
(Oligonucleotide-directed in vitro mutagenesis system version 2.1,

Amersham Co, Arlington Heights, IL).

Enzyme assays

For assay of pullulanase activity, 160 [.11 of 1.25% (w/v) pullulan (for
pullulanase activity) in 50 mM acetate buffer, pH 6.0, containingS mM CaC12
and 40 ul of enzyme solution were mixed and incubated at 60°C for 30 min.
The reaction was stopped by adding 0.8 ml of dinitro salicylate (DNS) solution
(Miller, 1959), and heated in a boiling water bath for 15 min. The samples
were cooled on ice and the absorbance of the reaction solution measured at
640 nm. One unit of pullulanase activity is defined as the amount of enzyme
which produces 1 pmol of reducing sugar (with glucose as the standard) per
min under the assay conditions.

For the assay of a-amylase activity, 160 pl of 1.25% (w/v) soluble
starch in 50 mM acetate buffer, pH 6.0, containing 5 mM CaClz and 40 pl of
enzyme solution were mixed and incubated at 60°C for 30 min. The reaction
was stopped by adding 0.8 ml of DNS solution, and heated in a boiling water
bath for 15 min. The samples were cooled on ice and the absorbance of the
reaction solution measured at 640 nm. One unit of a-amylase activity is
defined as the amount of enzyme that produces 1 pmol of glucose per min

under the assay conditions described.

1 3 8
Protein determination and gel electrophoresis

Protein concentrations were determined using the Bio-Rad protein
assay kits based on the dye binding assay of Bradford (1976) (Bio-Rad
Laboratories, Richmond, CA), or using bicinchoninic acid (BCA Assay Kit,
Pierce Co., Rockford, IL), using bovine serum albumin as standard. SDS-PAGE
was performed according to the method of Laemmli (1970) using 7.5%
polyacrylamide gels in a Mini-Protean II apparatus (Bio-Rad Laboratories),
and protein bands were visualized by staining with Coomassie Brilliant Blue
R-250. The molecular weights of the recombinant proteins were determined
using high range molecular weight standards (Bio-Rad Laboratories,
Richmond, CA) containing myosin (200,000), B-galactosidase (116,250),
phosphorylase b (97,400), bovine serum albumin (66,200), and ovalbumin
(42,700).

Preparation of E. coli competent cells and transformation

E. coli cells were made competent by the method by Hanahan (1983), as
described by Perbal (1988). Transformation of competent cells were carried out
as follows: 1 ul of the ligation reaction (10 ng of DNA) was mixed with 20 ul of
competent cells and incubated on ice for 30 min in 1.5 ml Eppendorf
microcentrifuge tubes. The mixture was heat shocked for 40 s at 42°C to 44°C
and placed on ice for 2 min. 80 ul of cold SOC medium (Hanahan, 1983) was
added and the transformation mixture incubated at 37°C for 1 h in a rotary
shaker at 225 rpm. Transformants harboring recombinant pUC 119 plasmids
were selected by plating the transformation sample onto 2 x YT agar
(Sambrook et al., 1989) containing 50 ug/ m1 ampicillin, 5-bromo-4—chloro-3-
indolyl-B-D-thiogalactopyranoside (X-gal) and isopropyl-B-D-thiogalacto
pyranoside (IPTG) as described by Rodriguez and Tait (1983).

1 3 9
Construction of nested deletion mutants for sequencing

pAPZ 72 DNA, which contained a 5.4 kbp apu gene fragment
(Mathupala and Zeikus, manuscript submitted for publication), was
linearized with XbaI and the 5.4 kbp fragment isolated from an agarose gel
(0.8% w/v) by electroelution. The 5.4 kbp DNA fragment was ligated into
phagemid vector pUC119 linearized with XbaI and transformed into E. coli
DHSaF‘ competent cells. Directional subclones of the insert within pUC119
were identified by restriction mapping using the unique SalI and SphI
restriction enzyme sites (originating from the respective multicloning sites
on the pUC18 donor plasmid and pUC119 recipient plasmid) within the
recombinant pUC119 vector. Inserts in opposite orientations were denoted
pAPZ118 and pAPZ119. The individual plasmids were double digested with
SSH and BamHI and used to create nested sets of deletion mutants (Ausubel et
al., 1988) (Fig. 1). Single stranded DNA generated from the deletion mutants
by superinfection of phagemid containing DH50LF' cells with helper phage
M13KO7, was used to deduce the DNA sequence of the amylopullulanase
gene by the dideoxy chain termination method (Sanger, 1977). The regions
external to the 5.4 kbp apu DNA fragment were sequenced by double stranded

DNA sequencing of pAPZ 71 plasmid DNA using synthetic oligonucleotides.

Production of single stranded DNA from phagemid vectors

Helper phage M13KO7 was grown and purified as described previously
(Sambrook et al., 1989). For production of single stranded DNA, a fresh
bacterial colony (grown on 2 x YT agar containing 50 ug/ ml ampicillin)
harboring pUC 118 or pUC 119 recombinant phagemid containing the 5.4 kbp
apu gene insert (from pAPZ 72) was suspended in a 15 ml culture tube

(Corning, New York, NY) containing 3 ml of 2YT (Sambrook et al., 1989)

Figure 1.

140

 

Hindlll
Banll
Hindlll
Pstl
Bqlll

Pvull
99m

Hindlll

 

G
c
..-
I

EcéRI

BamHl

Sstl

pAPZ 12-3

pUC119

Nested deletion mutant constructs used for
sequencing the apu gene. The hatched area

represents C. thermohydrosulfuricum 39E DNA insert;
mes = multicloning site. The solid areas represent part
of the C. thermohydrosulfuricum 39E DNA insert after

nested deletion mutations.

141
media. The medium was adjusted to 100ug/ ml with ampicillin and 30111 of

helper phage M13KO7 (2 x 109 pfu/ ml) was added to a final concentration of 2
x 107 pfu/ ml. The tubes were incubated at 37°C, at 250 rpm for 1.5 h, or until
slightly turbid. Kanamycin was added to a final concentration of 70ug/ ml and
incubation continued for a further 12 to 14 h more at 300 rpm, at 37°C. The
culture was centrifuged at 17,000 x g for 10 min at 4°C, and the supernatant
recovered. The phagemid particles were precipitated by adding to the 3 ml of
supernatant, 334 ml of 40% (w/v) PEG-8000 and 334 ml of 5 M sodium acetate
(pH 7.0). The phagemid was allowed to precipitate on ice for 15 min, and
collected by centrifugation at 17,000 x g for 10 min. The phagemid pellet was
resuspended in 0.3 ml TE buffer (10 mM tris, 1 mM EDTA, pH 8.0) and
extracted three times with an equal volume of phenol-chloroform (3:1 v/v)
by gentle mixing for 5 min. After a final extraction with an equal volume of
chloroform-isoamyl alcohol (24:1 v/v), the single stranded DNA was
precipitated with 0.1 volume of 3M sodium acetate and 2.5 volume of
ethanol, by chilling at -70°C for 30 min, and subsequent centrifugation for 30
min. The DNA was redissolved in 10 ul of water and used for DNA

sequencing or site directed mutagenesis.

DNA sequencing

Sequenase V.2.0 DNA polymerase and Sequenase V.2.0 sequencing kit
from United States Biochemicals were used for DNA sequencing. For single
stranded DNA sequencing, the protocol as described by the manufacturer was
used (Instruction manual, Sequenase V.2.0, United State Biochemicals). For
double stranded DNA sequencing, denaturation of double stranded plasmid
DNA was performed as described by Zhang et al., (1988). For sequencing

reactions where lac Z fusion constructs were involved (pAPZ 119), universal

1 4 2
M13/pUC forward sequencing primer was used. For double stranded DNA

sequencing, within the 5’ EcoRI-HindIII region of the 6.1 kb DNA insert of
the original recombinant plasmid pAPZ 71, synthetic oligonucleotides were

used as primers.

Sequence analysis

The amino acid sequence inferred from the amylopullulanase DNA
sequence was compared with the primary structures of tit-amylases and
pullulanases available through GenBank (IntelliGenetics Inc., Mountain
View, CA). GCG Sequence Analysis Software Package V.7.0 (Devereux et al.,
1984) was used in the analysis and multiple sequence alignment and

subsequent data manipulations.

Oligonucleotide directed mutagenesis of amylopullulanase gene
Oligonucleotide-directed in vitro mutagenesis system V.2.1 from
Amersham Co. (Arlington Heights, IL) was used in constructing single point
base mutants (Nakamaye 8: Eckstein, 1986). E. coli TG-l was transformed with
a nested deletion mutant, containing an apu gene fragment of 2.9 kb, denoted
pAPZ 12-3 (in pUC 119), which maintained both a-amylase and pullulanase
activities, as well as thermostability. This was used to generate single stranded
DNA, using M13KO7 helper phage, for use as the template for
oligonucleotide directed mutagenesis. The synthetic oligonucleotide primers
were designed to be complementary to the single stranded template DNA and
contained the appropriate single point base mismatches as shown in Fig. 2.
Following in vitro mutagenesis, plasmid DNA was isolated from E.
coli TG-1 transformants which did not express amylopullulanase activity. The

0.9 kbp KpnI-BglII DNA fragment was isolated from the plasmids by double

143

597
Template DNA 5’ C TGG AGA TTG GAE GTT GCA AA- --- -
pAPZ 76 Trp Arg Leu Asp val Ala
Primer Asp-Asn 3’ G ACC 'l‘C'l' AAC ETA CAR CGT TT- --- -
62 52
Primer Asp-Gln 3' - -CC TCT AAC CTI CAA CGT TTT A - —
62 69

626
Template DNA 5’ A ATG AT'J.‘ GCA GAB CTT TGG GGA GA
pAPZ 76 Met Ile Ala Glu Leu Trp Gly
Primer Gln-Gln 3’ T TAC TAA CGT QTT GAA ACC CC- --
62 56
Primer Glu-Asp 3’ — TAC TAA CGT CTA GAA ACC CCT CTA CGA
62 88

703

Template DNA 5' TTA GGT TCT CAT gag ACC ATG AGA ATA
PAPZ 76 Leu Gly Ser His Asp Thr Met Arg Ile
Primer Asp-Asn 3' --T CCA AGA GTA ITG TGG TAC TC- ---
62 55
Primer Asp-Gln 3’ --- ~CA AGA GTA CTQ TGG TAC TCT T--

62 68

3'

SI

SI

3!

SI

5!

3!

5'

SI

Figure 2. Synthetic oligonucleotide primers used in oligonucleotide
directed mutagenesis of active site amino acids. Plasmid
pAPZ 76 is identical to pAPZ 12-3 (in pUC 119) except for

growth in E. coli TG—l

144
digestion with KpnI and BglII, followed by agarose 1.2% (W/v) gel

electrophoresis and electroblotting onto NA-45 membranes (Schleicher 8:
Schuell, Inc., Keene, NH). pAPZ75 plasmid DNA lacking the 0.9 kbp BglII-
KpnI fragment was prepared by digestion with 33111 and KpnI, followed by
agarose gel electrophoresis and electroelution (Elutrap, Schleicher & Schuell).
The individual 0.9 kbp DNA fragments recovered from the site directed
mutants were ligated to the linearized vector pAPZ75 lacking the 0.9 kbp
DNA region, and transformed into E. coli TG-1. Single stranded DNA was
recovered from the transformants by infection with M13KO7 helper phage as
described previously. Nucleotide sequence of the single point base mutants
were confirmed by sequencing the 0.9 kbp KpnI-BglII region using synthetic

oligonucleotides.

Enzyme purification

E. coli TG-1 harboring the pAPZ 12-3 recombinant plasmids containing
the specific single point base mutations were grown at 37°C in 5.0 ml of LB
media containing ampicillin (50 ug/ml) and IPTG (1 mM). Cells were
harvested by centrifugation in a microcentrifuge (14,000 rpm x 1 min) and the
cell pellet sonicated in 1.0 ml of 50 mM acetate buffer (pH 6.0) containing 5
mM CaClz. The cell lysate was centrifuged (14,000 rpm x 5 min) and the
supernatant heat treated at 85°C for 5 min. After centrifugation (14,000 rpm x
5 min), the supernatant was recovered and tested for a-amylase and

pullulanase activity.

 

145

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2'? E
C .5
z 2'f_ :1:
E (if E 7‘; = = g z E
5; 3 °- 12 8 a a 5;
mmte
1 l pAPZ7l
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0
S: E — _
c: C c: "" E a:
Effi if i g: 8
DJ
pUC 119
E E
x a?
l l
Bglll/Kpnl Electroelute Site directed
mutagenesis
2% 2 E E 55; Select negative mutant
If!) I 0-\g' “(‘3'
| | pUC 119 l
lacZ L W @ - DAPZ76
Bglll/Kpnl
E E
x a?
isolate 0.9 kb fragment
by elecroblotting
ligate.

Figure 3.

Sequence Bglll/Kpnl fragment using synthetic oligonucleotides

Strategy for constructing oligonucleotide directed mutants.
The hatched area represents C. thermohydrosulfuricum 39E

DNA insert; apu represents the gene encoding
amylopullulanase activity.

RESULTS

Subcloning and construction of nested deletion mutants

In order to deduce the primary sequence of the amylopullulanase by
sequencing of the 6.1 kbp DNA insert containing the a pu gene, nested
deletion mutants were constructed from the 5’ and 3’ ends of the gene. The
nested deletion mutants constructed from subclones pAPle8 and pAPZlI9
are shown in Fig. 1. Since the Open reading frame of 5.3 kbp HindIII/XbaI
fragment of the apu gene is in the correct orientation to the lac Z promoter of
the pUC119 vector, and in the correct reading frame, amylopullulanase was
expressed by E. coli harboring the pAPZIl9 or harboring mutants constructed
by deletion from the 3’ end of the gene. In pAPZ118 however, the gene was in
the opposite orientation to the lac Z promoter, and therefore, the enzyme was

not expressed.

Nucleotide sequence of Clostridium thermohydrosulfuricum 39E apu gene.
In order to deduce the primary sequence of amylopullulanase, a 5 kbp
region within the 6.1 kbp chromosomal DNA insert was sequenced (Fig. 4).
The 5000 bp segment contained an open reading frame of 4443 bp, starting at
nucleotides 331 to 333 with a GTG codon, and terminated at 4773 bp, followed
by termination codons at 4774 bp (TGA) and at 4796 bp (TAA) positions. The
reading frame deduced from the DNA sequence was confirmed by N-terminal
amino acid sequencing, which was identical to the first seven amino acids of
the native enzyme (Mathupala and Zeikus, manuscript submitted for
publication). Therefore, the predicted primary sequence encoded by the open
reading frame corresponds to 1450 amino acids with an estimated molecular

weight of 162,780, which agrees closely with the M;- of 160,000 obtained by

146

Figure 4.

147

Nucleotide sequence and the deduced aminoacid sequence of apu
gene of C. thermohydrosulfuricum 39E. CAMP = sequence
resembling the consensus sequence for binding of CAMP-CAP
complex of E. coli. RBS = putative ribosome binding site. -35 =
sequence similar to -35 consensus promoter sequence of E. coli. -10
= sequence similar to -10 consensus promoter sequence of E. coli.
The initial underlined deduced amino acid sequence refers to the
putative signal sequence. Box represents the N-terminal amino acid
sequence of the processed protein.

 

-330

-279

-228

-177

-126

28

79

130

181

232

283

334

1418

TGTTCTTACTGTTTACAATACTTTTAGCAGAAAAAGCAGATAAGATGGAGA

TAACACAAATGTTAAAACAAATATGAATTTTTCAAAGAGGGATGCTTAIGI
CAMP

GQATCCTTTTCTTTTTTGCAAGGTAIIGAQAAAATATATGGGGTTGGCIAI
-35

AAIAACAATAAGGCAGCGAAAACGATTGCGCAAAAATTTGCACAAGGGATG
-10

TGTATTTATAAACACATTTATCTTTTTGCAATTTATGAAAGCGATTGCCTA
AAAGAAATGGGGGTGGTGTTGTAAAGGAGCTTTTCGTAAGATTTTTAAATA

AAAACATAGGIAAAGGGGQATGTAGTGTTTAAGAGGAGAACATTAGGCTTT
R B s MeLEheLxsargArgIhrLeufilXEhe

TTATTGTCATTTCTTTTAATTTATACAGCAGTGTTTGGCTCAATGCCTGTG
LeuLeuSerEheLenLeuIleIyrIhrAlayalEheGlxfierMeLErQMal

CAATTTGCAAAAGCTGAGACAGATACGGCGCCTGCTATAGCCAATGTTGTT

GlnEheAlaLyaAlaBluThrAspThrAlaProAlanleAlaAanalval
+1 10

GGCGATTTTCAATCAAAGATTGGAGATTCTGACTGGAATATAAACAGTGAC
GlyAspPheGlnSerLysIleGlyAspSerAspTrpAsnIleAsnSerAsp
20

AAAACAGTAATGACATATAAAGGTAATGGCTTTTATGAATTTACTACCCCA
LysThrValMetThrTerysGlyAsnGlyPheTyrGluPheThrThrPro
30 40

GTTGCGTTACCTGCAGGTGATTATGAGTATAAAGTTGCTCTTAATCATTCA
ValAlaLeuProAlaGlyAspTyrGluTerysValAlaLeuAanisSer
50 60

TGGGAAGGTGGAGGAGTTCCTTCACAAGGTAATTTAAGCTTGCATCTTGAT
TrpGluGlyGlyGlyValProSerGlnGlyAsnLeuSerLeuHisLeuAsp
70 80

TCAGATTCTGTAGTAACTTTTTATTACAACTATAATACTTCAAGTGTTACT
SerAspSerValValThrPheTeryrAsnTyrAsnThrSerSerValThr
90

-280

-229

-178

-127

-76

27

78

129

180

231

282

333

384

385

436

487

538

589

640

691

742

793

844

895

946

997

149

GATTCTACAAAATATACACCAATTCCGGAAGAAAAACTTCCAAGAATTGTA
AspSerThrLysTerhrProIleProGluGluLysLeuProArgIleVal
100 110

GGTACTATACAATCAGCAATAGGAGCAGGTGATGATTGGAAACCTGAAACA
GlyThrIleGlnSerAlaIleGlyAlaGlyAspAspTrpLysProGluThr
120 130

TCGACAGCTATAATGAGAGACTATAAGTTTAACAATGTTTACGAATACACT
SerThrAlaI1eMetArgAspTerysPheAsnAanalTyrGluTerhr
140

GCAAATGTTCCAAAAAGGTATTATGAGTTTAAAGTAACTTTAGGGCCCTCA
A1aAanalProLysArgTeryrGluPheLysValThrLeuGlyProSer
150 ' 160

TGGGATATAAATTATGGCTTAAATGGTGAACAAAATGGTCCAAATATTCCT
TrpAspIleAsnTyrGlyLeuAsnGlyGluGlnAsnGlyProAsnIlePro
170 180

TTGAATGTAGCCTATGATACTAAGATTACATTTTACTATGATTCGGTTTCA
LeuAanalAlaTyrAspThrLysIleThrPheTeryrAspSerValSer
190

CATAATATATGGACAGATTACAATCCACCTCTCACAGGGCCTGATAATAAC
HisAsnIleTrpThrAspTyrAsnProProLeuThrGlyProAspAsnAsn
200 210

ATATATTATGACGATTTAAAACATGACACCCATGACCCATTCTTCCGCTTC
IleTeryrAspAspLeuLysHisAspThrHisAspProPhePheArgPhe
220 230

GCTTTCGGTGCAATAAAAACAGGTGATACAGTGACTTTGAGGATACAGGCT
AlaPheGlyAlaI1eLysThrGlyAspThrValThrLeuArgIleGlnAla
240 250

AAAAATCATGACCTTGAGTCAGCTAAAATTTCTTATTGGGATGATATTAAA
LysAanisAspLeuGluSerAlaLysIleSerTerrpAspAsleeLys
260

AAAACAAGAACAGAAGTCCCGATGTATAAAATTGGTCAAAGTCCTGACGGG
LysThrArgThrGluValProMetTerysIleGlyGlnSerProAspGly
270 280

CAATATGAATACTGGGAAGTGAAGTTAAGCTTTGACTATCCCACAAGAATT
GlnTyrGluTerrpGluValLysLeuSerPheAspTerroThrArgI1e
290 300

TGGTATTACTTTATACTTAAAGACGGGACAAAAACTGCTTATTACGGAGAT
TrpTererheIleLeuLysAspGlyThrLysThrAlaTeryrGlyAsp
310

435

486

537

588

639

690

741

792

843

894

945

996

1047

1048

1099

1150

1201

1252

1303

1354

1405

1456

1507

1558

1609

1660

150

AACGATGAACAATTAGGTGGAGTAGGTAAAGCCACAGATACGGTAAATAAA
AsnAspGluGlnLeuGlyGlyValGlyLysAlaThrAspThrValAsnLys
320 330

GACTTTGAACTTACTGTATACGATAAAAATTTAGACACCCCTGATTGGATG
AspPheGluLeuThrValTyrAspLysAsnLeuAspThrProAspTrpMet
340 350

AAAGGGGCAGTAATGTATCAAATATTCCCAGATAGATTTTACAATGGTGAC
LysGlyAlaValMetTyrGlnI1ePheProAspArgPheTyrAsnGlyAsp
360

CCTTTAAATGACCGCCTAAAGGAATACAGTAGAGGTTTTGATCCTGTTGAA
ProLeuAsnAspArgLeuLysGluTyrSerArgGlyPheAspProValGlu
370 380

TATCATGACGACTGGTATGACCTTCCCGACAATCCGAATGATAAAGATAAA
TyrHisAspAspTrpTyrAspLeuProAspAsnProAsnAspLysAspLys
390 400

CCTGGATATACAGGGGATGGTATATGGAATAATGACTTCTTTGGTGGTGAT
ProGlyTerhrGlyAspGlyIleTrpAsnAsnAspPhePheGlyGlyAsp
410 420

TTACAAGGTATAAATGATAAATTGGATTATCTAAAAAACCTTGGAATATCA
LeuGlnGlyIleAsnAspLysLeuAspTereuLysAsnLeuGlyIleSer
430

GTTATTTATCTCAATCCAATTTTCCAATCACCTTCCAATCACCGATATGAT
ValIleTereuAsnProIlePheGlnSerProSerAanisArgTyrAsp
440 450

ACAACCGATTACACAAAGATAGACGAGTTATTGGGAGATTTAGATACATTT
ThrThrAspTerhrLysI1eAspGluLeuLeuGlyAspLeuAspThrPhe
460 470

AAAACACTTATGAAAGAAGCCCATGCAAGAGGAATTAAAGTAATACTTGAT
LysThrLeuMetLysGluAlaHisAlaArgGlyIleLysValIleLeuAsp
480

GGCGTCTTCAATCATACAAGTGATGATAGTATTTATTTTGATAGATACGGG
GlyValPheAanisThrSerAspAspSerIleTerheAspArgTyrGly
490 500

AAGTACTTGGATAATGAATTAGGTGCTTATCAAGCCTGGAAACAGGGAGAT
LysTereuAspAsnGluLeuGlyAlaTyrGlnAlaTrpLysGlnGlyAsp
510 520

CAGTCAAAATCTCCATACGGTGACTGGTACGAAATTAAGCCTGACGGTACC
GlnSerLysSerProTyrGlyAspTrpTyrGluIleLysProAspGlyThr
530

1098

1149

1200

1251

1302

1353

1404

1455

1506

1557

1608

1659

1710

1711

1762

1813

1864

1915

1966

2017

2068

2119

2170

2221

2272

2323

151

TATGAGGGCTGGTGGGGATTTGACAGCTTACCGGTAATAAGGCAGATAAAC
TyrGluGlyTrpTrpGlyPheAspSerLeuProValIleArgGlnI1eAsn
540 550

GGAAGTGAGTACAATGTAAAAAGTTGGGCAGATTTTATCATAAATAATCCT
GlySerGluTyrAanalLysSerTrpAlaAspPheIleIleAsnAsnPro
560 570

AATGCAATATCTAAGTATTGGTTAAATCCTGATGGGGATAAAGATGCAGGT
AsnAlaIleSerLysTerrpLeuAsnProAspGlyAspLysAspAlaGly
580 590

GCAGATGGCTGGAGATTGGATGTTGCAAATGAAATTGCTCACGATTTCTGG
AlaAspGlyTrpArgLeuAspValAlaAsnGluIleAlaHisAspPheTrp
600

GTTCATTTTAGAGCTGCAATTAATACTGTGAAACCAAATGCGCCAATGATT
ValHisPheArgAlaAlaIleAsnThrValLysProAsnAlaProMetIle
610 620

GCAGAACTTTGGGGAGATGCTTCATTAGATTTACTTGGAGATTCTTTTAAC
AlaGluLeuTrpGlyAspAlaSerLeuAspLeuLeuG1yAspSerPheAsn
630 640

TCTGTTATGAACTATCTTTTTAGAAATGCAGTTATTGATTTTATACTCGAT
SerValMetAsnTereuPheArgAsnAlaValIleAspPheIleLeuAsp
650

AAACAGTTTGATGATGGAAATGTGGTTCACAATCCTATAGATGCAGCAAAA
LysGlnPheAspAspGlyAanalValHisAsnProIleAspAlaAlaLys
660 670

CTTGACCAAAGGCTTATGAGCATATATGAGAGATATCCTCTTCCAGTATTT
LeuAspGlnArgLeuMetSerIleTyrGluArgTerroLeuProValPhe
680 690

TATTCTACTATGAACCTTTTAGGTTCTCATGACACCATGAGAATATTGACA
TyrSerThrMetAsnLeuLeuGlySerHisAspThrMetArgIleLeuThr
700

GTATTTGGATATAACTCTGCTAATGAAAATCAAAATTCTCAAGAGGCGAAA
ValPheGlyTyrAsnSerAlaAsnGluAsnGlnAsnSerGlnGluAlaLys
710 720

GACCTTGCAGTTAAGAGGCTTAAACTTGCCGCAATATTGCAAATGGGCTAT
AspLeuAlaValLysArgLeuLysLeuAlaAlaIleLeuGlnMetGlyTyr
730 740

CCGGGAATGCCTTCTATTTACTATGGTGACGAGGCAGGACAATCTGGTGGA
ProGlyMetProSerI1eTeryrGlyAspGluAlaGlyGlnSerGlyGly
750 760

1761

1812

1863

1914

1965

2016

2067

2118

2169

2220

2271

2322

2373

2374

2425

2476

2527

2578

2629

2680

2731

2782

2833

2884

2935

2986

152

AAAGACCCAGATAACAGGAGAACATTCTCTTGGGGAAGAGAAGATAAAGAT
LysAspProAspAsnArgArgThrPheSerTrpGlyArgGluAspLysAsp
770

CTGCAGGATTTCTTTAAGAAAGTCGTAAACATAAGGAATGAAAATCAAGTT
LeuGlnAspPhePheLysLysValValAsnIleArgAsnGluAsnGInVal
780 790

TTAAAAACAGGAGACCTTGAAACACTTTATGCAAATGGCGATGTTTATGCC
LeuLysThrG1yAspLeuGluThrLeuTyrAlaAsnGlyAspValTyrAla
800 810

TTTGGAAGAAGAATTATAAATGGAAAAGATGTATTTGGTAATTCTTATCCT
PheGlyArgArgIleIleAsnGlyLysAspValPheGlyAsnSerTerro
820

GACAGTGTAGCTATTGTTGTGATTAATAAAGGTGAGGCAAAGTCAGTACAA
AspSerValAlaIleValValI1eAsnLysGlyGluAlaLysSerValGln
830 840

ATAGATACTACTAAATTTGTAAGAGATGGAGTTGCTTTTACAGATGCCTTA
IleAspThrThrLysPheValArgAspGlyValAlaPheThrAspAlaLeu
850 860

AGTGGTAAGACATACACGGTTCGTGATGGACAAATTGTTGTAGAAGTTGTG
SerGlyLysThrTerhrValArgAspGlyGlnI1eVa1ValGluValVa1
870

GCATTGGATGGGGCTATACTCATTTCAGATCCAGGACAGAATTTGACGGCA
AlaLeuAspGlyAlaIleLeuIleSerAspProGlyGlnAsnLeuThrAla
880 890

CCTCAGCCAATAACAGACCTTAAAGCAGTTTCAGGAAATGGTCAAGTAGAC
ProGlnProIleThrAspLeuLysAlaValSerGlyAsnGlyGanalAsp
900 910

CTTTCGTGGAGTGCAGTAGATAGAGCAGTAAGTTATAACATTTACCGCTCT
LeuSerTrpSerAlaValAspArgAlaValSerTyrAsnIleTyrArgSer
920 930

ACAGTCAAAGGAGGGCTATATGAAAAAATAGCTTCAAATGTTACGCAAATT
ThrValLysGlyGlyLeuTyrGluLysIleAlaSerAanalThrGlnIle
940

ACTTATATTGATACAGATGTTACCAATGGTCTAAAGTATGTGTATTCTGTA
ThrTyrIleAspThrAspValThrAsnGlyLeuLysTeralTyrSerVal
950 960

ACGGCTGTAGATAGTGATGGAAATGAAAGTGCTTTAAGCAATGAAGTTGAG
ThrAlaValAspSerAspGlyAsnGluSerAlaLeuSerAsnGluValGlu
970 980

2424

2475

2526

2577

2628

2679

2730

2781

2832

2883

2934

2985

3036

3037

3088

3139

3190

3241

3292

3343

3394

3445

3496

3547

3598

3649

153

GCATATCCAGCATTTTCTATTGGTTGGGCAGGAAATATGAACCAAGTTGAT
AlaTerroAlaPheSerI1eGlyTrpAlaGlyAsnMetAsnGlnValAsp
990

ACCCATGTAATAGGCGTAAATAATCCAGTTGAAGTTTATGCTGAAATTTGG
ThrHisValI1eGlyValAsnAsnProValGluValTyrAlaGluIleTrp
1000 1010

GCAGAAGGATTAACAGATAAACCTGGCCAAGGGGAAAATATGATTGCCCAG
A1aGluGlyLeuThrAspLysProGlyGlnGlyGluAsnMetIleAlaGln
1020 1030

TTAGGATATAGGTATATTGGAGATGGTGGGCAAGATGCTACACGCAACAAA
LeuGlyTyrArgTyrIleGlyAspGlyGlyGlnAspAlaThrArgAsnLys
1040

GTAGAAGGTGTTGAAATAAATAAGGACTGGACATGGGTTGATGCACGGTAT
ValGluGlyValGluIleAsnLysAspTrpThrTrpValAspAlaArgTyr
1050 1060

GTAGGGGATTCTGGAAATAACGACAAATACATGGCTAAATTTGTACCTGAT
ValGlyAspSerGlyAsnAsnAspLysTeretAlaLysPheValProAsp
1070 1080

ATGGTAGGCACATGGGAATATATTATGAGATTTTCCTCTAACCAAGGACAG
MetValGlyThrTrpGluTyrI1eMetArgPheSerSerAsnGlnGlyGln
1090 1100

GATTGGACGTATACAAAAGGGCCAGATGGGAAAACAGATGAAGCAAAACAG
AspTrpThrTerhrLysGlyProAspGlyLysThrAspG1uAlaLysGln
1110

TTTATTGTCGTGCCATCAAATGATGTAGAACCACCTACAGCTCTAGGCTTA
PheI1eVa1Va1ProSerAsnAspVa161uProProThrAlaLeuGlyLeu
1120 1130

CAACAACCAGGAATTGAATCCTCAAGAGTTACACTTAACTGGAGTCTCTCA
GlnGlnProGlyI1eGluSerSerArgValThrLeuAsnTrpSerLeuSer
1140 1150

ACTGATAATGTAGCTATCTATGGCTACGAAATATACAAATCTTTAAGTGAA
ThrAspAanalAlaIleTyrGlyTyrGluIleTerysSerLeuSerGlu
1160

ACAGGACCATTTGTAAAGATTGCAACTGTGGCTGACACTGTGTATAACTAC
ThrGlyProPheValLysIleAlaThrValAlaAspThrValTyrAsnTyr
1170 1180

GTAGATACAGATGTAGTAAATGGAAAAGTGTACTATTATAAAGTTGTAGCA
ValAspThrAspValValAsnGlyLysVa1TerererysValValAla
1190 1200

3087

3138

3189

3240

3291

3342

3393

3444

3495

3546

3597

3648

3699

3700

3751

3802

3853

3904

3955

4006

4057

4108

4159

4210

4261

4312

154

GTTGATACTTCTTTTAACAGAACAGCATCAAATATAGTGAAAGCTACACCT
Va1AspThrSerPheAsnArgThrA1aSerAsnIleValLysAlaThrPro
1210

GATATAATACCTATCAAAGTGATATTTAATGTAACAGTCCCTGATTATACT
AsleeIleProIleLysValI1ePheAanalThrValProAspTerhr
1220 1230

CCTGATGACGGTGCAAATATTGCTGGAAACTTCCATGACGCTTTCTGGAAT
ProAspAspGlyAlaAsnIleAlaGlyAsnPheHisAspAlaPheTrpAsn
1240 1250

CCAAGTGCCCATCAGATGACAAAGACAGGACCTAACACTTACAGTATTACA
ProSerAlaHisGlnMetThrLysThrGlyProAsnThrTyrSerIleThr
1260 1270

TTGACTTTAAATGAAGGAACACAGCTTGAATATAAATATGCAAGGGGCAGC
LeuThrLeuAsnGluGlyThrGlnLeuGluTerysTyrA1aArgGlySer
1280

TGGGATAAGGTAGAAAAAGGTGAATATGGAGAGGAAATTGCAAATAGAAAA
TrpAspLysValGluLysGlyGluTyrGlyGluGluIleAlaAsnArgLys
1290 1300

ATAACTGTTGTCAATCAAGGTTCAAATACCATGGTGGTAAATGACACAGTG
I1eThrVa1ValAsnGlnGlySerAsnThrMetValValAsnAspThrVal
1310 1320

CAAAGATGGAGAGACTTACCAATATACATTTATTCTCCAAAAGATAATACT
GlnArgTrpArgAspLeuProIleTyrIleTyrSerProLysAspAsnThr
1330

ACAGTAGATGCAAATACAAACGAGATAGAGATTAAAGGCAATACCTATAAA
ThrValAspAlaAsnThrAsnGluIleGluIleLysGlyAsnThrTerys
1340 1350

GGTGCAAAAGTAACTATAAATGATGAATCTTTTGTACAACAAGAAAATGGC
GlyAlaLysValThrIleAsnAspGluSerPheValGlnGlnGluAsnG1y
1360 1370

GTATTTACAAAAGTAGTGCCTCTTGAATACGGTGTAAATACTACTAAAATA
ValPheThrLysValValProLeuGluTyrGlyValAsnThrThrLysI1e
1380

CATGTGGAGCCGAGTGGTGACAAGAATAATGAACTCACAAAAGATATAACA
HisValG1uProSerGlyAspLysAsnAsnGluLeuThrLysAspIleThr
1390 1400

ATAACTGTTATAAGAGAGGAGCCTGTCCAGGAAAAAGAACCAACTCCTACG
IleThrValIleArgGluGluProValGlnGluLysGluProThrProThr
1410 1420

3750

3801

3852

3903

3954

4005

4056

4107

4158

4209

4260

4311

4362

4363

4414

4465

4516

4567

4618

4669

155

CCAGAGTCTGAGCCAGCACCAATGCCTGAACCACAACCGACACCAACACCA
ProGluSerGluProAlaProMetProGluProGlnProThrProThrPro
1430 1440
GAACCACAGCCTTCTGCAATTATGGCATTGTGACTGCCTCAACACTTAATT
GluProGlnProSerAlaI1eMetA1aLeu *
1450

TAAGAGAAGGAGCAAGTATCACAAGTAAAATTATAGGTACTATCTGCTGGG
*

AAAGTTGTAAAATGGCTTGAGGAAGTGAATGGATGGTACAAAGTTGACTAT

AACGGCAAAGTAGGATATGTTTCAACCAAATATGTTTCTTCAGTGCCAGAT

CCATCAAAGGTAACCGTTGCAAAATCAGTGAAAGTTATAGTGAAGAGCGGA

TT 4670

4413

4464

4515

4566

4617

4668

1 5 6
SDS-PAGE analysis for the enzyme expressed in E. coli, although it is about 22

kDa higher than the 140,000 mw determined for the native enzyme (Saha et
al., 1988). The .GTG initiation codon is preceded with a spacing of 5 bp, by a
putative ribosomal binding site (5'-AAAGGGGG-3’) exhibiting strong
similarity to the 16S rRNA of Bacillus subtilis (McConnell et al., 1986). The
structural gene is preceded by a putative promoter sequence, similar to the
consensus promoter sequences of E. coli and B. subtilis (Rosenburg and Court,
1979; McConell et al., 1986). A region (5’-TATAAT-3’) similar to the -10
consensus promoter sequence of E. coli can be identified 158 bp upstream of
the ribosome binding site, preceded by a region (5’-TTGACA-3’) similar to the
-35 consensus promoter sequence of E. coli. Also recognizable upstream of
these regions is a sequnce resembling the consensus sequnce for binding of
the CAMP-CAP complex of E. coli (Crombrugghe et al., 1984 ), with the
sequence 5’-TGTGC-3’. The N-terminal of the deduced amino acid sequence
contained a 31 amino acid signal sequence putatively involved in cell-surface
localization of the enzyme in C. thermohydrosulfuricum 39E. Hydropathy
profile analysis (data not shown) indicated the putative signal sequence as the
most hydrophobic region within the deduced peptide sequence. The 31 amino
acid sequence had strong similarity to typical secretion signal sequences

(McConnell et al., 1986).

Sequence analysis

In order to identify the conserved regions, and the putative catalytic
residues of amylopullulanase, the deduced sequence was aligned with
known amino acid sequences of a-amylases and debranching enzymes,
available through GENBANK (Intelligenetics, Inc., Mountain View, CA).

Amino acid sequence comparisons of the conserved regions between the

1 5 7
deduced sequence of amylopullulanase with the sequence of enzymes capable

of hydrolyzing (It-1,4 bonds in polysaccharides are shown in Fig. 5. Amino acid
sequence comparisons of the deduced sequence of amylopullulanase with
conserved regions within the sequence of enzymes hydrolyzing a-1,6 bonds
in polysaccharides are presented in Fig. 6. It can be seen that the sequence of
amylopullulanase contains four motifs, DGVFNH, DGWRLDVA, AELWG,
LLGSHD, which are aligned with the consensus sequence motifs of enzymes
capable of tat-1,4 or tit-1,6 glucosidic bond hydrolysis. Previous studies
(Mathupala and Zeikus, manuscript submitted for publication) using group
specific chemical modification of amylopullulanase had indicated the
involvement of acidic amino acids in catalysis. Therefore, aSpartate and
glutamate residues within these regions were targeted for site directed

mutagenesis studies to identify those involved in catalysis.

Identification and modification of the putative catalytic residues on
amylopullulanase

In order to test the hypothesis for a single active site imparting dual
activity, residues tentatively identified using the alignment data for 0:-
amylase of A. oryzae, were modified by site directed mutagenesis.

Using the four conserved regions of a-amylases as a template, similar
regions were identified on amylopullulanase from C .
thermohydrosulfuricum 39E. Most of the residues identified in tit-amylase of
A. oryzae to be involved in substrate binding or catalysis were also conserved
in amylopullulanase. The catalytic residues of A. oryzae tat-amylase (taka-a-

amylase; TAA) reported to be Glu230

297

in the third conserved region, and

Asp in the fourth conserved region (Matsuura et al.,1984), or as Aspzm in

the second conserved region and Asp297 in the fourth conserved region

Figure 5.

158

Multiple sequence alignment of deduced amino acid sequence of
apu from C. thermohydrosulfuricum 39E with sequences of a-
amylases using ’Pileup’ computer program (Devereux et al., 1984).
The legend at the bottom of the figure identifies the organisms from
which the sequences are represented. The four regions indicated by
Roman numerals represent sequence motifs identified by alignment
with the four conserved regions of a-amylase. Putative catalytic
residues targeted for site directed mutagenesis on
amylopullulanase are represented by closed circles. The dots
represent regions within the sequences where an overall alignment
could not be found.

 

 

159

I
39E 465 LGDLDTFKTLMKEAHARGIKVILDGVFNHT 494
Tea O94 YGTADDLKALSSALHERGMYLMVDVVANHM 123
Cloeml 138 FGSFTDFQNLINTAHAHNIKVIIDFAPNHT 167
Stmamyl O93 LGDRAAFKSMVDTCHAAGVKVVADSVINHM 122
Bacamyla 078 YGTKAQYLQAIQAAHAAGMQVYADVVFDHK 107
II.
39E 582 NPDG.DKDAGADGWRLDVANEIAHDFWVHF 610
Taa 190 WVGSLVSNYSSDGLRIDTVKHVQKDFWPGY 219
Cloeml 241 AIKVWLD.MGIDGIRLDAVKHMPFGWQKNF 269
Stmamyl 191 YLNDLLS.LGVDGFRIDAAKHMPAA.DLTA 219
Bacamyla 218 WGKWYVNTTNIDGFRLDGLKHIKFSFFPDW 247
, III
39E 611 RAAINTVKPNAPMIAELWGDASLD ...... 634
Taa 220 NKA....AG.VYCIGEVLDGDPAYTCPYQN 244
Cloeml 270 MDSILSYRP.VFTFGEWFLGTNEIDVNNTY 299
Stmamyl 220 IKAKVGNGS.TYWKQEAIHGAGEAVQPSEY 249
Bacamyla 248 SYVRSQTGKPLFTVGEYWSYDINKLHNYIT 278
Iv ,
39E 685 ERYPLPVFYSTMNLLGSHDTMRILTVFGYN 714
Tea 289 .......... LGTFVENHDNPRFASYTNDI 308
Cloeml 348 .......... MVTFIDNHDMDRFYNGGSTR 367
Stmamyl 290 .......... SAVFVDNHDTER. . .GGDTL 306
Bacamyla 324 ........... VTFVDNHDTNPAKRCSHGR 342

39E Clostridium thermohydrosulfuricum 39E amylopullulanase
Taa Aspergillus oryzae a-amylase
Cloeml C.thermosulfurogenes EM1 a-amylase
Stmamyl Streptomyces limosus a-amylase
Bacamyla Bacillus stearothermophilus liquefying a-amylase

Figure 6.

160

Multiple sequence alignment of the deduced amino acid
sequence of apu with sequences of tit-1,6 hydrolyzing enzymes
using the ’Pileup’ computer program (Devereux et al., 1984). The
legend at the bottom of the figure identifies the organisms from
which the sequences are represented. The four regions indicated by
Roman numerals represent sequence motifs identified by algnment
with the four conserved regions of a—amylases. Putative catalytic
residues are represented by closed circles. The dots represent
regions within the individual sequences where an overall
alignment could not be found.

 

 

161

I

393 473 LMKEA. HARGIKVILDGVFNHTSDDSIYFD 501
Neo 234 LIDRC. HEKGIRVMLDAVFNHC. .. 254
Anglu 084 LLHEM. HERNMKLMMDLVVNHTSDEHNWFI 113
Isoam 304 MVQAF.HNAGIKVYMDVVYNHTAEGGTWTS 332
Kaepula (n4) MIQAIKQDLGMNVIMDVVYNHTNAAGP.TD 638
II
39E 585 G. .DKDAGADGWRLDVANBIA ......... 603
Neo 321 TYWIREFDIDGWRLDVANEID ......... 340
A16glu 192 ..... EKGIDGFRMDVINFISKEEGLPTVE 215
Isoam 387 AYWANTMGVDGFRFDLASVLGN....SCLN 412
Kaepula 686 AVWTTDYKIDGFRFDLMGYHPKAQILSAWE 715
, III
39E 615 NT...VKPNAPMIAELWGDASLDLLGDSFN 641
Neo 353 KA...LKPDVYILGEIWHDAMPWLRGDQFD 379
AIGglu 247 ....LSHYDIMTVGEMPGVTTEEAKLYTGE 272
Isoam 415 ...HASAPNCPNGGYFNDAADSNVAINRIL 441
Kaepula 718 ...KALNPDIYEFGEGWDSNQSD..REFIA 742
Iv ,

39E 686 RYPLPVFYSTMN..LLGSHDTMRILTVFGY 713
Neo 415 SYPNNVNEAAFN..LLGSHDTSRILTVCGG 442
AIGglu 312 KWQKALEHTGWNSLYWNNHDQPRVVSRFGN 341
Isoam 515 LFQSSGRSPWNSINFIDVHDGMTLKDVYSC 544
Kaepula 809 LGMAGN...LADFVLIDK.DGAVKRGSEID 834

391-3 Clostridium thermohydrosulfuricum 39B amylopullulanase
Neo Bacillus stearothermophilus neopullulanase
1116qu B. cereus oligo 1,6 glucosidase
Isoam Pseudomonas amylodermosa isoamylase
Kaepula Klebsiella aerogenes pullulanase

l 6 2
(Buisson et al., 1987), were used in identifying the putative catalytic residues

of amylopullulanase from C. thermohydrosulfuricum 39E. Mutagenesis of
targeted residues, Asp597, Glu626, and Asp703, resulted in both a—amaylse and
pullulanase activities being lost almost completely, indicating that Asp597 of
amylopullulanase, corresponding to Asp2m of TAA in the second conserved

region, Glu626 230

corresponding to the Glu in the third conserved region of
TAA, and Asp7(B corresponding to Asp297 of the fourth conserved region of
TAA, are tentatively involved in catalysis (Table 1). The thermostability of
the mutants was verified by heat treatment of the cell lysates and testing for
the mutant proteins by SDS-PAGE.

Alignment of the sequence of amylopullulanase with those of oz-
amylases, pullulanases and glucoamylases is shown in Fig. 7. The apu
sequence of C. thermohydrosulfuricum 39E displayed similarities to a-
amylase-pullulanase of C. thermohydrosulfuricum E101 (82%), a-amylase of
A. oryzae (48%), neopullulanase of B. stearothermophilus (60%), and
pullulanases of Klebsiella spp. (44%). However, the overall similarity towards
glucoamylases of Aspergillus spp. was much less, with a similarity of 40%.

It can be seen that the four conserved regions of all the amylases are
located in the center of the encoded peptide. It is interesting to note that the
Mr 100,000 peptide encoded by the 2.9 kbp nested deletion mutant constructed
from the 4.4 kbp apu gene is located towards the center of amylopullulanase

primary sequence, in alignment with the conserved sequence motifs of other

amylases.

163

Table 1. Activity bof oligonucleotide directed mutant constructs of apu gene.

 

 

Enzyme Mutation Activity (U /m1)a
pAPZ75 none 4.200 :t 0.07 (100%)
D597N ASp597->Asn 0.006 :I: 0.002 ( 0.14%)
D597E Asp597->Glu 0.008 :t 0.003 (0. 19%)
E626Q Glu626—>G1n 0.004 :t 0.002 (0.10%)
E626D Glu626—>Asp 0.006 :1: 0.002 (0.14%)
D703N Asp703->Asn 0.005 t 0.002 (0.12%)
D703E ASP703->Glu 0.004 :1: 0.003 (0.10%)

 

aadjusted to OD660 = 1.0 for all subclones

bpullulanase activity is shown. In all mutants, concomitant loss of a-amylase
activity was also observed.

164

 

200 400 600 300 I 000 l 200 1 400
l 1 1 1 I I n l 4 l l l a a
I-l-l Wm...
El 01 a-amylase
l--| www-
I

 

 

 

 

Figure 7.

TAA saccharifying a-amylase
B.amy liquefying a-amylase
Aniger glueoamy lase

A.ory zae glucoamy lase

K .39 pollulanase

Kpn pullulanase

Overall alignment of the deduced sequence of

amylopullulanase of C. thermohydrosulfuricum 39E with

amylases from microbial and fungal origin. 39E = C.
thermohydrosulfuricum 39E; E 101 = C. thermohydrosulfuricum E
101; TAA = Aspergillus oryzae; B.amy = Bacillus amyloliquefaciens;

K. ae = Klebsiella aerogenes; K. pn = K. pneumoniae ; The open boxes

represent regions putatively identified on all sequences based on

the four conserved regions of a-amylase of A. oryzae. The 100 kDa

thermostable amylopullulanase fusion protein encoded by the 2.9

kbp apu gene fragment, is shown by the shaded area on

amylopullulanase.

DISCUSSION

This is the first report which identifies the catalytic amino acids of
amylopullulanase, an enzyme capable of hydrolyzing oc-I,6 and tit—1,4 bonds of
polysaccharides, as well as demonstrating categorically that a single active site
is responsible for the dual activities. In this report, we present evidence for
the true nature of the catalytic site of the dual substrate specific
amylopullulanase type enzymes, indicating the involvement of an additional
acidic residue in the general acid-base type catalytic mechanism proposed for
a-amylase. The extra acidic residue may be involved in catalysis by either
enhancing or modifying the ionization state of the two other residues
involved in catalysis, or by being involved in stabilizing the transition state
intermediate. Multiple sequence alignment of the deduced sequence of C.
thermohydrosulfuricum 39E showed greatest similarity towards a-amylase-
pullulanase of C. thermohydrosulfuricum DSM 3783 (87% identity) and
pullulanase of C. thermosulfurogenes (69% identity). Analysis of the 5’
region of apu gene enabled the identification of a sequence motif for binding
of the CAMP-CAP complex of E. coli (Crombrugghe et al., 1984) for regulation
of gene expression, which may be involved in catabolite (glucose) repression
of pullulanase activity, reported previously for C. thermohydrosulfuricum
39E (Hyun and Zeikus, 1985).

In order to clarify the true nature of catalysis, and to locate the apu gene
region encoding the catalytic domain, we proceeded to restrict the gene from
the N- terminal end and the C- terminal end, testing each nested deletion
mutant quantitatively for both a-amylase and pullulanase activities, as well
as for thermostability. The smallest gene fragment, capable of expressing both

activities and thermostability was 2.9 kbp in length and contained sequence

165

l 6 6
motifs similar to each of the four conserved regions identified on (it-amylases,

indicating that only one active site is present on apu gene. The 2.9 kbp gene
fragment was identified within the apu gene by sequencing the 5' and 3’
regions of the nested deletion mutant. This region corresponded to a
polypeptide containing 955 amino acid residues, which was encoded by a
region 2865 bp in length.

The catalytic mechanism of tit-amylases (Matsuura et al., 1984) has been
modeled after the acid-base catalytic mechanism proposed for lysozyme
(Walsh, 1979), where a glutamate, Glu35, acts as a general acid catalyst while
an aspartate, Asp52, acts as a general base, stabilizing the glycosyl-
oxocarbonium transition state intermediate. a-Amylases contain four highly
conserved regions, which have been proposed to form the active site, the
substrate binding sub-sites and the sites for calcium binding, based on X-ray
crystallography data obtained for the a-amylase of A. oryzae (Matsuura et al.,
1984) and porcine liver pancreatic a-amylase (Buisson et al., 1989).

In the present study, alignment of the sequences of amylopullulanase
from C. thermohydrosulfuricam 39E with tat-amylase of Aspergillus oryzae
was used to identify the location of the catalytic residues in amylopullulanase.

597 597 and Asn7(I3

respectively, by single point base mutations of the apu gene, and Glu626was

Asp and Asp7(B of amylopullulanase were changed to Asn
changed to Gln626. Although loss of activity is expected when only two of the
three residues are individually modified, complete loss of a-1,6 and a-1,4
cleavage activity was detected when all three residues were mutated
individually. Further resolution of which aspartate or glutamate is essential
for catalysis and proof of the catalytic mechanism requires X-ray

crystallographic structure analysis.

l 6 7
In the 3-D structure of tit-amylase from A. oryzae, the residues Asp205,

Glu230, and Asp297, which align with Asp597, Glu626, and Asp703 of
amylopullulanase, are located within close proximity to each other, within
the catalytic center, indicating that the three catalytic residues identified in
amy10pullulanase are also closely positioned, forming a single active site for
the dual activities. Therefore, the data suggest that amylopullulanase of C.
thermohydrosulfuricum 39E contains a single active site for dual activities, in
contrast to the dual active sites proposed for the a-amylase-pullulanase of C.
thermohydrosulfuricum E101 (Melasniemi et al., 1990) and amylase-
pullulanase of B. circulans F-2 (Sata et al., 1989).

The loss of activity upon change of Asp597 to Glu597, Asp703 to Glu703
or Glu626 to Asp626 indicates that the geometric alignment of the catalytic
residues within the active center of amylopullulanase is critical, in order to
bring about catalysis. This contrasts with the flexibility shown by
amylopullulanase towards a wide range of oligosaccharides and
polysaccharides containing (it-1,6 and tat-1,4 linkages, which may be related to
flexibility of the substrate binding residues rather than of the catalytic
residues.

Amylopullulanase of C. thermohydrosulfuricum 39E is an
enzyme with greater flexibility towards a wide variety of polymeric substrates,
in contrast to the amylases reported from other prokaryotic or eukaryotic
organisms, which are highly specific towards either (1-1,4 or a-1,6 linkages for
catalysis.

Amylopullulanase may be considered as a non-specific amylase which
has the ability to cleave both (It-1,4 and a-l,6 linkages, rather than as an a-
amylase or a pullulanase which had acquired the ability to hydrolyze a-1,6 or

(it-1,4 bonds respectively. Since amylopullulanase was isolated from a

168
thermophilic anaerobe, which are believed to be among the first

microorganisms to evolve on earth, the broad substrate specificity of this
enzyme suggests that a-amylases and pullulanases may have evolved from
an amylopullulanase type precursor enzyme.

Therefore this enzyme may be of great value in protein engineering
studies to alter the substrate or product specificity of amylases, by changing the

substrate binding or catalytic residues.

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Buisson, G., Duee, E., Haser, R., and Payan, F. (1987) Three dimensional
structure of porcine pancreatic a—amylase at 2.9A resolution. Role of calcium

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Carraway, K.L., and Koshland, D.E.Jr. (1972) Carbodiimide modification of
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Coleman, R.D., Yang, 5.5., and McAllister, M.P. (1987) Cloning of the

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Crombrugghe, B., Busby, S., and Buc, H. (1984) Cyclic AMP receptor protein:

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Devereux, J., Haeberli, P., and Smithies, O. (1984) A comprehensive set of

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Doi, R.H., (1983) in Recombinant DNA Techniques: An Introduction

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Hanahan, D. (1983) Studies on the transformation of Escherichia coli with

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Hyun, H.H., and Zeikus, J.G. (1985) General biochemical characterization of
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thermohydrosulfuricum. Appl. Environ. Microbiol. 49, 1168-1173.

Imanaka, T., and Kuriki, T. (1989) Pattern of action of Bacillus

stearothermophilus on pullulan. I. Bacteriol. 171, 369-374.

Kennedy, J.F., Cabral, J.M.S., Sa-Correia, 1., and White, CA. (1987) in Starch:
Properties and Potential (Galliard, T., ed) pp.122-130, John Wiley and Sons,
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Kuriki, T., Takata, H., Okada, S., and Imanaka, T. (1991) New type of

pullulanase from Bacillus stearathermophilus and molecular cloning of the

gene in Bacillus subtilis. ]. Bacterial. 173, 6147-6152.

Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of
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Mathupala, S.P., Saha, B.C., and Zeikus, J.G. (1990) Substrate competition and
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thermahydrasulfuricum. Biochem. Biophys. Res. Commun.166, 126-132.

Matsuura, Y., Kusunoki, M., Harada, W., and Kakudo, M. (1984) Structure and

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McConnell, D.J., Cantwell, B.A., Devine, K.M., Forage, A.J., Laoide, B.M.,
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Melasniemi, H. (1988) Purification and some properties of the extracellular 0t-
amylase-pullulanase produced by Clostridium thermahydrosulfuricum.

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Melasniemi, H., Paloheimo, M., and Hemio, L. (1990) Nucleotide sequence of
the a-amylase-pullulanase gene from Clostridium thermahydrosulfuricum.

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1 7 2
Miles, B.W. (1977) Modification of histidyl residues in proteins by

diethylpyrocarbonates. In Methods. Enzymol. 47, 431-442.

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Nakamage, K.L., and Eckstein, F. (1986) Inhibiton of restriction endonuclease
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directed mutagenesis. Nucl. Acids Res. 14, 9679-9698.

Perbal, B. (1988) in A Practical Guide to Molecular Cloning. 2nd Ed. John
Wiley 8: Sons, New York.

Plant, A.R., Clemens, R.M., Daniel, R.M., and Morgan, H.W. (1987)
Purification and preliminary characterization of an extracellular pullulanase

from Thermoanaerobium Tok6-31. Appl. Microbial. Biotechnol. 26, 427-433.

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Rosenberg, M., and Court, D. (1979) Regulatory sequences involved in the
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319-353.

Saha, B.C., Mathupala, S.P., and Zeikus, J.G. (1988) Purification and
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1 7 3
Sakano, Y., Hiraiwa, S., Fukushirna, J., and Kobayashi, T. (1982) Enzymatic

properties and action patterns of Thermoactinomyces vulgaris at-amylase.

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Sambrook, J., Fritch, ER, and Maniatis, T. (1989) in Molecular Cloning: A
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Sata, H., Umeda, M., Kim, C.-H., Taniguchi, H., and Maruyama, Y. (1989)
Amylase-pullulanase enzyme produced by Bacullis circulans F-2. Biochim.

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Spreinat, A., and Antranikian, G. (1990) Purification and properties of a
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Freeman and Co., San Francisco, CA.

CHAPTER VI

CONCLUSIONS AND PERSPECTIVES

174

CONCLUSIONS AND PERSPECTIVES.

The studies described in this dissertation on the biochemical
characterization of an amylase from Clostridium thermahydrasulfuricum 39E has
contributed to the information available on these unique thermostable enzymes
found in thermophilic, anaerobic bacteria. The studies provided information on;
the unique catalytic properties of this amylase, named amylopullulanase, from C.
thermahydrasulfuricum 39E; the biochemical relationship of amylopullulanase to
known microbial pullulanases and at-amylases; cloning of the amylopullulanase
gene into E. coli to investigate the molecular biological features of the gene in
relationship to amylases reported from thermophiles, and their gene
organization; methods for overexpression and excretion of amylopullulanase
from C. thermohydrasulfuricum 39E; recognition of regions within the
amylopullulanase gene that encodes the peptide region of amylopullulanase
essential for thermostability and catalysis; identification of aspartate and
glutamate residues essential for catalysis; expression of the amylopullulanase
gene in B. subtilis for potential use in biotechnological processes; identification of
potential use of amylopullulanase in starch industry by biochemical
manipulation of the enzyme using protein engineering for improved or altered
catalytic properties.

Thermostable starch hydrolyzing enzymes, i.e., glucoamylase,
pullulanase, and a-amylase play an important role in the starch processing
industry. These enzymes, are used in solubilization and liquefaction of starch for
the production of maltodextrin and conversion syrups. Several thermophilic
anaerobic bacteria have been investigated with respect to their ability to produce
extracellular saccharidases, and in the ability of such enzymes isolated from these

organisms for their potential for use in applied biotechnological processes. The

175

176
present studies were initiated after investigations into a thermostable pullulanase

produced by C .thermahydrasulfuricum 39E indicated the ability of this enzyme to
cleave (at-1,4 glucosidic bonds in starch. From a biochemical standpoint, the
studies were important due to the potential insights the biochemical analysis of
this unique enzyme would give with regard to the mode of catalysis and nature
of thermostability.

For potential biotechnological applications, the study was of applied
importance because of the potential for use of this enzyme, both as a solubilizing
and debranching enzyme functioning at higher temperatures and at lower pH, in
starch liquefaction and saccharification.

Initial investigations identified the high degree of thermostability, lower
pH optimum for activity and the dual at-amylase and pullulanase activities of the
purified enzyme. Subsequent characterization identified the biochemical
relationship of amylopullulanase to pullulanase and a-amylase isolated from
microbes, with respect to affinity for substrates, inhibition by cyclodextrins,
product formation upon action against oligosaccharides and branched
polysaccharides. For isolation and purification of amylopullulanase, an effective
purification scheme was developed using an affinity matrix. Manipulation of the
culture conditions with respect to substrate provided a method for
overexpression and excretion of amylopullulanase from C. thermahydrasulfuricum
39E. Kinetic analysis of the purified enzyme verified the unique nature of
amylopullulanase, and conclusively showed that the a-amylase and pullulanase
activities are maintained in the same enzyme.

For detailed analysis of amylopullulanase, the gene encoding the enzyme
was cloned and expressed in E. call. For potential use of the enzyme in starch
industry, the gene was subcloned and expressed extracellularly in B. subtilis.

NMR analysis of the recombinant enzyme conclusively showed the ability of

177

amylopullulanase to hydrolyze at-1,6 branch points in polysaccharides.
Overexpression of amylopullulanase in E. coli and B. subtilis provided a
simplified purification process for large scale isolation of amy10pullulanase.

Analysis of the deduced amino acid sequence of amylopullulanase with
sequences from microbial at-amylases provided further insights into the primary
structure and identifiedthe regions within the enzyme involved in catalysis.
Conservation of thermostability in the recombinant enzyme expressed in E. coli,
and lack of cysteins in the deduced sequence of amylopullulanase indicated that
the glycan moiety detected in the native enzyme or disulfide bonds are not
involved in maintaining thermostability.

Identification of the residues essential for catalysis conclusively showed
that a single active site is involved in both a—amylase and pullulanase activities of
amylopullulanase. The identificationof a single active site supported the
biochemical evidence obtained previously for this enzyme, and suggests the
possibility of using this enzyme in structure, function studies in to the catalytic
and substrate binding sites of amylases. Location of the esssential catalytic
residues by sequence analysis identified the region within the enzyme involved
in catalysis and substrate binding, giving potential insights for future research in
to altering the catalytic and substrate specificity by using protein engineering
approaches. Further studies using X-ray crystallography are necessary to identify
the residues involved in catalysis, and to identify the residues involved in
substrate binding to bring about the broad substrate specificity observed for this
enzyme.

In perspective, the results obtained upon detailed biochemical
characterization of this unique thermostable amylase from Clostridium
thermohydrosulfuricum 39E, has indicated it’s potential for use as a novel

biocatalyst, for use in maltodextrin and conversion syrup production where this

178

acid stable thermophilic enzyme can be substituted for fungal at-amylase and
bacterial pullulanase used at present. The process can be operated at higher
temperatures, and thus enable the use of higher concentrations of raw starch in
the process.

Using genetic engineering techniques, it was possible to restrict the
amylopullulanase by a third, giving rise to a truncated but thermostable 100,000
molecular weight enzyme which maintained both a-amylase and pullulanase
activities. This suggested the possibility for protein engineering of fusion
proteins, where the 2.9 kbp gene region encoding the dual activities of
amylopullulanase can be fused to a gene encoding thermostable glucoamylase, to
engineer an amylase fusion protein with at-amylase, pullulanase, and
glucoamylase activities, for single step conversion of raw starch into glucose.
Also evident from this study is the possibility for nested deletion of genes from
other thermostable amylases to identify truncated protein products that are
thermostable and active, for use in protein engineering of fusion proteins.

Since this enzyme was isolated from a thermophilic anaerobe, which are
believed to be among the first microorganisms to evolve on earth, the broad
substrate specificity of this enzyme suggests that oc-amylses and pullulanases
may have evolved from an amylopullulanase type precursor enzyme.
Possibilities exist for protein-engineering of the substrate binding and catalytic
sites of amylopullulanase to generate unique amylases with novel substrate

specificities and product profiles.

APPENDIX

PRELIMINARY CHARACTERIZATION OF CELL SURFACE
MICROSTRUCTURES OF Clostridium thermahydrosulfuricum
39E, IN RELATION TO LOCATION OF AMYLOPULLULANASE.

179

ABSTRACT

Cationized ferritin was used to visualize the cell-surface of C.
thermahydrasulfuricum 39E, using scanning electron microscopy. The cell-
surface structure of the organism under continuous culture lacked any micro-
structure, while under batch culture, cells showed significant cell-surface
heterogeneity in the form of blebs or granular structures. The presence of
these cell surface structures coincided with predominantly cell associated
enzyme activity, in contrast to mainly extracellular activity found in cells

lacking these protruberances.

INTRODUCTION

In Clostridium thermacellum, 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 (Bayer and Lamed, 1986; Lamed and Bayer, 1988). The
cellulosome constitutes the majority of the endogluconase 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
(Lamed et al., 1987).

Cationized ferritin (Erdos, 1986) has been used for the visualization of
exocellular structures and for general enhancement of the resolution of

surface topology of single cells (Lamed et al., 1987). With this technique,

180

 

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negatively charged cell surface structures are labelled and can be observed

using scanning electron microscopy.

The purpose of this study was to examine the ultrastructure of the cell
surface of the cells grown under continuous culture and under batch culture,
in order to identify any morphological differences which could be correlated
with the cellular location of the enzyme, and to tentatively identify the
presence of a multienzyme complex formed and released to the medium by C.
thermohydrasulfuricum 39E, which may be involved in complete

degradation of any starch related polysaccharide into glucose.

MATERIALS AND METHODS

Chemicals and gases

All chemicals used were reagent grade and obtained from either Sigma
Chemical Company (St. Louis, Mo) or Aldrich Chemical Company
(Milwaukee, Wi). Nitrogen used was 99.9% pure and was made free of oxygen

by passage over heated (370°C) copper filings.

Organism and culture conditions

C. thermohydrasulfuricum 39E (ATCC 33223) was grown at 60°C under
anaerobic conditions on TYE medium (Zeikus et al, 1980), supplemented
with 1% (w/v) soluble starch, maltose or glucose as the substrate. Batch
culture studies were carried out using pressure tubes containing 10 ml of
medium and samples were taken at mid to late exponential phase for electron

microscopy studies.

 

 

1 8 2
Affinity cytochemistry

C. thermahydrosulfuricum 39E was grown on glucose, maltose, or
soluble starch to mid-exponential phase in anaerobic tubes (having
previously subcultured twice in medium containing the same substrate).
Cells grown under continuous culture were harvested directly from the
chemostats grown in media containing maltose.

Cells were treated with cationized ferritin as described previously
(Bayer et al., 1985). 10 to 50 ml of cell culture was washed twice with 0.9%
(w/v) NaCl, and centrifuged at 750 xg for 10 min at 4°C. The cells were
resuspended in 5.0 ml of the same solution with 2.0 ml of 2.5% (v/v)
glutaraldehyde in 0.1 M NazHPO4-KH2PO4 buffer (pH 7.2). After incubation
for 20 min at room temperature, the cells were washed three times with
0.9% (w/v) NaCl and finally resuspended in 3.0 m1 of saline. 5.0 ml of the
glutaraldehyde-phosphate fixative was added to 1.5 ml of this cell suspension,
and stored at 4°C, for use as negative controls for cationized ferritin staining.
To the remaining 1.5 ml of the cell suspension, 0.25 ml of cationized ferritin
(1 mg/ ml in 0.9%(w/v) NaCl; Sigma) was added, and the solution incubated
at room temperature for 1 hour. The cells were then centrifuged and washed
twice with 0.9%(w/v) N aCl and resuspended in 6.5 m1 of the glutaraldehyde-

phosphate fixative.

Scanning electron microscopy

For scanning electron microscopy, the cells were deposited on 13mm
diameter polyester membrane, pore size 0.4 pm (Nucleopore, Pleasenton, CA)
by suction, immersed in fixative, and stored overnight at 4°C. Dehydration of
the cells was carried out in an ethanol series of 25% to 100%(v/v), where the

membranes with deposited cells were immersed sequentially in 25%, 50%,

 

1 8 3
75%, 90%, and 100% ethanol (v/v in water), for 10 minutes, and finally stored

in 100%(v/v) ethanol. Critical point drying and gold coating of the cells were
carried out as described previously (Klomparens et al.,1986), in an Emscope
Sputter Coater model SC 500 to a thickness of 28 nm. Scanning electron
micrographs were obtained in a JEOL JSM-35CF microscope at an accelerating

voltage of 15 kV, and at a magnification of 20,000.
RESULTS

Scanning electron microscopy

As the cellular location of tat-amylase and pullulanase activities varied
according to the growth condition, cells were examined for cell surface
integrity and for cell associated structures, using cationized ferritin and gold
labelling to identify negatively charged structures, with scanning electron
microscopy. In the absence of cationized ferritin, cell surface structures were
not evident on cells of C. thermohydrasulfuricum 39E grown on glucose,
maltose, or soluble starch, either under batch culture or under continuous
culture conditions (Fig. 1 and 2). Under batch culture, the cell surface
morphology of glucose grown cells treated with cationized ferritin appeared
similar to non-treated cells, with the cell surface remaining smooth in
appearance (Fig. 1). This differed from cells grown on maltose, or soluble
starch. Treatment of these cells with cationized ferritin revealed the presence
of protruberances on the cell surface (Fig. 1 and 2). The concentration of
maltose in the growth medium appeared to influence the surface topology of
the cell. Cells of C. thermahydrasulfuricum 39E grown at higher

concentrations of maltose (0.5% w/v) in batch culture were covered with

l 8 4
protruberances, and such structures were almost absent on cells grown under

maltose limitation (0.2% w/v) in continuous culture.

DISCUSSION

This study describes the influence of culture conditions on the presence or
absence of cell surface structures (protruberances) visualized using scanning
electron microscopy.

This study provides preliminary evidence to correlate the cell-surface
structures to the possible presence of multienzyme complexes, on the cell
surface of C. thermohydrasulfuricum 39E, using immuno-labelling methods.
Since C. thermahydrosulfuricum 39E can efficiently degrade starch and
related polysaccharides, these studies will indicate whether the organism
harbors multienzyme complexes on the cell surface to ensure effective
hydrolysis of starch, similar to the cellulosome, a multienzyme complex, that
has been isolated from the cell-surface of C. thermocellum (Lamed et al., 1987;
Lamed and Bayer, 1988), for the efficient degradation of cellulose polymers.
This multifunctional, multienzyme complex is capable of efficient
degradation of cellulosic substrates. The localization of these enzymes in the
form of a high molecular weight complex was envisaged as a major
contributing factor in the synergistic action of the cellulase system of C.
thermacellum, for the efficient degradation of cellulosic polymers.

An tat-glucosidase (Saha and Zeikus, 1991) which can efficiently cleave
small oligosaccharides into glucose, and a cyclodextrinase (Saha and Zeikus,
1990) which can hydrolyze cyclic dextrins, have been reported from C.
thermahydrasulfuricum 39E. Therefore, an amylolytic system similar to the

cellulosome may be present in C. thermahydrasulfuricum 393, for the

l 8 5
efficient degradation of glucose polymers, since the enzymes isolated so far

are synergistically capable of breaking down starch related polymers into
glucose. Polyclonal antibodies raised against each enzyme will be used in
immunolabelling studies to identify the location of each enzyme type under
different growth stages.

The preliminary studies on C. thermahydrasulfuricum 39E cells from
batch culture and continuous culture using scanning electron microscopy
after treating with cationized ferritin have shown that the surface
microstructure was structurally different between cells from batch culture and
continuous culture. Cationized ferritin labelled structures were found on
batch culture cells, while cells grown under continuous culture lacked any
structures.

Another possible explanation for the release of cell-bound enzyme to
the medium upon substrate limited chemostat culture may be disruption of
the S-surface layer (Hollaus and Sleytr, 1972; Sleytr and Messner, 1983) of this
organism. The 8 layer, found as the outermost surface layer of archaebacteria
and most eubacteria, including C. thermohydrosulfuricum (Sleytr and
Messner, 1983), is inferred to be directly involved in the interaction of these
cells with their environment. It can be speculated that the porous
glycoprotein (Messner et al., 1992) meshwork of the 5 layer may entrap or
anchor a segment (or domain) of the extracellular-but cell—bound enzyme,
while the remainder of the enzyme containing the domains responsible for
substrate binding and catalysis are exposed to the external medium so that
large polymers such as polysaccharides are readily accessible and degradable by
the organism. The S-layer may be disrupted or differentially synthesized
under the physiologically stressed conditions present in the chemostat,

releasing the enzyme to the medium, while the inner cell-wall and cell-

1 8 6
membrane layers remain intact. Therefore, evidence for the involvement of

the S layer in the localization of the enzyme can be obtained by immuno-gold
labelling studies using polyclonal antibodies raised against the S layer
glycoprotein, for which isolation protocols are already available (Heckels and

Virji, 1988).

LIST OF REFERENCES
Bayer, E.A., Setter, E., and Lamed, R. 1985. J. Bacteriol. 163 : 552-559
Bayer, E.A., and Lamed, R. 1986. J. Bacteriol. 167 : 828-836

Erdos, G.W. 1986. in Ultrastructure Techniques for Microorganisms (Eds.
Aldrich, H.C., and Todd, W.J.) Plenum Press, New York, NY. pp. 401-402

Heckels, J.E., and Virj i, M. 1988. in Bacerial Cell-Surface Techniques (Eds.
Hancock, 1C., and Poxton, I.R.) John Wiley and Sons, New York, NY.pp. 102-
104

Hollaus, F., and Sleytr, U.B. 1972. Arch. Mikrobiol. 86 : 129-146

Klomparens, K., Fleger, S.L., and Hooper, G.R. 1986. in Procedures for
Transmission and Scanning Electron Microscopy for Biological and medical
Science (2 ed). Ladd Research Industry, Burlington, VT.

Lamed, R., and Bayer, EA. 1988. Adv. Appl. Microbiol. 33 : 1-46

Lamed, R., and Bayer, EA. 1987. in Biochemistry and Genetics of Cellulose
Degradation (Eds. Aubert, J.-P., Beguin, P., and Millet, J.) Acad. Press, London.

pp. 101-116

Lamed, R., Naimark, J., Morgenstern, E., and Bayer, EA. 1987. J. Microbiol.
Methods 7: 233-240

187

Messner, P., Christian, R., Kolbe, J., Schultz, G., and Sleytr, U.B. 1992. J.
Bacteriol. 174, 2236-2240

Saha, B.C., and Zeikus, J.G. 1991. Appl. Microbiol. Biotechnol. 35 : 568-571
Saha, B.C., and Zeikus, J.G. 1990. Appl.Environ. Microbiol. 56 : 2941-2643
Sleytr, U.B., and Messner, P. 1983. Ann. Rev. Microbiol. 37 : 311-339

Zeikus, J.G., Ben-Bassat, A., and Hegge, P.W. 1980. J. Bacteriol. 143 : 432-440

188

ISKU 828888 2813137

 

 

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Fig. 1 Scanning electron microscopy of gold coated cells of C.
thermohydrosulfuricum 39E, grown on maltose (0.5% w/v) in batch
culture (A,B), and on maltose 0.2% (w/v) in continuous culture
(C,D).

Figures A and C are of cells in the presence of cationized ferritin,
and figures 3 and D are of untreated cells.

189

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15K” 828888 1884 1 8U [€892

15lH US$888

Fig. 2 Scanning electron microscopy of gold coated cells of C.

thermohydrosulfuricum 39E grown on glucose (0.5% w/v)

in batch culture (E,F), and on soluble starch (0.5%w/v) in
batch culture (G,H).

Figures E and G are of cells in the presence of cationized
ferritin, and figures F and H are of untreated cells.