THESIS

llll"Hill”Ill!Ill“”1””!!!le”ll”!JIIIWIHWIHII

31293 01698 7392

This is to certify that the

dissertation entitled

Characterization of an a-L-
Arabinofuranosidase and other Components of
the Xylanase System of Cytophaga xylanolytica

presented by
Michael Jay Renner I

has been accepted towards fulfillment \
of the requirements for 1

Doctor of Philosophy Microbiology
degree in

 

 

 

Date Dec. 2, 1997

 

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1/90 mumps-p.14

CHARACTERIZATION OF AN a-L-ARABINOFURANOSIDASE AND OTHER
COMPONENTS OF THE XYLANASE SYSTEM OF Cytophaga xylauolytica

By

Michael Jay Renner

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Microbiology

1 997

ABSTRACT

CHARACTERIZATION OF AN a-L-ARABINOFURANOSIDASE AND OTHER
COMPONENTS OF THE XYLAN ASE SYSTEM OF Cytophaga xflanolytica

By

Michael Jay Renner

All land plants, and most aquatic plants and algae, contain some form of xylan, a
plant cell wall hemicellulose second only to cellulose as the most abundant
polysaccharide on earth. Although xylans are abundant and widely distributed, studies of
their degradation have been rather limited and almost entirely restricted to cellulose-
degrading aerobic bacteria or fiingi and rumen anaerobes, microorganisms that usually
secrete their polysaccharide-degrading enzymes. Research described herein examined the
largely cell-associated xylan—degrading enzyme system of Cytophaga xylanolytica, a
ubiquitous anaerobic gliding bacterium found in anoxic freshwater and marine sediments.

An a-L-arabinofuranosidase (Arfl) was purified 85-fold from cells of Cytophaga
xylanolytica strain XM3. The native enzyme had a p1 of 6.1 and an apparent molecular

mass of 160-210 kDa, and it appeared to be a tn'mer or tetramer consisting of 56 kDa

subunits. The enzyme exhibited a Km of 0.504 mM and a Vmax of 3 19 umol - min'l - mg

protein'l, and it had optimum activity at pH 5.8 and 45°C. Arfl was relatively stable over
a pH range of 4 to 10 and at temperatures up to 45°C. The enzyme released arabinose
from a variety of arabinose-linked artificial and natural substrates and was specific for the
a-linked furanose form. Arfl interacted synergistically with three partially purified

endoxylanase fiactions fiom C. xylanolflica in hydrolyzing rye arabinoxylan. However,

cell fractionation studies revealed that Arfl was largely, if not entirely, cytoplasmic, so its
activity in vivo is probably most relevant to hydrolysis of arabinose-containing
xylooligosaccharides small enough to pass through the cytoplasmic membrane.
Antibodies prepared against purified Arfl also cross-reacted with arabinofuranosidases

from other freshwater and marine strains of C. xylanolytica.

DEDICATION

This thesis is dedicated to my good fiiend and colleague Dr. Richard M. Behmlander,

whose untimely death at such a young age shocked us all. I will remember you as one of

the finest friends and scientists I ever knew.

iv

ACKNOWLEDGMENTS

To accomplish a Ph.D., one must endure many long hours of hard work and study,
have patience and luck, but more importantly, have the support and companionship of
good scientists and friends. I feel fortunate to have had the support and friendship of so

many wonderful people.

I am especially grateful to the members of my guidance committee, Drs. P. J.
Oriel, R. P. Hausinger, J. G. Zeikus, and C. A. Reddy, for their patience, guidance, and
encouragement. I am also thankful to Drs. R. P. Hausinger, R. B. Hespell, M. Mulks, and
W. Godchaux III, for the use of laboratory equipment, supplies, and technical knowledge

throughout various stages of my research.

I want to thank members of our laboratory, Kwi Kim, Tim Lilburn, Andreas
Brune, Edourd Miambi, Sheridan Haack, and Dave Emerson for making the lab a fun
place to be. I am especially thankful to Jared Leadbetter for being a supportive classmate

and good fi'iend. You made life, school, and science all the more enjoyable.

I would like to thank Dr. John A. Breznak for allowing me the opportunity to

work and grow in his laboratory. Thanks John for the chance to learn and have fun at the

same time. Scientifically speaking, you made me feel like a kid all over again. I hope I

have developed and matured into the scholarly scientist you expected.

I am thankful to my parents, Marjorie and George Renner, for their support
throughout all my endeavors in life. I also want to thank the faculty of the University of
Wisconsin-La Crosse Microbiology Department (especially Drs. Mike Winfrey, Matty
Venneman, and Bob Burns) for my solid start in microbiology. I also want to thank my
first boss and mentor, Dr. Al Wortman, for being a good fi'iend and teacher. A]
encouraged me to continue my education and made every effort to pass on his enormous

wealth of scientific knowledge.

Finally, I want to thank my beautiful wife, Beth, for her patience and support
through this difficult, yet very fulfilling start to my scientific journey, and also for

providing me with the most precious gift of all, our daughter Emily.

TABLE OF CONTENTS

LIST OF TABLES ............................................................................ viii
LIST OF FIGURES ............................................................................ ix
CHAPTER 1. INTRODUCTION AND BACKGROUND ................................. 1
INTRODUCTION AND BACKGROUND ............................................. 2
CHAPTER 2. PURIFICATION AND PROPERTIES OF AN a-L-
ARABINOFURANOSIDASE FROM C ytophaga xylanolytica ........................... 32
ABSTRACT ................................................................................ 33
INTRODUCTION ......................................................................... 35
MATERIALS AND METHODS ......................................................... 37
RESULTS ................................................................................... 52
DISCUSSION .............................................................................. 79
APPENDIX. a-L-ARABINOFURANOSIDASE-ENCODING GENES FROM
C ytophaga xylanolytica ......................................................................... 84
LIST OF REFERENCES ................................................................... 100

vii

LIST OF TABLES

TABLE 1. ORIGIN AND PROPERTIES OF ENZYMES WITH
a-L-ARABINOFURANOSIDASE ACTIVITY ............................................. 12

TABLE 2. SUMMARY OF PURIFICATION OF (xi-ARABINOFURANOSIDASE
FROM C. xylanolytt'ca .......................................................................... 56

TABLE 3. PROPERTIES or PURIFIED a-L-ARABINOFURANOSIDASE
(Arfl) .............................................................................................. 60

TABLE 4. ENHANCEMENT BETWEEN Arfl AND ENDOX FRACTIONS OF C.
xylanolytica IN HYDROLYSIS OF RYE ARABINOXYLAN ........................... 66

TABLE 5. DISTRIBUTION OF Arfl AND ENDOX ACTIVITIES IN CELLS OF
C ytophaga xylanobztica .......................................................................... 68

viii

LIST OF FIGURES

FIGURE 1. MICROTITER PLATE ASSAY OF GLYCOSIDASE
ACTIVITIES ..................................................................................... 53

FIGURE 2. ZYMOGRAMS OF NATIVE PAGE GELS OF Arfl AFTER
EACH PURIFICATION STEP ................................................................ 54

FIGURE 3. SDS-PAGE GEL OF Arfl AFTER EACH PURIFICATION STEP ...... 58
FIGURE 4. IEF GEL OF PURIFIED Arfl .................................................... 59

FIGURE 5. THIN-LAYER CHROMATOGRAM OF VARIOUS
ARABINOXYLAN S AND AN ARABINAN BEFORE AND AFTER EXPOSURE

TO PURIFIED ArfI .............................................................................. 62
FIGURE 6. ZYMOGRAM AND PROTEIN STAIN OF NATIVE PAGE GELS OF

PARTIALLY PURIFIED ENDOX POOLS .................................................. 64
FIGURE 7. SPECIFICITY OF ANT I-Arfl ANT ISERUM ................................ 71

FIGURE 8. OAT SPELT ARABINOXYLAN ZYMOGRAM OF NATIVE PAGE
GEL SHOWING THE DISTRIBUTION OF ENDOX COMPONENTS IN CELLS
AND CULTURE FLUIDS OF C. xylanolytica STRAIN XM3 ............................ 73

FIGURE 9. Arfl-LIKE PROTEINS AND ENZYME ACTIVITY OF VARIOUS
FRESHWATER AND MARINE ISOLATES OF C. xylanolytica AFTER GROWTH
IN FRESHWATER (FW) OR MARINE (m4) MEDIUM ................................ 77

FIGURE 10. NUCLEOTIDE SEQUENCE OF my] AND THE DEDUCED AMINO
ACID SEQUENCE OF Arfl FROM C. xylanolytica ........................................ 86

FIGURE 11. NUCLEOTIDE SEQUENCE OF 0771 AND THE DEDUCED AMINO
ACID SEQUENCE OF A PUTATIVE a-L-ARABINOFURANOSIDASE FROM C.
xylanobztica ....................................................................................... 91

ix

FIGURE 12. CLUSTAL W COMPARISON OF THE DEDUCED AMINO ACID
SEQUENCES OF am AND arflI To SIMILAR ARAFs FROM OTHER
ORGANISMS .................................................................................... 96

CHAPTER 1

INTRODUCTION AND BACKGROUND

INTRODUCTION AND BACKGROUND

Plant cell walls consist mainly of cellulose, hemicelluloses, and lignin. Cellulose,
an unbranched polysaccharide chain consisting of B-l,4—linked glucose units, is the main
structural polymer in plant cell walls. This homopolysaccharide can form [insoluble
microfibril bundles through hydrogen bonding of numerous single chains having degrees
of polymerization averaging 14,000 glucose units in length (McNeil, et a1. 1984).
Hemicellulose is a generic term, first introduced by Schulze in 1891, to describe the
alkali extractable carbohydrate fraction remaining after hot and cold water extraction of
plant material (Schulze 1891). Hemicelluloses are typically branched

heteropolysaccharides that are classified according to their resident backbone sugar
moieties. For example, xylans are composed of a B-l,4—linked xylose backbone that can
be up to 200 units in length and contain side groups of arabinose, galactose, glucuronic
acid, and acetate. Similarly, there are xyloglucans (B-1,4-linked glucose backbone with
some B-l,6-linked xylose side chains), mannans (B-l,4—linked mannose backbone),
arabinans (or-1,5-linked arabinose backbone With or—l,2- and Ot-l,3-linked arabinose side
groups), and arabinogalactans (OI-1,3-linked galactose backbone with Ot-l,6-linked

galactose and a-l,3-linked arabinose side groups) (Aspinall 1980).

Cellulose microfibers are thought to be encased by hemicellulose polymers in the

maturing secondary plant cell walls, possibly through hydrogen bonds and van der Wahl

3

forces (Atkins 1992, Wong, et al. 1988). In turn, this complex is then thought to combine
with lignin (an amorphous, complex polyphenolic polymer) and other minor components
such as pectins (polygalacturonic acid backbone), and proteins (hydroxyproline-rich,
arabinose substituted extensins; expansins; and other glycosylated proteins and enzymes),
through covalent cross-linkages (to arabinose side groups) and hydrogen bonds (McNeil,
et al. 1984, Monro, et al. 1976, Shcherban, et al. 1995). The resulting insoluble matrix is
thought to provide plants with their structural rigidity and cell wall impermeability

(Whistler and Richards 1970).

Xylan, the most common hemicellulose, is second only to cellulose as the most
abundant polysaccharide on earth (Biely 1985), and it has been estimated that 1010 metric
tons are recycled annually, with the degradative arm occurring largely through the action
of microbes (Wilkie 1983). Xylans are found in the growing primary and mature
secondary cell walls of all terrestrial, and nearly all marine plants and algae (W ilkie 1983,
Wong, et al. 1988). The amount of xylan found in plants can vary from 35% to as little as
7% of the dry weight of birchwood and gymnosperms, respectively (Aspinall 1959,
Timell 1967). Xylans consist of a [MA-linked xylopyranose backbone, to Which are
often attached side groups of a-l,2- and/or a-1,3-linked arabinose, or-l,2-linked 4-0-
methyl-glucuronic or glucuronic acid, and ester- or ether-linked ferulic, p-coumaric
and/or acetic acid side groups depending on the plant source (Aspinall 1959, Timell
1967). Xylans can vary in structure from plant to plant, from tissues within the same
plant, and even within the same tissue at different grth stages (W ilkie 1983, Wong, et
a1. 1988). They can be found in various forms from simple, unbranched linear xylans

(e.g., Esparto grass and tobacco stalks) to low and high branching arabinoxylan with

4
arabinose side groups (e.g., monocots and grains) to glucuronoxylan with 4-0-methyl-
glucuronic and glucuronic acid side groups (e.g., soybeans, hardwoods, and legumes); to
the most complex glucuronoarabinoxylans containing arabinose, glucuronic acid, and
galactose side groups (e.g., sofiwoods and dicots) (Joseleau, et al. 1992). These various
xylans can have fiIrther complexity by the addition of short chain oligosaccharides of

various composition (Zinbo and Timell 1965).

In 1889, Hoppe-Seylor set out to determine the microbes present in anoxic river
mud enrichments that could degrade hemicellulose (Hoppe-Seylor 1889). The evolved
gases he observed could have been due in part to the action of cytophagas, since they are
widely distributed in nature and are well known, but poorly studied, for their biopolymer-
degrading capabilities (Reichenbach 1989). The usually saprophytic organisms in the
genus Cytophaga are classified as aerobic, Gram-negative, unicellular gliding rods, that
form no endospores, microcysts, or fiuiting bodies (Reichenbach 1992). They are the
most commonly found gliding bacteria and typically inhabit organically rich marine,
freshwater, and terrestrial environments (e.g., soils (Reichenbach and Dworkin 1981),
decaying plant material (Liao and Wells 1986), and sewage treatment plants (Gude
1980)). The cytophagas were originally classified, in part, on the basis of their ability to
degrade cellulose (Christensen 1977, Christensen 1974, Christensen and Cook 1972,
Colwell 1973, Goodfellow 1967, Mitchell, et al. 1969, Reichardt 1974, Reichenbach and
Dworkin 1981, Reichenbach and Kleinig 1972, Soriano 1973, Weeks 1969), but the
original classification scheme has been modified to include non-cellulose degraders
(Jooste, et al. 1985, Lysenko, et a1. 1995, Reichenbach 1989, Reichenbach 1992,

Schindler and Metz 1989). A large group of non-cellulose degradcrs include the

~ 5

Cytophaga—like bacteria (CLB), which are typically pathogens of fish and shellfish
(Amin, et al. 1988, Anderson and Conroy 1969, Bemardet and Kerouault 1989, Bragg
1991, Brown, et al. 1996, Cipriano, et al. 1995, Dungan, et al. 1989, Gennari and
Tomaselli 1988, Hansen, et al. 1992, Jack, et al. 1992, Kent, et al. 1989, Kueh and Chan
1985, Lee and Pfeifer 1977, Lee and Preifer 1975, Murchelano and Bishop 1969, Nomura
1997, Pacha 1968, Potin, et al. 1991, Shotts 1991, Sinskey, et al. 1967, Speare, et al.
1995, Speare and Mirsalimi 1992, Vasconcelos and Lee 1972). More recent studies
involving 168 rRNA sequences have strengthened the classification of the Cytophaga
branch, but have also confirmed the diversity within the genus (Manz, et a1. 1996,

Nakagawa and Yamasato 1993).

The only known strictly anaerobic C ytophaga described to date is Cytophaga
xylanolytica (Haack and Breznak 1993). Other unusual characteristics of this bacterium
include its inability to degrade cellulose and its ability to grow under either low or high
salt conditions (e.g., freshwater and marine media); two traits that are atypical for
bacteria in this genus. C. xylanolytica was originally isolated in Germany fi'om
methanogenic and sulfidogenic enrichment cultures. These enrichments contained
powdered xylan as sole fermentable substrate and were inoculated with fi'eshwater
sediments. Similar organism(s) were also isolated, using the above methods, fiom
fieshwater sediment samples fi'om several locations on the campus of Michigan State
University and from samples of fieshwater and marine sediments found in Woods Hole,
Massachusetts (Haack and Breznak 1993). Luxuriant growth of C. xylanobztica was seen
on xylan, and it was observed that gliding cells were attached in masses to the xylan

particles. When examined further, it was found that greater than 90% of xylan-degrading

6
enzymes of C. xylanolytica were cell-associated (Haack and Breznak 1992). Although
polysaccharide-degrading enzymes have been described for a number of fiingi and
bacteria [e.g., (Anderson 1995, Bachmann and McCarthy 1991, Castanares, et al. 1995,
Costa-Ferreira, et al. 1994, Flipphi, et al. 1994, Gasparic, et al. 1995, Gasparic, et al.
1995, Harada 1983, Kimura, et al. 1995, Lee, et al. 1993, Luonteri, et al. 1995, Ransom
and Walton 1997, Salyers 1993, Schwarz, et al. 1990, van der Veen, et a1. 1991, Wood
and McCrae 1996)]. the information available on xylan-degrading microorganisms has
been more limited [e.g., (Bachmann and McCarthy 1991, Belancic, et al. 1995,
Castanares, et al. 1995, Clermont, et al. 1970, Costa-Ferreira, et al. 1994, Fernandez, et
al. 1995, Gasparic, et al. 1995, Gasparic, et al. 1995, Haack and Breznak 1992, Lee, et a1.
1993, MacKenzie, et al. 1989, Saraswat and Bisaria 1997, Schwarz, et a1. 1990, Sunna

and Antranikian 1997, Whitehead and Hespell 1990)].

Studies of polysaccharidases from cytophagas have been mostly limited to
agarases, cellulases, amylases, carrageenases, and pectinases (Bacon, et al. 1970, Bacon,
et a1. 1970, Chang and Thayer 1977, Charpentier 1965, Clermont, et al. 1970, Duckworth
and Turvey 1969, Duckworth and Turvey 1969, Duckworth and Turvey 1968, Duckworth
and Turvey 1969, Jeang, et al. 1995, Li, et al. 1996, McKay 1991, Osmundsvag and
Goksoyr 1975, Potin, et al. 1991, Sarwar, et al. 1987, Sarwar, et al. 1985, Sundarraj and
Bhat 1972, Sundarraj and Bhat 1971, Turvey and Christison 1967, van der Meulen and
Harder 1976, van der Meulen and Harder 1975, van der Meulen and Harder 1976, van der
Meulen, et al. 1974). Two cellulases, one secreted and one cell-associated, were found to
be produced by Sporocytophaga myxococcoides (Osmundsvag and Goksoyr 1975). A

cellulolytic soil Cytophaga, sp. LX-7, secreted a cellobiose-oxidizing enzyme (Li, et al.

7

1996) whereas another soil species secreted an amylase (Jeang, et al. 1995). A mostly
cell-associated amylase was described for Cytophaga johnsonae (McKay 1991). C.
johnsonii (sic) produced a cell-associated dextranase (Jansen 1975). The most thorough
study of a Cytophaga species (Chang and Thayer 1977) described a completely cell-
associated cellulose-degrading system with cellulases found in the periplasm, cytoplasm, '
and also membrane-associated. However, the only known studies of xylanases of
cytophagas are those of Haack and Breznak with C. xylanolytica (Haack and Breznak
1993, Haack and Breznak 1992) and Clermont et al. with Sporocytophaga

myxococcoides (Clermont, et al. 1970).

Microorganisms degrade plant material by a variety and/or combination of
strategies. The most well known strategy described is that of enzyme secretion. Examples
include the polysaccharidases of most fungi and actinomycetes (McCarthy 1987,
McCarthy and Ball 1987, Ramachandra, et al. 1987, Wood 1989). Although this strategy
allows organisms to degrade polysaccharides from a distance, it also enables other
organisms to compete for the breakdown products. A second strategy is to keep the
enzymes on or near the outer surface so that the breakdown products released are more
readily accessible for uptake by the degrading organism. The most thoroughly studied
example of this strategy is the cellulose-degrading “cellulosome” of Gram-positive
Clostridium spp. This large cell surface-associated complex is composed of numerous
polysaccharidases and structural proteins (Beguin 1996, Beguin and Aubert 1994,
Lamed, et al. 1987, Lamed, et al. 1983, Lamed, et al. 1983, Leschine 1995) and is
thought to be anchored to the crystalline S-layer located just external to the

peptidoglycan. The cellulosome also appears to help anchor the bacterium to cellulose,

8

allowing prolonged, intimate contact and therefore enhancing its competition for this
substrate. The third, and the most recently described, strategy for polymer degradation is
exemplified by Gram-negative bacteria of the genus Bacteroides (Reeves, et al. 1997,
Salyers, et al. 1996). B. thetaiotaomicron appears to bind starch or maltooligosaccharides
(up to 7 glucose units in length) to its outer surface where they are then projected into the
polysaccharidase-containing periplasmic space. This strategy is unusual since it was
previously thought that only small, e.g., mono— and disaccharides could penetrate outer
membranes. In addition to the three main examples above, there are also organisms that
use combinations of the above strategies (Salyers, et a1. 1996). Although some of these
enzyme systems have only recently been described, others have been studied for over
thirty years (e. g., the cellulase system of C. thermocellum), yet no one system has been

completely defined.

Owing to the complexity of xylans, it is generally assumed that a microorganism
would require a suite of enzymes working in a synergistic/cooperative manner to
completely degrade these heteropolysaccharides to their monomeric constituents. The
degradation of a typical xylan is believed to be accomplished through the action of
endoxylanases (1,4-B-D-xylan xylanohydrolase; EC 3.2.1.8) which cleave randomly
within the xylan backbone, usually at unsubstituted B-1,4-xylosyl linkages; B-xylosidases
(1,4-B-D-xylan xylohydrolase; EC 3.2.1.37) which release xylose through exo-acting
activity on xylooligosaccharides and xylobiose; and the debranching enzymes, which
remove side chain substituents. Debranching enzymes include a-L-arabinofuranosidases
(or-L-arabinofiiranoside arabinofiiranohydrolase; EC 3.2.1.55) which hydrolyze non-

reducing terminal a-1,2- and or-l,3-linked arabinofuranose side groups, glucuronidase

9
(EC 3.2.1.31) that releases a-1,2-1inked glucuronic and (4-0-methyl) glucuronic acid
residues, and xylan acetylesterase or other esterases (EC 3.2.1.6) that remove acetic,
ferulic, or p-coumaric acid 0-1,2- or 0—1,3-linked groups (Biely 1985, Wong, et al.

1988)

Xylanases have been used in studies of plant polysaccharide structure (Nishitani
and Nevins 1989), as animal feed supplements (Bedford 1995, Lewis, et al. 1996,
Selinger, et al. 1996), in bread making (McCleary 1994, Rouau 1993), in paper and pulp
production (Jimenez, et al. 1997, Paice, et al. 1995), for fruit juice clarification (Brillouet,
et al. 1996, Gueguen, et al. 1995), and in nutraceuticals and chemical feedstock
production (Prade 1996, Wong, et al. 1988). Extensive reviews have been written on the
sources and activities associated with xylanases (Bajpai 1997, Biely 1985, Dekker 1985,
Dekker and Richards 1976, Prade 1996, Reilly 1981, Sunna and Antranikian 1997,
Thomson 1993, Wong, et al. 1988), but most authors recognize the lack of information
available on the debranching enzymes, even though all acknowledge the important role

debranching enzymes play in xylan degradation.

The only review on arabinosidases was written in 1984 (Kaji 1984). At that time,
less than a dozen arabinosidase enzymes had been purified and none of the genes
encoding them had been cloned. The arabinosidases were described and classified (in
Enzyme Nomenclature) into two main groups: (i) those enzymes that hydrolyze from the
nonreducing, terminal end of the substrate molecule; and (ii) those enzymes that act

randomly at internal sites. The enzymes of the first group are the a-L-

arabinofuranosidases (a-L-arabinofuranoside arabinofuranohydrolase; EC 3.2.1.55) that

{10

have activity against low molecular weight substrates [e.g., the synthetic substrates
para-nitrophenyl- and 4-methylumbelliferyl-or-L-arabinofirranosides (pNP-AF and MU-
AF, respectively), and arabinooligosaccharides], containing or-l,2- and or-l,3-linked (to
carbons C2 and C3 of the backbone residue, respectively) arabinose side groups. The
second group of arabinosidases are the a-1,5-endoarabinases (EC 3.2.1.99) that effect I

random hydrolysis of the internal Ot-l,5-linked backbone of arabinans.

Kaji further classified the a-L-arabinofirranosidases (ARAFS) into two groups: (i)
the Aspergillus niger type that releases arabinosyl residues from arabinans, arabinoxylan,
and arabinogalactan, but is also active towards the Simple synthetic substrates pNP-AF
and MU-AF and has low or-1,5-endoarabinase activity on arabinans; or (ii) the
Streptomyces putpurascens type that acts on low molecular weight synthetic substrates
and arabinooligosaccharides, but does not act on arabinans, arabinoxylans, or

arabinogalactans.

The above classification was based on information only available for a relatively
few purified ARAFS, and mostly those secreted by, and purified fi'om, actinomycetes and
firngi. Since then, numerous other ARAFs have been purified and/or cloned from
mesophilic and thermophilic bacteria and fungi, as well as several plants (Table 1).
Characteristics of these newer ARAFS have clouded the classical groupings of or-L-
arabinofuranosidases. In particular, a previously purified ARAF (AXH; described as a B-
1,4-D-arabinoxylan arabinofirranohydrolase) from Aspergillus awamori showed
arabinose-releasing activity with arabinoxylooligosaccharides, but had no activity

towards the synthetic substrate pNP-AF, nor did it act on arabinans or arabinogalactans

 

.ll

(Kormelink, et al. 1991). The enzyme did possess weak activity towards arabinoxylans,
but only after prolonged incubations. The authors also described a second, unpurified
ARAF from A. awamori, which had no activity on pNP-AF but did have significant
activity on arabinoxylans. A recently purified ARAF fiom Bifidobacterium adolescentis,
also showed no activity on pNP-AF, nor did it act on sugar beet arabinan, soy
arabinogalactan, arabinooligosaccharides, and arabinogalactooligosaccharides (Van
Laere, et al. 1997). However, the enzyme did possess activity on wheat flour
arabinoxylan, but only on terminal C3-linked arabinosyl groups from double-substituted,
and not single-substituted, xylose residues derived from arabinoxylans. These same
authors describe preliminary findings of enzymes that only release Cz-linked arabinosyl
groups from single-substituted xylose residues and similar activities in T richoderma

reesei (to be published by K. Rakasi).

As is evident fi'om the above discussion, sorely-needed new information about
ARAFS has been slowly accumulating since the last review by Kaji in 1984 (Kaji 1984).
In general, almost all ARAFS have activity on synthetic substrates (typically only the
furanoside configuration) and numerous plant polysaccharides (although with a narrow
substrate specificity). However, as a group they display a variety of pIs, molecular
weights and subunit compositions, pH and temperature optima, K... and V...“ constants,
and cellular location(s). The following review consolidates the information currently
available on ARAFs (Table 1), and the organisms producing them are discussed in more

detail.

12

 

 

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22

Bacillus. Species of the genus Bacillus (Gram-positive endospore-forming bacteria)
secrete a large number of industrially relevant enzymes, including cellulases and
hemicellulases (Priest 1977). However, few have been examined for their xylanolytic
enzymes (Coughlan and Hazlewood 1993). Of those studied thus far, all but one secrete
their arabinofuranosidase enzymes. As examples, ARAF I purified from Bacillus sp. no.
430 was found in the spent growth medium and was able to liberate arabinose from pNP-
AF, sugar beet arabinan, arabinogalactan, and arabinoxylan, thus placing it with the A.
niger type of ARAFs (Yasuda, el al. 1983). The secreted AF 166 purified from B.
polymyxa was a 166 kDa polypeptide thought to consist of two 66 kDa and one 33 kDa
subunits (Morales, et al. 1995). The enzyme had activity on pNP-AF and MU-AF
substrates, but not on arabinoxylans, arabinans, or arabinogalactans, thus placing it with
the S. purpurascens type ARAFS, B. polymyxa also contains a broad range of xylanases
that have either been cloned and/or purified (Sandhu and Kennedy 1986, Yang, et al.
1988), along with a cloned endoxylanase (xynD) that contained arabinofuranosidase
activity (Gosalbes, et al. 1991). The xynD gene product has recently been identified with
the purified ARAFs (AF64 and AF53) of B. polymyxa (Morales, et a1. 1995). These latter
two ARAFs had identical N—terminal amino acid sequences that coincided with the
deduced amino acid sequence from xynD afler processing of the putative peptide signal
sequence. In addition, the authors speculated that AF 53 was a post-translationally
modified product of AF64 created by removal of 100 amino acids from the C-terminal
end. Both enzymes had activity on pNP-AF and MU-AF along with oat spelt- and wheat
flour arabinoxylans, but neither was active on arabinoxylooligosaccharides, birchwood

xylan, arabinan, or arabinogalactan. Studies with two purified xylanases from

23

B. pob/myra (Morales, et al. 1995), in combination with either ARAF, yielded greater

overall xylanase activities, but no net increase in arabinose release.

B. subtilis F-ll secreted two ARAFs and an endoarabinase when grown on sugar
beet residue (Weinstein and Albersheim 1979). One ARAF (II) was purified and shown
to possess activity on pNP-AF, as well as an exo-activity on branched arabinans, but no 1
activity upon a multitude of other synthetic or natural substrates (tested substrates
included polysaccharides typically found in plant cell walls or synthetic mimics of their
linkages). A secreted ARAF purified from B. subtilis 3-6 was specific for the fiiranoside
linkage and released arabinose from arabinan and two of three highly purified
arabinoxylooligosaccharides [i.e., xylobiose (A1X2) and xylotriose (A1X3), but not
xylotetraose (A1X4); (Kaneko, et al 1994)]. The authors noted that the enzyme did not fit
well into either of the two ARAF classes. They suggested additional research was
needed with previously purified ARAFs to determine their activity on more defined

substrates in order to establish a more refined ARAF classification scheme.

A thermophilic Bacillus, B. stearothermophilus L1, produced an ARAF that had a
temperature optimum of 70°C, a native molecular mass of 110 kDa, and which consisted
of 52.5 and 57.5 kDa subunits; and is only the second ARAF determined to consist of
heterogeneous subunits (Bezalel, er al. 1993). When the ARAF from strain Ll was
combined with a purified xylanase from strain Ll, synergy was demonstrated on
softwood kraft pulp by the 25% increase in the release of lignin, as compared to that
released by the sum of the individual activities. B. stearothermophilus T-6 produced a
mostly cell-associated ARAF with a native molecular mass of 256 kDa that was

composed of four identical 64 kDa subunits (Gilead and Shoham 1995). The temperature

 

24

optimum for the T-6 ARAF was the same as that from B. stearothermophilus L1, but the
first 50 N-terminal amino acids had 45.8% identity with an N-terminal region of an
ARAF encoded by the abfA gene of Streptomyces lividans 66 (accession no. U04630).
An unusual aspect of the T-6 ARAF was its weak activity on carboxymethylcellulose
[(CMC), a soluble derivative of cellulose]. A T-6 xylanase exhibited synergy with the
ARAF during hydrolysis of oat spelt arabinoxylan. About 60% of the ARAF activity was
cell-associated, and only 9% of the cytoplasmic marker, isocitrate dehydrogenase, was
found in the culture supernatant. Apparently, cell lysis was not the only explanation for

the extracellular ARAF activity.

Bacteroides. The genus Bacteroides are Gram-negative anaerobes that have been studied
mostly for their plant polysaccharide fermenting capabilities (Salyers, er al. 1977). Three
xylan-degrading genes were cloned fi'om B. ovatus V975 and found to cluster in a 3.8
kilobase region of the genome (Whitehead and Hespell 1990). A xylosidase with ARAF
activity was partially purified fi'om the cytoplasmic fraction of an E. coli JM83 clone.
The enzyme was thought to be the first bifunctional xylan-degrading enzyme, i.e.,

possessing both xylosidase and arabinofuranosidase activities.

Rumen bacteria. The rumen anaerobe, Bacteroides wkmolyticus XS-l, produces a
mainly cell-associated ARAF that consisted of six identical 61 kDa subunits forming a
364 kDa native enzyme (Schyns, et al. 1994). The purified enzyme had activity on short
arabinooligosaccharides, but not oat spelt arabinoxylan, arabinogalactan, or other aryl-
glycosides. The anaerobe Butyrivibrio fibrisolvens GSI 13 produces an entirely
cytoplasmic ARAF (Hespell and O'Bryan 1992) with a native molecular mass of 240 kDa

and composed of eight identical 31 kDa subunits. The enzyme had activity on oat spelt-

.25

and corn endosperm xylan, and sugar beet arabinan, but not arabinogalactan. The
cytoplasmic location for ARAF suggested that its role was in removal of arabinose from
arabinose-containing xylooligosaccharides generated by the action of other xylan-

degrading enzymes and subsequently taken up into the cytoplasm.

A close phylogenetic relative of the Bacteroides, F ibrobacter succinogenes, is an
anaerobic bacterium that produces a number of secreted xylan-degrading enzymes
(Forsberg, et al. 1981). A purified debranching endoxylanase that contained ARAF
activity had no activity on arabinans or synthetic ARAF substrates, but did release
arabinose from oat spelt- and rye arabinoxylans and apparently did so before hydrolysis
of the xylan backbone (Matte and Forsberg 1992). Ruminococcus albus 8 secretes a
glycosylated ARAF (qt-AF) of 305-310 kDa that consists of 75 kDa subunits (Greve, et
al. 1984). a— AF was shown to act synergistically with a xylanase from R. albus 8 in

hydrolyzing alfalfa cell walls.

Clostridium. The genus Clostridium includes anaerobic Gram-positive, endospore-
forming, rod-shaped bacteria well known for their polysaccharide degrading capabilities
(Bronnenmeier 1993, Hazelwood 1993). C. acetobutylicum ferments simple sugars to the
solvents acetone, isopropanol, butanol, and ethanol. The organism also produces a variety
of xylan-degrading enzymes including a secreted ARAF that consists of a single 94 kDa
polypeptide (Lee and Forsberg 1987). The ARAF had no activity on
arabinooligosaccharides with degrees of polymerization from 2 to 6. The enzyme
released arabinose fi'om arabinan, but had no activity on larchwood-, oat spelt-, and rye

arabinoxylans, CMC, and arabinogalactan. Cooperativity with a xylosidase and/or

 

26

xylanase from the same strain was demonstrated with ARAF when used individually or
in combination. A thermophilic Clostridium, C. stercorarium, produces a number of
xylanolytic enzymes including two ARAFs encoded by genes arfA and aer (Schwarz, et
al. 1990). The arng gene product was bifunctional, possessing both ARAF and xylosidase
activities, whereas the aer gene product was an ARAF specific for the furanoside ‘
configuration and was found to be the major ARAF activity in culture supernatants

(Schwarz, et al. 1995).

Pseudomonads. An interesting feature of the ARAF encoded by xynC cloned from the
soil-saprophyte Pseudomonas fluorescens subspecies cellulosa was that it contained a
cellulose-binding domain [CBD; (Kellett, er al. 1990)]. Cellulose-binding domains have
been described for a number of cellulases and xylanases (Ferreira, et al. 1993, Gilkes, et
al. 1991, Kellett, et al. 1990), and although their role has not been entirely defined, CBDs
are believed to enhance contact of the enzyme with cellulose. Although a number of
organisms in Table 1 have been found to produce cellulases with cellulose-binding
domains (e. g. , F ibrobacter succinogenes, Thermomonospora fusca, T richoderma reesei,
and P. fluorescens (Kellett, et al. 1990), the xynC gene product is the only ARAF so far
described to contain a CBD. The author’s speculate that the CBD could promote longer
contact between the secreted ARAF and cellulose where it would have access to any

nearby arabinose-containing polysaccharides.

Actinomycetes. Various actinomycetes have been recognized and studied for their xylan-
degrading enzymes. The genus Streptomyces contains filamentous prokaryotes found

mainly in soils and decomposing vegetation that produce a wide variety of

2'7

hemicellulolytic enzymes (McCarthy 1987, McCarthy and Ball 1987, Zimmerman 1989).
Of the many Streptomyces possessing ARAF activities, eight strains were studied in
detail and most were found to produce multiple ARAFs. Ten ARAFs were purified
and/or cloned, including one debranching endoxylanase (Table l). Streptomyces sp. no.
17-1 produced a secreted ARAF, whereas S. diastatochromogenes 065 produced two
ARAFS, one secreted and one intracellular [(Higashi, et al. 1983, Kaji, et al. 1981),
respectively]. S. diastolicus produced an apparent multiplicity of secreted ARAFS, two of
which were purified and characterized (Tajana, et al. 1992). Two ARAF-encoding genes
(abfA and abe) were cloned from S. lividans strain 1326 [(Manin, et al. 1994, Vincent, et
al. 1997), respectively]. The cloned abfA gene, which lacked a deduced putative signal
peptide, was expressed in S. lividans IAF 10-164 (3 cellulase-, xylanase-, and
arabinosidase-negative mutant) and purified from the cytoplasmic fraction. It was found
to be a 380 kDa polypeptide consisting of 69 kDa subunits. Its role was thought to
remove arabinose fiom arabinose-containing xylooligosaccharides produced by the action
of other xylan-degrading enzymes and taken up into the cytoplasm. The abjB gene was
also expressed in the same host system as abfA and was secreted into the culture
supernatant (Vincent, et al. 1997). The deduced amino acid sequence showed that the
enzyme contained a catalytic domain and a xylan-binding domain (XBD). XBDs have
only recently been described for xylanases and the abjB gene is the first ARAF
determined to contain one (Black, et al. 1995). Other actinomycetes that also produced
secreted ARAF activities include Streptomyces massasporeus [an A. niger type of ARAF;
(Kaji, et al. 1982)], S. purpurascens [the largest reported ARAF of 495 kDa, consisting

of 62 kDa subunits; (Komae, et al. 1982)], S. roseiscleroticus [a debranching

28

endoxylanase; (Grabski and Jeffries 1991)], and Thermomonospora fitsca [producing
multiple xylanases with cooperative/synergistic activities; (Bachmann and McCarthy

1991)].

Fungi. Fungi are by far the largest group of ARAF-producing organisms represented in
Table 1. They have long been known for their hydrolytic activities, and in 1928, Ehrlich
and Schubert reported the presence of arabinose-releasing activity in Takadiastase
(Ehrlich and Schubert 1928). In 1967, the first ARAF was purified and characterized
from Aspergillus niger (Kaji, et al. 1967). Since then, at least 32 cloned and/or purified
ARAF enzymes have been described from a number of different fiingi (Table 1). As was
stated before, all but one of the ARAFs were secreted. Two ARAFs were purified from A.
awamori, including the one described before for its inability to act on pNP-AF
(Kormelink, er al. 1991). The second enzyme showed two isozymic forms by isoelectric
focussing, acted synergistically with an endoxylanase purified from the same strain, and
has the unique distinction of being the only known ARAF to release feruloyl- and p-
coumaroyl-linked arabinose substituents from wheat straw arabinoxylan (Wood and
McCrae 1996). Two strains of A. nidulans produced two different ARAFs. Strain WGO96
produced an ARAF B that was antigenically similar to the ARAF B of A. niger N400
(Ramon, et al. 1993). Other species and strains of Aspergillus had ARAFs with a range of
sizes and activities (Table 1). Comparison of some of these show striking similarities
such as those between the AXH of A. awamori and the AXH of A. tubingensis
[(Gielkens, et al. 1997, Kormelink, et al. 1993), respectively]. All the ARAFs produced
by these fungi were secreted, except that of A. niger 5-16. The ARAF from this organism

was found intracellularly and had activity on pNP-AF and A1X2, but not on gum arabic,

29

arabinoxylan, A1X3, or AK. (Kaneko, et al. 1993). Examination of Table 1 shows the
enzyme also to have similar properties to the ARAF B of A. niger N400 and ARAF B of
A. nidulans WGO96. All the ARAFs from fungi showed acidic pH optimums and nearly
all had acidic or neutral pls with the exception of A. terreus. Three ARAFs were purified

from A. terreus and all three were shown to possess basic pls (Luonteri, er al. 1995).

Several ARAFs were purified and cloned from various fungi including a secreted
ARAF fi'om the plant pathogen Cochliobolus carbonum that, when purified, digested
with a proteinase, and the resulting fragments sequenced, yielded two peptide fragments
with amino acid sequence identities of 56 and 64% to a cloned
xylosidase/arabinofuranosidase from Bacteroides ovatus (accession no. U04957;
(Ransom and Walton 1997)). Other secreted ARAFs purified and characterized from
firngi include those from the plant pathogens Monilinia fiuctigena (Kelly, et al. 1987),
Phytophthora palmivora (Akinrefon 1968), Sclerotiniafmctigena (Laborda, et al. 1973 ),
and Sclerotim'a sclerotiorum (Baker, et al. 1979), the white-rot fungus Dichomitus
squalens (Brillouet and Moulin 1985), and a partially purified ARAF from the

thermophilic fungus Mermaascus aurantiacus (Roche, et al. 1994).

Yeast. A secreted ARAF was purified and characterized fi'om the yeast, Rhodotorula
flava, and found to have activity on pNP-AF, branched and linear beet arabinans, and

arabinoxylan (Uesaka, et al. 1978).

Plants. A few ARAFs have been purified from carrot cell cultures, germinating soybean
and lupin cotyledons, Japanese pears, radish seeds, and partially purified from callus

tissue. The polysaccharidases found in plants are thought to be involved with seed

,30

germination, cell growth, expansion, and maturation, although their biological role has
yet to be determined (Cleland 1981). The most thoroughly investigated ARAF was that
purified from a cell homogenate of carrot cell culture (Konno, et al. 1987). The enzyme
possessed arabinose-releasing activity on pNP-AF, arabinans, and carrot pectic
polysaccharide fractions (containing arabinosyl residues), but not on larch
arabinogalactan, and had physicochemical characteristics similar to ARAFs from other
organisms (Table 1). Examination of the ARAF activity present in germinating soybean
cotyledons revealed several isozymic forms and it was suggested that these isoenzymes
may contribute to the metabolism of seed stored- or structural polysaccharides through
their different characteristics and compartmentalization (Hatanaka, et al. 1991). One
isoenzyme was purified and had substrate specificity towards pNP-AF, arabinan, and
arabinose-containing soybean polysaccharides, but had no activity towards
arabinogalactans or arabinoxylans. On germination of lupin seeds, the growth of
cotyledons is associated with the depletion of intercellular- and cell wall polysaccharides
[in particular, those containing arabinose and galactose constituents (Matheson and Saini
1977)]. Two cell-associated ARAFs (I and H) were purified from germinating seed
extracts and found to be specific for the furanoside configuration of pNP-AF and MU-AF
substrates and released arabinose from several lupin polysaccharide fractions. The cell-
associated ARAF purified from extracts of Japanese pear fruits had the second highest K...
reported for all purified ARAFs (Table l), and its activity increased dramatically during
fi'uit ripening. The ARAF activity purified from radish seed extracts was a bifunctional
xylosidase/ARAF that was thought to use a single catalytic site for both activities (Hata,

el al. 1992). The only plant thought to secrete an ARAF was Scopoliajapanica, since its

31

ARAF was partially purified fi'om suspension culture medium of the calluses of the plant.
ARAF activity was detected in the roots, stems, and leaves of the plant during flowering.
The apparently secreted ARAF was in contrast to the other intracellular
polysaccharidases and was thought to be involved in cell wall softening during cell

growth.

From the above discussion, one can deduce that the substrate specificities of
purified ARAFs are complex and varied. Comparisons of physical attributes of ARAFs
have been relatively straight forward, but comparisons of their activities have been much
more difficult. This is due in part to their varying substrate preferences, and also to the

fact that different researchers rarely test purified ARAF s on exactly the same substrate(s).

In as much as little is known about complex, cell-associated xylanase systems, or
about biopolymer-degrading enzymes in cytophagas (a group of bacteria widely
recognized for such activity), the aim of the present study was to characterize some of the
major components of the cell-associated xylanase system from Cytophaga xylanolytica.
Herein I report on the isolation, characterization, and localization of a major
a-L-arabinofuranosidase component (Arfl) and its interaction with partially-purified
endoxylanase components. Also included is the amino acid sequence of Arfl, as deduced
from the nucleotide sequence of the cloned gene (arfl') encoding it, as well as the deduced
amino acid sequence of a second ARAF (ArflI) encoded by a gene (arfll) not expressed

by C. xylanolytica under our grth conditions.

CHAPTER 2

PURIFICATION AND PROPERTIES OF AN uni-ARABINOFURANOSIDASE
FROM C ytophaga xylanolytica

32

33

ABSTRACT

An a-L-arabinofirranosidase (a-L-arabinofuranoside arabinofuranohydrolase;

EC 3.2.1.55; referred to herein as Arfl) was extracted from cells of Cytophaga
xylanolytica strain XM3 by using Triton X-100 and was purified 85-fold by anion-
exchange and hydrophobic interaction column chromatography. The native enzyme had a

p] of 6.1 and an apparent molecular mass of 160-210 kDa, and it appeared to be a trimer

or tetramer consisting of 56 kDa subunits. With p-nitrophenyl-a-L-arabinofuranoside as
substrate, the enzyme exhibited 3 Km of 0.504 mM and a Vmax of 3 l9 umol - min'l - mg

protein“, and it had optimum activity at pH 5.8 and 45°C. Arfl was relatively stable over
a pH range of 4 to 10 and at temperatures up to 45°C, and it retained nearly full activity
when stored at 4°C for periods as long as 24 months. The enzyme also released arabinose
from 4-methylumbelliferyl-a-L-arabinofuranoside, as well as from rye-, wheat-, corn
cob-, and oat spelt arabinoxylans and sugar beet arabinan, but not arabinogalactan. Arfi
showed no hydrolytic activity towards a range of p-nitrophenyl- or 4-methylumbelliferyl-
glycosides other than arabinoside, for which it was entirely specific for the
a-L—furanoside configuration. Arfl interacted synergistically with three partially purified
endoxylanase fi'actions from C. xylanolytica in hydrolyzing rye arabinoxylan. However,
cell fi'actionation studies revealed that Arfl was largely, if not entirely, cytoplasmic, so its
activity in vivo is probably most relevant to hydrolysis of arabinose-containing
oligosaccharides small enough to pass through the cytoplasmic membrane. Antibodies

prepared against purified Arfl also cross-reacted with arabinofuranosidases from other

34

freshwater and marine strains of C. xylanolytica. To our knowledge, this is the first

a-L-arabinofiiranosidase to be purified and characterized from any gliding bacterium.

,35

INTRODUCTION

Xylans are included within the family of plant cell wall heteropolysaccharides
referred to as hemicelluloses. They consist of a B-l,4-linked xylopyranose backbone, to
which are often attached side groups of arabinose, (0-methyl-) glucuronic acid, ferulic or
p-coumaric acid, and/or acetate, depending on the plant source (Aspinall 1959, Timell
1967). Next to cellulose, xylans are the most abundant polysaccharides on earth (Biely
1985), and it has been estimated that 1010 metric tons are recycled annually, with the

degradative arm occurring largely through the action of microbes (W ilkie 1983).

In an effort to increase our understanding of the microbiology and biochemistry of
xylan degradation, we initiated a study of the xylan-hydrolyzing multienzyme system
(i.e., the "xylanase system") of a new, anaerobic gliding bacterium, Cytophaga
xylanolytica strain XM3 (Haack and Breznak 1993). Strain XMB and other fieshwater
and marine strains similar to it were isolated fi'om freshwater sediments on the basis of
their ability to adhere to, and dominate the initial fermentation of, insoluble xylan
particles in sulfidogenic and methanogenic enrichment cultures. Unlike the secreted
xylanases found with most other organisms, the xylanase system of C. xylanolytica was
almost entirely cell-associated, but it can be easily extracted fi'om whole cells by using
0.2% Triton X-100. Such Triton extracts were shown to possess activities important for
xylan hydrolysis, including endo-1,4-B-D-xylanase (EC 3.2.1.8), B-D-xylosidase (EC
3.2.1.37), or-L-arabinofirranosidase (EC 3.2.1.55), and or-D- and B-D-glucopyranosidases
(EC 3.2.1.20 and EC 3.2.1.21, respectively) (Haack and Breznak 1992). Triton extracts
were remarkably stable, retaining full xylanolytic activities for more than 6 months when
stored at 4°C; however, they exhibited no activity toward microcrystalline cellulose, ball-
milled Whatman no. 1 cellulose filter paper, or carboxymethyl cellulose (CMC) (Haack
and Breznak 1992). The latter observations were consistent with the inability of cells to

grow on cellulose or CMC.

,36

Efforts to resolve the nature and number of components of the xylanase system
yielded several column chromatography fractions each enriched with multiple
endoxylanase (ENDOX) activities, as well one fraction enriched with a single
arabinofuranosidase (ARAF) activity. ARAFs are important, because they catalyze the
hydrolysis of arabinofuranosyl residues from hemicellulosic polysaccharides (Kaji 1984).
Such residues are typically attached by a-l,2- and/or a-l,3-linkages to the backbones of
arabinoxylans, arabinans, and arabinogalactans (Biely 1985, Dekker and Richards 1976),
and their removal by ARAFs usually facilitates the attack of the xylan backbone by
ENDOXs (Greve, et al. 1984, Lee and Forsberg 1987, Puls and Poutanen 1989, Wong, et
al. 1988). Here we report the purification and characterization of the apparently only
ARAF (designated herein as Arfl) produced by C. xylanolytica XM3 when it is growing
on arabinoxylan. Also, included are the results of studies performed to (i) determine the
cellular location of Arfl, (ii) examine synergy between Arfl and ENDOXs of
C. xylanolytica, and (iii) evaluate the occurrence of Arfl (or antigenically similar
enzymes) in other freshwater and marine strains of C. xylanolytica. (A preliminary report

of the findings has been presented previously [Renner and Breznak 1995]).

37

MATERIALS AND METHODS

Growth of cells and preparation of cell extracts. C. xylanolytica strain XM3 (= DSM
6779) and other fi'eshwater strains were grown anoxically at 30°C in rubber-stoppered
2 L Pyrex bottles nearly filled with Nags-reduced, COz/bicarbonate-bufi‘ered freshwater ‘
mineral medium no. 2 containing 0.2% (wt/vol) oat spelt arabinoxylan (preextracted with
70% [vol/vol] ethanol) as the sole fermentable substrate (Haack and Breznak 1993).
Marine strains were grown in a similar manner, except that oat spelt arabinoxylan was
used as substrate in marine medium (Haack and Breznak 1993).

For enzyme purification, cells of strain XMB were harvested from late-
exponential- to early-stationary-phase batch cultures by centrifugation at 10,000 x g for
20 min at 4°C. The cell pellets were resuspended in 0.2% (wt/vol) Triton X-100 to 1/40th
of the original culture volume and stirred for 2 h at 4°C. The treated cells were then
removed by centrifugation at 10,000 x g for 30 min at 4°C, and the resulting supernatant
fluid was firrther centrifuged at 100,000 x g for 2 h at 4°C. The supernatant fluid from the
latter centrifugation step was placed into a 3500 molecular weight cut-ofi‘ (MWCO)
dialysis membrane and dialyzed against 50 mM N-[2-hydroxyethyl]piperazine-N'-2-
ethanesulfonic acid (HEPES) buffer (pH 8.0) (ca. 4 L total) at 4°C. The dialyzed material,
hereafter referred to as the ”Triton extract," was stored at 4°C until use.

Sonicated cell extracts were prepared from cells harvested by centrifugation at
18,000 x g and then resuspended in 10 mM HEPES buffer (pH 6.8). Sonication was for
eight 30 s bursts [output 5 and 50% duty from a 1/8” diameter tapered horn; Branson

Sonifier model 450 (Danbury, CT)], followed by recentrifugation.

38

Purification of Arfl. Purification of Arfl from Triton extracts was done at room
temperature (RT) by a low-pressure column chromatography procedure performed with

an Econo System apparatus (Bio-Rad Laboratories, Hercules, CA) operating at a flow

rate of 1 ml - min‘l (unless otherwise noted). Eluted fractions were monitored for protein
content, by measuring the A230 and for ARAF activity by using a semi-quantitative
microtiter plate assay (see below). Sequential column chromatography was performed as
follows: (i) Triton extract (204 ml, 430 mg protein) was applied to a column of DEAE

cellulose (2.5 x 50 cm) previously equilibrated with 50 mM HEPES buffer (pH 8.0). The

column was washed with 1 L of equilibration buffer at a flow rate of 2 ml ° min], and
elution of Arfl was achieved by using a linear gradient of 0 to 1 M NaCl in the same
buffer. Fractions containing Arfl eluted fi'om the column between 50 and 200 mM NaCl
and were pooled. The pooled material was concentrated by ultrafiltration through a YM
10 membrane having a 10 kDa MWCO (Amicon Co., Danverse, MA), and in the process
the buffer was changed to 20 mM HEPES (pH 8.0); (ii) The Arfl pool from step (i) (75
ml, 180 mg of protein) was applied to a DEAE Sephadex A-50 column (1.5 x 50 cm)
previously equilibrated with 20 mM HEPES (pH 8.0), and elution was performed with
linear gradient of 0 to IM NaCl in the same buffer. Fractions containing Arfl eluted from
the column between 0.1 to 0.4 M NaCl: these were pooled, dialyzed, and concentrated as
before, except that the dialysis buffer was 20 mM potassium phosphate (pH 6.5); (iii) The
Arfl pool from step (ii) (75 ml, 74 mg of protein) was applied to a hydroxylapatite
column (1.5 x 50 cm) previously equilibrated with 20 mM potassium phosphate buffer
(pH 6.5), and chromatography was performed with a linear gradient of 20 to 300 mM

potassium phosphate. Fractions containing ArfI eluted from the column between 170 to

39

225 mM phosphate and were pooled; (iv) A solution of 5 M NaCl was added to the Arfl
pool from step (iii) (43 ml, 7.7 mg of protein) to achieve a final concentration of l M
NaCl. The pool was then applied to a Phenyl-Sepharose CL-48 column (1.0 x 50 cm)
previously equilibrated with 1 M NaCl in 5 mM 3-[N-morpholino]propanesulfonic acid
(MOPS) buffer (pH 6.5). Elution of Arfl was performed with a simultaneously decreasing
linear gradient of 1 to 0 M NaCl and an increasing pH gradient of 6.5 to 8.0 in 5 mM
MOPS buffer. Arfl activity eluted between 250 to 0 mM NaCl and pH 7.5 to 8.0 and
relevant fractions were pooled; (v) The ArfI pool from step (iv) was concentrated by
ultrafiltration (as above), and the buffer was changed to 20 mM MOPS (pH 7.5). This
concentrated pool of Arfl (74.5 ml, 1.7 mg of protein) was then applied to a Macro Prep
SOQ column (1.0 x 50 cm) previously equilibrated with 20 mM MOPS (pH 7.5).
Chromatography was performed with an increasing linear gradient of 0 to l M NaCl in
the same buffer. Active fractions eluted between 50 to 100 mM NaCl and were pooled;
(vi) The Arfl pool from step (v) was dialyzed, as before, against 20 mM MOPS (pH 8.0)
containing 0.5 M NaCl, and the dialyzed pool (91 ml, 1.6 mg of protein) was
rechromatographed on a column of Phenyl-Sepharose CL-4B (1.5 x 50 em), but this time

with a decreasing linear gradient of 0.5 to 0 M NaCl in the same buffer and a flow rate of
0.5 ml - min". Active fractions eluted between 25 and 0 mM NaCl and were pooled.
Ultrafiltration was then used to concentrate the final pool to 28.8 ml (1.2 mg of protein),

which was stored at 4°C.

Partial Purification of ENDOXs. Components from three major electrophoretically-
separable zones of ENDOX activity [i.e., top, middle, and bottom zone after native

polyacrylamide gel electrophoresis (PAGE) gels of Triton extracts (see Results)] were

40

partially purified from a Triton extract by using a four step procedure. The first three
steps involved sequential column chromatography on the following matrices/eluants by
using conditions as described above for ArfI: (i) DEAE cellulose/T[NaCl]; (ii)
Hydroxylapatite/T[PO4'3]; and (iii) Macro Prep 50Q/T[NaCl]. A significant amount of
ENDOX activity in the major peak eluting from step (i) did not bind to the '
hydroxylapatite column of step (ii) and eluted in the void volume. This material (referred
to as ENDOX 11) was enriched with ENDOX components that migrated in the middle
zone of native PAGE gels and was saved. Steps (ii) and (iii) were effective in removing
some additional contaminating proteins from the remaining ENDOX activity (which
consisted of components from the “top” and “bottom” zones of native PAGE gels), but
did not resolve these. Therefore, an additional step (step iv) was included and involved
preparative native PAGE of the remaining ENDOX activity through a 28 mm (inside
diameter) Model 491 Prep-Cell column (Bio-Rad) containing a 2 cm (4% [wt/vol]
polyacrylamide) stacking gel and 10 cm (10% [wt/vol] polyacrylamide) resolving gel
(Davis 1964). Electrophoresis was done according to the manufacturer’s
recommendations, and fiactions were screened for ENDOX activity by release of
reducing sugar from xylan (see below) and by native PAGE slab gels subsequently
overlaid with cat spelt arabinoxylan zymogram gels (see below). Step (iv) separated the
remaining ENDOX components into a group that migrated furthest during native PAGE

(referred to as ENDOX III) and a group that migrated least (referred to as ENDOX I).

Enzyme assays. ARAF activity was determined semi-quantitatively by a modification of
the Bachmann and McCarthy method (Bachmann and McCarthy 1991), as follows: 1.0 ul

portions of enzyme (e.g., column chromatography fractions) were added to separate wells

.41

of a 96 well microtiter plate (Corning Cell Wells; Corning Glass Works, Corning, NY)

which also contained 90 ul of 4-methylumbelliferyl-a-L—arabinofirranoside (MU-AF)

solution (10 ug - ml") per well. After various periods of incubation (from 10 min to 24 h
at RT, depending on the relative activity) the microtiter plates were placed on a UV
transilluminator, and active fractions were identified by the intense fluorescence of
liberated methylumbelliferone.

Quantitative assays for ARAF activity were performed as described by Greve et
al. (Greve, et al. 1984) with 1 ml total volume reaction mixtures containing 1 mMpara-
nitrophenyl-or-L-arabinofuranoside (pNP-AF) in 50 mM 2-(N-morpholino)ethanesulfonic
acid (MES) buffer (pH 6.0) plus enzyme. Reaction mixtures lacking enzyme were pre-
warmed to 45°C, and reactions were started by the addition of 20 ul of appropriately
diluted enzyme (0.24 to 42 mg of protein) also pre-warmed in the same buffer (the actual
pH of the reaction mixture at 45°C was determined to be 5.8). After 1 min, the reaction
was terminated by the addition of 2 ml of 1 M NI-LOH, and the A...» of the resulting
solution was measured with a Bausch & Lomb Spectronic 20 colorimeter. Absorbance
readings were converted to micromoles of p-nitrophenol by comparison to a stande
curve. One unit of ARAF activity was defined as the amount of enzyme that produced 1
umol of p-nitrophenol per min under the assay conditions. Catalytic constants of purified
ArfI were determined in a similar way, but by using triplicate reaction mixtures each
having a total volume of 4.5 ml and containing: 50 mM MES (pH 6.0); pNP-AF ranging
in concentration from 25 uM to 5 mM; and 0.06 Units (U) of Arfl in 50 mM MES [pH
6.0]. Periodically during incubation, 1 m1 samples were removed and added to a separate

tube containing 2 ml of 1 M NI-LOH, after which the A405 was determined as above.

42

Michaelis-Menten kinetic parameters were determined by using the method of Wilkinson
(Wilkinson 1961).

Glycosidase activities other than ARAF, as well as acetyl esterase, were
determined by using other p—nitrophenyl- or 4-methylumbelliferyl- derivatives as
substrates and were assayed either quantitatively, as described above for pNP-AF, or
semi-quantitatively by the microtiter plate assay as described above for MU-AF. The p-
nitrophenyl- derivatives tested included (each at 2.5 mM final concentration): a—L- and B-
L—arabinopyranoside; or-D- and B-D-glucopyranoside; a—D— and B-D-galactopyranoside; or-
L-rhamnopyranoside; a-L-fucopyranoside; B-D-lactopyranoside; B-D-xylopyranoside; B-
D-cellobioside; a-D-mannopyranoside; B-D-glucuronide; and acetate. The 4-
methylumbelliferyl- derivatives (each at 5 mM final concentration) included: or-L-
arabinopyranoside; or-D- and B-D-glucoside; or-D- and B-D-galactoside; B-D-glucuronide;
B-D-cellobioside; and acetate.

The ability of purified Arfl to release arabinose from hemicellulose substrates was
tested by incubating (40°C for 48 h) 0.52 U of enzyme in 1 ml reaction mixtures buffered
with MOPS (10 mM, pH 6.5) and containing (10 to 25 mg per reaction): Lenzing
beechwood-, rye-, wheat-, and cat spelt (arabino)xylans; three corn cob arabinoxylan
fractions (CCXA [containing 5.6% arabinose, 87.7% xylose, 3% glucose, 0.1% galactose,
and 3.6% other], CCXB [containing 15.3% arabinose, 77.1 % xylose, 2.7% galactose,
and 4.9% glucose], and CCX [containing 34.8% arabinose, 57.3% xylose, 7.2%
galactose, 0.5% glucose, and 0.2% other] all corn cob arabinoxylan fi’actions were
prepared and analyzed at the Agricultural Research Service, U. S. Department of

Agriculture, Peoria, IL); arabinogalactan; and sugar beet arabinan. Enzyme-free substrate

43

controls were also incubated under identical conditions. At the end of incubation, reaction
mixtures were centrifuged at 11,000 x g for 15 min to sediment particulate material. The
resulting supernatant fluids were then brought to 70% ethanol by the addition of 100%
absolute ethanol, and recentrifirged. These second supernatants were removed to fresh
microcentrifuge tubes and lyophilized. When dried, the samples were redissolved in 50 ul
of H20, and 1-5 [11 of each was spotted onto a 20 x 20 cm thin-layer chromatography
plate pre-coated with a 250 um layer of Whatman silica gel (K5) 150A. The plate was
placed in a glass enclosed tank for chromatography with a solvent mixture of n-butanol:
acetic acid: H20 (2:1:1; vol/vol/vol). Lanes containing authentic arabinose, xylose,
xylobiose, xylotriose, xylotetraose, and xylopentaose (10 ug each) were included as
standards. Alter chromatography, the plate was air-dried, then sprayed uniformly with ca.
4 ml of aniline-diphenylamine reagent (Sigma) and developed by heating at 85°C for 20
min. Sugars gave blue-green or brown spots.

ENDOX (or any other enzyme activity capable of hydrolyzing xylan, or portions
of xylan) was assayed by measuring the release of reducing sugar from Lenzing
beechwood-, oat spelt-, and rye (arabino)xylan (0.5 mg - ml'l [wt/vol] final
concentration). Reducing sugar was quantified by the method of Nelson (Nelson 1944),
as modified by Somogyi (Somogyi 1952), with xylose as standard. Samples were boiled
for 30 min with the c0pper reagent for optimal detection of reducing sugars. One unit of
activity is defined as the amount of enzyme liberating 1 mo] of reducing sugar
equivalent (as xylose) per minute under the assay conditions. Synergy between Arfl and

ENDOX pools was determined by using reaction mixtures containing rye arabinoxylan [2

mg - ml", in 10 mM HEPES buffer (pH 6.8) containing 0.5 mM CaClg) and various

44

amounts of each enzyme (pool). Assays were run for 90 min, during which time aliquots
were removed for determination of reducing sugar as described above.
Glyceraldehyde-3-phosphate dehydrogenase was determined by the method of
Velick (Velick 1955), as modified by Hespell and Canale-Parola (Hespell and Canale-
Parola 1970), by using 1 m1 reaction mixtures containing: 150 mM HEPES (pH 8.4); 2
mM nicotinamide adenine dinucleotide (NAD); 10 mM D,L-glyceraldehyde 3-phosphate;
50 mM KHzAsO4; 10 11M dithiothreitol (DTT); and appropriately diluted enzyme. The
reaction (at 25°C) was started by the addition of enzyme and followed continuously for 2
min at 340 nm in a Perkin-Elmer Lambda 14 spectrophotometer (Norwalk, CT) equipped
with a thermal jacketed cuvette holder. The level of reduced NAD was estimated using
the molar extinction coefficient (1 cm light path) of 6.22 x 103 at 340 nm (Horecker

1948)

Determination of pH and temperature optima and stability. The pH optimum for Arfl
activity was determined by using reaction mixtures buffered over the range indicated (in
0.5 pH increments) with: sodium citrate, pH 3 to 6; MES, pH 5.5 to 6.5; and MOPS, pH
6.5 to 8.0. Pre-wanned (45°C) reaction mixtures (4 ml, final volume) contained 3.575 ml
of 50 mM buffer and 400 pl of 10 mMpNP-AF and were initiated by adding Art] (25 ul
containing 0.026 U in 2 mM MES buffer [pH 6.0], and 1 ml aliquots were removed at 2
min intervals for assay of p-nitrophenol (above). A similar procedure was used to
determine the temperature optimum of Arfl (5 i 0.2°C increments over the range of
temperatures from 5°C to 70°C). Final concentrations of reactants in these 2.5 m1 reaction

mixtures were: 10 mM MOPS (pH 6.5); 2 mMpNP-AF; and 0.065 U enzyme.

, 45

To determine the stability of Arfl when exposed to various pHs, 50 ul of purified
Arfl (0.26 U in 10 mM MES [pH 60]) was added to 50 ul of the following buffers (all at
50 mM concentrations and in 0.5 pH increments): acetate, pH 4.0 to 5.5; MES, pH 5.5 to
6.5; MOPS, pH 6.5 to 8.0; N-tris[hydroxymethyl]methyl-3-arninopropanesulfonic acid
(TAPS), pH 8.0 to 9.0; and 2-[N-cyclohexylamino]ethanesulfonic acid (CHES), pH 9.0 to
10.0. Control tubes containing 100 pl of 10 mM MES buffer (pH 6.0) and 100 pl of each
50 mM test buffer were checked to ensure that ArfI was being exposed to the intended
pH. Tubes were incubated at RT, and after 24 and 72 h residual activity was determined
by the routine pNP-AF procedure (above). To determine the stability of ArfI to various

temperatures, 400 [.11 samples of purified Arfl (0.52 U in 10 mM MES [pH 6.0]) were

incubated for 1 h at temperatures from 25 to 65°C in 5 :1: 0.2°C increments. After
incubation, residual enzyme activity was determined by using the pNP-AF procedure

(above).

Slab Gel Electrophoresis and Zymograms. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) was performed by using 4% (wt/vol) polyacrylamide
stacking gels and 12.5% (wt/vol) polyacrylamide resolving gels (Laemmli 1970). Native
PAGE (4% [wt/vol] polyacrylamide stacking gel; 10%, 12%, or 7.5 to 18% gradient
[wt/vol] polyacrylamide resolving gel) was performed without SDS and 2-
mercaptoethanol, and without pre-exposure of sample proteins to
100°C (Bachmann and McCarthy 1991). Unless otherwise noted, all native and SDS-
PAGE gels were stained with a Silver Stain Plus kit (Bio-Rad) according to the
manufacturer's instructions. Isoelectric focussing (IEF) of proteins was performed with a

model 11] mini IEF cell (0.4 mm thick mini-gels; Bio-Rad), and pH 5 to 7 ampholytes

46

according to manufacturer's instructions. Unless otherwise noted, all electrophoresis gels
were prepared with GelBond PAG films (for native and SDS-PAGE; FMC BioProducts,
Rockland, MB) or Gel Support Films (for IEF; Bio-Rad) to facilitate handling.

Slab gels for detection of nucleic acids were prepared with 0.8% agarose in Tris-
borate-EDTA (TBE) buffer. Electrophoresis conditions, and ethidium bromide staining,
were performed according to previously published methods (Sarnbrook, et al. 1989).

Zymograms were prepared by overlaying native PAGE or IEF gels with 7.5%
polyacrylamide gels containing the desired substrate (Biely 1985, Royer and Nakas
1990). Zymogram gels for detecting ENDOX activity contained 1% (wt/vol)
pre-extracted oat spelt arabinoxylan in 50 mM HEPES (pH 6.8) containing 0.5 mM C30;
and were poured with GelBond Film. After an oat spelt arabinoxylan zymogram gel was
placed on a native PAGE gel, both were wrapped together in Sealwrap (Borden

Chemical, North Andover, MA) and incubated overnight at 40°C. After incubation, the

zymogram gel was stained with Congo red (10 mg - ml") for 2 h and destained for ca. 30
min with several changes of 1 M NaCl until bands of hydrolysis were visible and the
destaining solution was fairly clear. The zymogram was then treated with 0.1% acetic
acid, which converted Congo red-stained xylan to a dark purple color that enhanced the
contrast of hydrolysis zones for photography.

Zymogram gels for detecting ARAF were prepared in a similar manner, but

contained MU-AF (200 ug - ml") instead of xylan and were incubated for only 20 min at

room temperature before photography on a UV transilluminator.

Antibody Production and Western blotting. Polyclonal anti-Arfl antiserum was

produced by injection of a female New Zealand White Rabbit with 100 ug of purified

47

enzyme emulsified in TiterMax (Cthx Corporation; Norcross, GA) according to the
method of Harlow and Lane (Harlow and Lane 1988). A booster injection (100 ug,

prepared as above) was administered at 28 days and serum was collected 14 days later.

Anti-Arfl antiserum titer was determined by using 1 ug - well'1 of purified Arfl in a dot-
blot apparatus (Bio-Rad) containing an Immobilon PSQ nylon membrane (Millipore).
Development was with a goat anti-rabbit IgG (H + L) alkaline phosphatase immun-blot
assay kit (Bio-Rad) according to manufacturer’s instructions. Western blots were
performed by using 10% (wt/vol) polyacrylamide native PAGE gels, without GelBond
film, blotted onto Immobilon PSQ nylon membranes in a Trans-blot Cell apparatus (Bio-
Rad) according to the method of Matsudaira (Matsudaira 1987). Detection of Art] was
performed as above with 120,000 diluted anti-Arfl antiserum. Preimmune rabbit serum

(1 : 1000 dilution) was used to screen a duplicate blot for non-specific binding.

Fractionation of cells for Arfl localization. Cells fi'om a 2 L culture were divided into
equal portions and centrifisged at 10,000 x g, as above. After centrifugation, the
supernatant (secreted enzyme fraction) was pooled and concentrated (10,000 MWCO
membrane).

One cell pellet (containing cell-associated enzymes) was resuspended in 15 ml of
10 mM Tris buffer (pH 7.6) and subjected to sonication (above), followed by
centrifugation (30,000 x g for 30 min at 4°C) to yield a particulate enzyme fiaction and a
soluble (i.e., cytoplasmic + periplasmic) enzyme fraction.

A second cell pellet was subjected to an osmotic shock procedure similar to that
described by Godchaux and Leadbetter (Godchaux and Leadbetter 1988). First, the pellet

was thoroughly resuspended in 5 ml of ice cold 10 mM Tris buffer (pH 7.6) containing

48

0.3 M NaCl, and then rapidly warmed to 25°C in a 40°C water bath and incubated at 25°C
for 5 min. The suspension was next chilled on ice and 0.75 mg of lysozyme (75 [.11 of 10
mg/ml stock solution) was added with rapid stirring. The lysozyme-treated cells were
then subjected to a slow addition of 10 ml ice cold 10 mM Tris buffer (pH 7.6) and
incubated for 2.5 min on ice. The suspension was then warmed as before to 25°C and
kept at that temperature until >90% of cells were converted into spheroplasts (as
determined by phase contrast light microscopy). Spheroplasts were centrifuged (30,000 x
g for 30 min at 4°C) to yield a soluble periplasmic enzyme fraction (supernatant). The
spheroplast pellet was then resuspended in 10 ml of 10 mM Tris buffer (pH 7.6),
sonicated, and recentrifuged to yield a soluble cytoplasmic enzyme fraction (supernatant).
A portion of the resulting pellet was treated with 0.2% Triton X-100 for 2 h at room
temperature, then recentrifuged to yield Triton-extractable enzymes associated with

particulate cell material.

Amino acid sequences of Arfl peptides. For partial amino acid sequence determination,
purified Arfl (ca. 15 pg per lane) was electrophoresed on a 16% (wt/vol) polyacrylamide
SDS-PAGE resolving gel with a 4% (wt/vol) polyacrylamide stacking gel and blotted
onto Immobilon PSQ membrane as described previously. After blotting, the membrane
was rinsed with H2O, soaked in 100% methanol, then stained with 0.2% Amido Black in
40% methanol for 30 seconds, and destained with multiple changes of H20. The band
corresponding to the position of Arfl in each lane on the membrane was excised with a
clean, sterile razor blade and placed in a sterile Eppendorf tube. The individual ArfI-
containing membrane fi'agments were sent to the Worcester Foundation for Biomedical

Research (Shrewsbury, MA) for sequence determination. Upon receipt, ArfI was digested

49

with trypsin and the oligopeptide fi'agments that were released were purified by reversed-
phase high performance liquid chromatography (RP-HPLC). The N-terminal amino acid
sequence of three such fragments was then determined by the Edman degradation
method.

Alternatively, 48.0 ug of Arfl was digested with 3.125 ug of Endoproteinase
Lys C (Boehringer Mannheim; Indianapolis, IN) at 37°C overnight according to the
manufacturer’s instructions. After digestion, sample was run on SDS-PAGE gel, blotted,
and Art] fragments excised, as above. ArfI-containing membrane fragments were
sequenced, as above, at the Michigan State University Macromolecular Sequence

Facility.

Examination for glycosylation. Glycosylation of purified Arfl was examined by using
the Digoxigenin (DIG) Glycan Detection Kit according to the manufacturer's instructions
(Boehringer Mannheim; Indianapolis, IN). Samples of Arfl (24 and 48 ug) were
subjected to SDS-PAGE as described before, but without GelBond PAG film, then
electroblotted onto an Immobilon PSQ membrane (Millipore) by the method of
Matsudaira (Matsudaira 1987). Positive (Transferrin) and negative (Creatinase) controls
were included and were supplied by the manufacturer. The blotted membrane was
washed with phosphate buffered saline (PBS), and then treated with sodium
metaperiodate. The membrane was then washed again in PBS and treated with DIG-0-3-
succinyl-e-aminocaprioc acid hydrazide, washed with Tris-buffered saline (TBS), and
stained with Ponceau S. After location of protein bands by the Ponceau S stain, the
membrane was soaked in blocking solution (which removed Ponceau S stain) and washed

with TBS. Next, the membrane was incubated with an anti-digoxigenin alkaline

50

phosphatase (AP) conjugated antibody. The membrane was then washed with TBS and

glycosylated proteins were detected by incubation with AP substrate.

Other analytical procedures. The native molecular masses of purified Arfl and partially
purified ENDOX pools were determined by size-exclusion chromatography with a
Pharmacia fast protein liquid chromatography (FPLC) system equipped with either a
Superose 6 (Arfl) or Superose 12 (Arfl and ENDOX pools) HR 10/30 columns

(Pharmacia). The columns were pre-equilibrated with 20 mM MOPS (pH 6.5) containing

50 mM NaCl and run at a flow rate of 0.5 ml - min'l. Column effluents were monitored
for absorbance at 280 nm, as well as for enzyme activity by using the MU-AF plate assay
(Arfl) or reducing sugar assay and native PAGE zymograms (ENDOX pools).
Size-exclusion chromatography standards for Superose 6 included: blue dextran 2000
(2000 kilodaltons [kDa]); bovine thyroglobulin (670 kDa); ferritin (440 kDa); catalase
(232 kDa); and aldolase (158 kDa). Size-exclusion chromatography standards for
Superose 12 included: thyroglobulin (670 kDa); gamma globulin (158 kDa); ovalbumin
(44 kDa); myoglobin (17 kDa); and vitamin B-12 (1.35 kDa).

Protein was determined as described by Smith (Smith, et al. 1985) using the
Micro Bicinchoninic Acid (BCA) Protein Assay Reagent Kit (Pierce, Rockford, IL) or by

the Bradford assay (Bradford 1976) with bovine serum albumin as standard.

Chemicals and other materials. Oat spelt arabinoxylan, arabinogalactan, xylose,
p-nitrophenyl derivatives, 4-methy1umbelliferyl derivatives, DEAE Sephadex A-50,
nicotinamide adenine dinucleotide, D,L-glyceraldehyde 3-phosphate; KH2AsO4,

dithiothreitol and bovine thyroglobulin were obtained from Sigma Chemical Co.

_51

(St. Louis, MO). Phenyl-Sepharose CL-4B, blue dextran 2000, ferritin, catalase, and
aldolase were obtained from Pharmacia Biotech (Uppsala, Sweden). Low molecular
weight SDS-PAGE standards, IEF Standards, FPLC size-exclusion standards, pH 5 to 7
ampholytes, hydroxylapatite, and Macro-Prep 50Q were obtained fi'om Bio-Rad
Laboratories (Hercules, CA). DEAE cellulose was obtained from Whatman Specialty
Products Inc. (Fairfield, NJ). Xylobiose, xylotriose, xylotetraose, xylopentaose, rye- and
wheat arabinoxylans, and sugar beet arabinan were obtained fi'om Megazyme, Inc.
(Sydney, Australia). SPECTRA/POR molecularporous membrane tubing (45mm wide;
3500 molecular weight cutoff [MWCO]) was from Spectrum Medical Industries, Inc.
(Los Angeles, CA). Lenzing beechwood xylan and three corn cob arabinoxylan fractions
were kind gifts fi'om Dr. Robert B. Hespell (Agricultural Research Service, U. S.
Department of Agriculture, Peoria, IL). Other chemicals were of reagent grade and were
obtained from commercial sources. All H2O used was double distilled (Millipore Corp;

Bedford, MA).

52

RESULTS

Enzyme Activities in Crude Extracts. Triton extracts of C. xylanolytica contained a
variety of enzymatic activities important for hydrolysis of xylans and other saccharides,
including a-L-arabinofirranosidase (but not or-L-arabinopyranosidase), B-D-xylosidase,
or-D- and B-D-glucosidases, or-D- and B-D-galactosidases, and acetylesterase (Figure 1).
Likewise, native PAGE of Triton extracts revealed an array of electrophoretically
separable proteins, including approximately 15 with ENDOX activity and distributed
within a top (least migrating), middle, and bottom (furthest migrating) zones of the gel
and detectable by zymograms with cat spelt arabinoxylan (Figure 2A and C; lane 1). By
contrast, analogous zymograms with MU-AF revealed that ARAF activity was associated
with only a single band, and this was referred to as Arfl (Figure 2B; lane 1). No apparent
activity was seen for B-D-glucuronidase or B-D-cellobiosidase when assayed by using

MU-linked substrates in microtiter plate wells (Figure 1).

Purification and Physicochemical Properties of Arfl. ArfI was purified 85-fold to
homogeneity from Triton extracts by using anion exchange and hydrophobic interaction
column chromatography (Table 2; Figures 2 and 3). With these procedures, 23.6% of the
original activity in Triton extracts was recovered. A major step in the purification
procedure was chromatography on hydroxylapatite, which alone yielded an 8-fold
increase in purity. Although a slight decrease in specific activity was seen after Macro

Prep SOQ chromatography (presumably owing to some inactivation of Arfl), this step

53

ArfI

Control

 

Triton extract

Figure l. Microtiter plate assay comparing the activity of purified Arfl (1st row) to
activities present in Triton extract (3rd row) on the following 4-methylumbelliferyl
derivatives (all at 5 mM final concentration in 25 mM MES, pH 6.0): l, B-D-galactoside;
2, or-D-galactoside; 3, or-L-arabinopyranoside; 4, or-L-arabinofuranoside; 5, a-D-
glucoside; 6, B-D-glucoside; 7, B-D-glucuronide; 8, B-D-cellobioside; 9, acetate; 10, B-D-
xyloside. Each well of purified Arfl contained 48 ng of protein (equivalent to 10.6 U of
ARAF activity). Each well of Triton extract contained 4.2 ug of protein (equivalent to
0.011 U of ARAF activity). Control (2nd row) contained test substrate, but lacked

enzyme. Incubation was for 12 h at room temperature.

54

Figure 2. Zymograms and a protein stain of native PAGE gels of Art] after each
purification step. A. Oat spelt arabinoxylan zymogram (stained with Congo red) of
endoxylanase activity. B. MU-AF zymogram showing ARAF activity as a UV
fluorescent band. C. Native PAGE (silver-stained) gel. Lanes 1-7 correspond to fractions
obtained after each purification step listed in Table 2 and contained 105; 123; 49; 9.1;
1.1; 0.9; and 1.2 ug of protein, respectively (B & C). The gel for panel A was loaded with
3x as much protein per lane. Arrow indicates position of Arfl. Note: Panels B & C are of

higher magnification than A.

 

doom cause—ESQ some coca 3 he flow maxed 3%: mo mEEwofixN .N 0.5»:—

56

Table 2. Summary of Purification of or-L-arabinofuranosidase from C. xylanolytica.

 

Purification Total Total Purif-
Step‘ _ protein activity Sp act ication Yield
(mg) (U) (U - mg") (fOId) (°/°)

 

1. none (Triton Extract) 429.9 1,118‘ 2.6 1.0 100.0
2. DEAE Cellulose 183.9 1,067 5.8 2.2 95.4
3. DEAE Sephadex 73.1 951 13.0 5.0 85.1
4. Hydroxylapatite 7.7 808 104.9 40.3 72.3
5. Phenyl Sepharose 1.7 347 204.1 78.5 31.0
6. Macro Prep 500 1.6 291 181.9 70.0 26.0
7. Phenyl Sepharose 1.2 264 220.0 84.6 23.6

 

‘ See Materials and Methods for details.

b As measured with pNP-AF as substrate.

,57

nevertheless eliminated a significant amount of contaminating protein (compare lanes 5

and 6 of Figures 2C and 3).

Size-exclusion FPLC of purified Arfl yielded a single, symmetrical peak
corresponding to a native molecular mass of 210 kDa with Superose 6 and 160 kDa with
Superose 12 (not shown). However, SDS-PAGE revealed that ArfI consisted of subunits
of 56 kDa (Figure 3, lane 7), implying that the native enzyme was a trimer or tetramer.
IEF of purified Arfl yielded a single protein band with a pI of 6. 1, which corresponded to
the only ARAF activity in the IEF gel detectable by zymogram analysis (Figure 4). At no
time during the purification of Arfl was more than a single activity region observed to
elute from chromatographic columns, or more than a single band of activity observed on
zymograms of native PAGE gels (Figure 2B). The major physical properties of Arfl are

included as part of Table 3.

Enzymatic Activity and Stability of Arl'I. With p-nitrophenyl-a-L-arabinofuranoside as

substrate, ArfI had optimum activity at pH 5.8 and 45°C and exhibited a Km of

0.504 mM and a Vmax of 3 l9 umol - min'l - mg protein". Arfl was highly specific for the
a-L—arabinofuranoside linkage, as no hydrolytic activity was seen with the following pNP
derivatives: a-L- and B-L-arabinopyranosides; a-D- and B-D-glucopyranosides;
a-D- and B-D-galactopyranosides; or-L-rhamnopyranoside; or-L-fucopyranoside;
B-D-lactopyranoside; B-D-xylopyranoside; B-D-cellobioside; or-D-mannopyranoside;
B-D-glucuronide; and acetate. This specificity was also seen when testing MU derivatives

of many of these same compounds (Figure 1).

58

1 2 3 4 5 6 7 Std Mr
(kDa)
4—97.4
+662

4— 45.0

4— 31.0

«21.5

 

+ 14.4

Figure 3. SDS-PAGE (silver-stained) gel of Arfl after each purification step. Lanes 1-7
correspond to fractions obtained alter each purification step listed in Table 2 and
contained 10.5, 9.8, 4.9, 3.6, 2.3, 1.7, and 2.4 ug protein, respectively. Std, molecular
weight markers in kilodaltons (kDa): 97.4, rabbit muscle phosphorylase B; 66.2, bovine
serum albumin; 45.0, hen egg white ovalbumin; 31.0, bovine carbonic anhydrase; 21.5,

soybean trypsin inhibitor; 14.4, hen egg white lysozyme.

59

——- +6.0
.. +6.5

 

Figure 4. [BF gel of purified Arfl (1 pg protein per lane): stained with crocein scarlet
(A); or overlaid with a MU-AF zymogram gel (B). Lane: I, purified Art]; 2, purified
Arfl plus IEF standards (pl standards: 5.1, B-lactoglobulin B; 6.0, bovine carbonic

anhydrase; 6.5, human carbonic anhydrase).

60

Table 3. Properties of Purified or-L-arabinofirranosidase (Arfl).

 

Mr (kDa)
Native protein 160-210
Subunns 56
pI 6.1
Glycosylation not detected
Km' 0.504 1 0.034 mM
Vmaxa 319 :1: 6.6 umol - min"° mg"
Optima‘
pH (stability)b 5.8 @ 45°C (pH 4.10; 24 hr) .
temperature (stability)° 45-50°c (5 50°C; 1 hr)

 

' As measured with pNP-AF as substrate.

° Retention of 280% activity, after exposure to conditions indicated.

61

Small amounts of reducing sugar were liberated when Arfl was incubated with cat
spelt arabinoxylan, and this was due primarily to the release of arabinose (see below).
However, bands of electrophoretically separated Arfl also coincided with a faint zone of
Congo red non-binding that was present in zymogram gels designed to screen for
ENDOX activity (Figure 2A, arrow). The basis for such band formation is not known, but

does not appear to represent ENDOX activity associated with Arfl (see below).

A sensitive TLC assay was used to demonstrate the ability of Arfl to liberate
arabinose from rye-, wheat-, corn cob-, and oat spelt arabinoxylans and sugar beet
arabinan (Figure 5). Although the spot on TLC plates corresponding to arabinose was
usually the most intense fi’om all such digests, there was also a region of less intensely
stained material below the arabinose spot in digests of corn cob- and oat spelt
arabinoxylans (Figure 5; lanes 10, 12, 14, and 16). The nature of this material is
unknown, but it migrated a distance between that of xylobiose and xylotriose standards
and may be a branched oligosaccharide. The barely detectable arabinose spot seen with
Arfl digests of corn cob arabinoxylan fraction A (Figure 5; lane 10) was not surprising,
inasmuch as this substrate contained only 5.6% arabinose by weight. ArfI displayed no
apparent hydrolytic activity on arabinogalactan or Lenzing beechwood xylan (not

shown).

Whether present in Triton extracts or in purified form, Arfl was quite stable in

solution. Essentially full ARAF activity was retained in Triton extracts when stored at

4°C for periods as long as 24 months. In fact, zymograms of native PAGE gels of Triton

62

X1 ‘ U
A o. . . ' a . a

X2 ‘ . ‘
x3 0
x4 '
x5 9 .

o --$‘Oood
12 345 678910111213141516171819

Figure 5. Thin-layer chromatogram of various arabinoxylans and an arabinan before (i. e.,
controls) and after exposure to purified Arfl. Lane: 1 and 19, Xr-Xs xylooligosaccharide
standards; 2 and 18, arabinose standard; 3, rye arabinoxylan (RAX) + Arfl; 4, RAX
control; 5, wheat arabinoxylan (WAX) + Arfl; 6, WAX control; 7, sugar beet arabinan
(SBA) + Arfl; 8, SBA control; 9, A + X1 standards; 10, corn cob xylan A (CCXA) +
Arfl; ll, CCXA control; 12, corn cob xylan B (CCXB) + Arfl; l3, CCXB control; 14,
corn cob xylan (CCX) + Arfl; 15, CCX control; 16, oat spelt arabinoxylan (OSAX) +
Arfl; 17, OSAX control. Symbols: A = arabinose; X2 = xylose; X2 = xylobiose; X3 =

xylotriose; X4 = xylotetraose; X5 = xylopentaose.

63

extracts stored at 4°C for up to 48 months had ARAF and ENDOX activities that were
only slightly less than that seen with fresh extracts (not shown). Likewise, purified Arfl
retained 2 80% of its activity when exposed at RT to pHs of 4 to 10 for 24 h, or to pHs
from 6 to 10 for 72 h. Full activity was retained after exposure to temperatures up to
45°C for 1 h; however, activity began to decline sharply on exposure to temperatures ’
>50°C and was completely lost at 65°C. The stability and catalytic properties of Arfl are

included as part of Table 3.

Interaction of Ad] with Endoxylanases. The documented ability of many ARAFs to
interact synergistically with ENDOXs (Bachmann and McCarthy 1991, Greve, et al.
1984, Kormelink, et al. 1991, Lee and Forsberg 1987, Martin, et al. 1994, Poutanen 1988,
Vincent, et al. 1997, Wood and McCrae 1996), prompted us to explore this phenomenon
with the analogous enzymes of C. xylanolytica. ENDOX components fi'om three main
zones separable by native PAGE of Triton extracts (Figure 2A) were partially purified
and referred to as ENDOX I, ENDOX II and ENDOX 111 (Figure 6). The ENDOX II pool
consisted of at least four ENDOX enzymes, including a small but significant amount of a
component(s) present in ENDOX HI. In contrast, the ENDOX I and ENDOX III pools
each contained only one or perhaps two electrophoretically-resolvable ENDOX
components. Each ENDOX pool, however, contained far less non-ENDOX Coomassie
blue-stainable material than crude Triton extracts. Size-exclusion FPLC of each ENDOX
pool yielded a single activity peak that eluted at a position corresponding to 130 kDa for

ENDOX I, 63 kDa for ENDOX II, and 43 kDa for ENDOX III.

1234

 

Figure 6. Zymogram and protein stain of native PAGE gels of partially purified ENDOX
pools. A. Oat spelt arabinoxylan zymogram (stained with Congo red). B. Native PAGE
(Coomassie blue-stained) gel. Lane: 1, ENDOX I; 2, ENDOX II; 3, ENDOX III; and 4,

Triton extract (0.24, 1.03, 1.30, and 110.9 ug of protein, respectively).

65

When Arfl was mixed with the ENDOX I, II, or III pool, the rate of hydrolysis of
rye arabinoxylan was 22 to 46% greater than the sum of the rates for each component
acting alone (Table 4). The increased rate of hydrolysis was apparently due to catalytic
synergy, as opposed to enhanced stability of one enzyme component conferred by the
presence of the other, because inclusion of an equivalent amount of bovine serum
albumin (BSA) as a surrogate for any one of the enzyme components in the two-
component reaction mixtures did not afiect the rate of hydrolysis catalyzed by the

remaining component acting alone.

Cellular Distribution of Art] and ENDOX activities. The relatively high molecular
weight of most arabinoxylans (Aspinall 1980, McNeil, et al. 1984), requires that they
undergo hydrolysis to fragments small enough to pass through the cell membrane before
dissimilation of the monomeric units can take place by cytoplasmic enzymes. Inasmuch
as most of the enzymes of the xylanase system of C. xylanolytica are cell-associated, it
therefore seemed likely that some or all of them, including ArfI, might be on or in the
outer membrane or periplasm of this Gram-negative bacterium, where they would have
access to the native substrate or to large fragments released from it. This notion was
supported by the ready extractability of the xylanase system fi'om whole cells with a low
concentration (0.2%) of the detergent Triton X-100, a treatment which caused little or no
obvious disruption of cells as assessed by phase-contrast and electron microscopy (Haack
and Breznak 1992). However, Triton extracts were found to exhibit glyceraldehyde-3-
phosphate dehydrogenase (GAP) activity (a typically cytoplasmic enzyme), and 0.8%

agarose gel electrophoresis of Triton extracts revealed the presence of ethidium bromide

66

Table 4. Enhancement Between Arfl and ENDOX Fractions of C. xylanolytica in
Hydrolysis of Rye Arabinoxylan.

 

 

Enzyme Reducing sugar Enhancement"

released‘ (%)

Arfl 2.41

ENDOX I 0.30

Arfl + ENDOX I 3.42 26.2

ENDOX II 2.02

ArfI + ENDOX II 6.45 45.6

ENDOX 111 2.33

ArfI + ENDOX 111 5.79 22.2

 

' As umoles of xylose equivalent ‘ min" ' ml".

° Values represent the % increase in reducing sugar release above that expected
from the action of Arfl alone + relevant ENDOX fraction alone. Controls for
catalytic synergy consisted of an amount of bovine serum albumin equivalent

in protein content to that of Arfl or the ENDOX fi'action used in the reaction

mixture.

67

stained material that migrated to a position typical of that of RNA
(not shown). These observations suggested that Triton X-100 was in fact disrupting the
cytoplasmic membrane enough to liberate cytoplasmic components. Therefore, a cell
fractionation procedure was used to determine more precisely the cellular location of Art]

and other enzymes of the xylanase system.

Sonication of xylan-grown cells, followed by centrifirgation, yielded a soluble
supernatant fraction that contained almost all of the Arfl and GAP activities (Table 5).
This indicated that Ad] resided in the cytoplasm and/or periplasm. When cells were
subjected to an osmotic shock, little or no Art] or GAP activities were released into the
shock fluid (Table 5), suggesting that each was located primarily, if not entirely, in the
cytoplasm. Unfortunately, a legitimate positive control could not be included in this
experiment, as no authentic periplasmic enzymes have yet been documented for
C. xylanolytica, and alkaline phosphatase (a typically periplasmic enzyme in many
Gram-negative bacteria) was almost undetectable in this bacterium. However, the ability
of exogenously added lysozyme to induce spheroplast formation during the osmotic
shock procedure implied that the outer membrane was being disrupted enough to allow
proteins such as lysozyme to pass through it. Therefore, if Arfl was located in the
periplasm a significant amount of it should have been released into the post-shock
extracellular fluid, but that was not the case. Rather, most of the Arfl and GAP activities
from such spheroplasts were liberated into the soluble fraction following sonic disruption,
a result entirely consistent with these enzymes being cytoplasmic. The relatively high

proportion of GAP associated with the particulate fraction of spheroplasts (18.5%) is

68

Table 5. Distribution of Arfl and ENDOX Activities in Cells of Cytophaga xylanobrtica.

 

 

 

Activity or Protein
(96 of total)‘
Treatment/Cellular Arfl ENDOX GAP Protein
Fraction

Sonicated Cells

soluble fraction 96.6 60.3 99.9 79.7

particulate fraction 3.4 39.7 <0.1 20.3
Osmotic Shocked Cells

extracellular shock fluid 1.8 8.7 <0.1 2.3

soluble (cytoplasmic) fraction 87.0 72.5 57.2 77.4

particulate fraction z._6 m 18,§ 13.1

Total recovery” 91.4 100.9 75.7 . 92.

 

' Total amounts present in 1 x 1010 sonicated cells of C. xylanolytica are as follows: Arfl,
0.36 U; ENDOX 0.36 U; GAP, 0.66 U; and protein 1.33 mg.

° Based on values given in footnote ‘.

69

curious and may reflect a change in cells resulting from osmotic shock, as virtually all the

GAP is routinely recovered in the soluble fraction of non-osmotically-treated cells.

In each cell fraction in which Arfl activity was detected, the activity could be
attributed to the Arfl protein, based on zymograms prepared from native PAGE of
homologous fractions (Figure 7A). In this experiment, virtually no Arfl (or any ARAF)
activity was detected in concentrated samples of cell-free spent growth medium (Figure
7A; lane 5), although such samples contained a variety of proteins (Figure 7B; lane 5)
including some ENDOX components (see below). However, a trace amount of ARAF
activity observed in culture fluids in previous experiments eluted in the same position as
Art] during column chromatography and had similar pI values in IEF gels (not shown).
Attempts to confirm further the primarily cytoplasmic location of Arfl, by immunogold-
labelling followed by electron microscopy, were unsuccessful, presumably because the
absolute amount of Art] protein in the cytoplasm was too dilute to be detected in thin
sections. However, the polyclonal anti-Arfl antiserum found use in screening other

strains of C. xylanolytica for cross-reactive proteins in Western immunoblots (see below).

In contrast to Arfl, ENDOX activity was more widely distributed in
C. xylanolytica, a result not surprising given the diversity of electrophoretically-separable
ENDOX proteins produced by cells (e.g., Figure 2A; lane 1). After sonication of whole
cells, about 60% of the ENDOX activity was recovered in the soluble fraction (Table 5),
which was rich in a variety of components representing the ENDOX I class, as well as
two fairly discrete components representing the ENDOX H class (Figure 8; lanes 2 and

3). Triton X-100 extraction of the ENDOX activity associated with particulate fraction

70

revealed that it also contained components from ENDOX I, as well as a band of activity
from the ENDOX 11 region that appeared to be different from each of those present in the

soluble fraction of sonicated cells (Figure 8; lane 4).

When cells were subjected to osmotic shock, about 9% of the total ENDOX
activity was released into the shock fluid (Table 5) and presumably represented ENDOX
components present in the periplasm. Zymograms of this material (Figure 8; lane 5) were
similar to that of Triton-extracted particulate cell material obtained after sonication of
whole cells (Figure 8; lane 4) suggesting that some of the ENDOX activity associated
with the latter (Table 5) was due to periplasmic ENDOXS that were not completely
liberated by sonication of cells and, hence, remained entrapped within the particulate
fi'action. Most of the ENDOX activity remaining in the lysozyme-induced spheroplasts
formed during the osmotic shock procedure (ca. 73% of total) appeared to be of
cytoplasmic origin, as it was released as soluble activity following sonication of the
spheroplasts (Table 5). As expected, its zymogram profile (Figure 8; lane 6) was virtually
identical to that obtained in the soluble fraction of sonicated whole cells (Figure 8; lane
2). Triton X-100 extraction of the particulate fraction of spheroplasts contained about
20% of the total activity and yielded a zymogram pattern (Figure 8; lane 7) similar to that
of material extracted with Triton from the particulate fraction of whole cells and that

released by osmotic shock (Figure 8; lanes 4 and 5, respectively).

Although cell-free spent growth medium generally contained 3 5% of the total

ENDOX activity of cultures, zymograms of this material after native PAGE (Figure 8;

71

Figure 7. Specificity of anti-Art] antiserum. A. MU-AF zymogram of native PAGE gel
(viewed by UV light). B. Native PAGE (silver-stained) gel. C. Western blot with anti-
Arfl antiserum. Lane: 1, purified Arfl; 2, soluble cell-free extract (obtained by sonication
and centrifiJgation); 3, Triton extract of particulate cell material (obtained by sonication
and centrifugation); 4, Triton extract of whole cells; and 5, concentrated cell-free growth

medium (0.6, 75.0, 72.5, 60.0, and 72.0 ug of protein, respectively).

72

 

.EEomccu 34:8 mo .3690on .b Ping

73

 

1;. a; 4. ma; «mi-2mm

Figure 8. Cat spelt arabinoxylan zymogram of native PAGE gel showing the distribution
of ENDOX components in cells and culture fluids of C. xylanolytica strain XM3. Lane:
1, concentrated cell-free growth medium; 2, soluble cell-free extract; 3, soluble cell-free
extract plus Triton X-100; 4, Triton extract of particulate cell material; 5, osmotic shock
fluid (= cell periplasm fiaction); 6, soluble extract of osmotically shocked cells
(= soluble, cytoplasmic fraction); 7, Triton extract of particulate fraction from
osmotically shocked cells; 8, Triton extract of untreated cells (0.014, 3.7, 3.7, 2.8, 0.094,

5.1, 1.8, and 4.4 mg protein, respectively).

74

lane 1) revealed activities of similar electrophoretic mobility to those seen in
cell-associated ENDOXs including component(s) from ENDOX H (in particular, the
ENDOX II component released by osmotic shock and by Triton extraction of particulate
cell material (Figure 8; lanes 4, 5 and 7), and a component from ENDOX III that was

seen in cytoplasmic contents (Figure 8; lanes 2 and 6).

Arl'I-like Enzymes in Other Strains of C. xylanolytica. Polyclonal antiserum produced
against purified Arfl was specific for Art], as judged by Western immunoblot analysis of
native PAGE gels containing purified Arfl, or various cell fractions containing other
proteins as well (Figure 7C). As with the zymograms, Western immunoblots failed to

demonstrate Arfl in cell-free spent culture fluids.

The antiserum was used to determine whether Arfl-like proteins were present
in other freshwater and marine strains of C. xylanobrtica. Zymograms of native PAGE
gels containing soluble, cell-free extracts from sonicated cells of various freshwater and
marine strains of C. xylanolytica (Haack and Breznak 1993) revealed that most of the
strains possessed an ARAF, which appeared as a single band of activity (Figure 9A).
However, three marine strains did not appear to express any ARAF activity (Figure 9A;
lanes 11-13), and the ARAF activity bands for the two other marine strains were very
faint (Figure 9A; lanes 9 and 10). Zymograms prepared from Triton extracts of the
particulate fraction of sonicated cells yielded patterns that were essentially identical to
those seen for the soluble cell fraction (not shown). Western immunoblots showed that
ARAFs produced by various freshwater (but not marine) strains cross-reacted with the

anti-Arfl antiserum (Figure 9B), although some of these did so only weakly (e.g., Figure

75

9B; lanes 4-6). The immunoblots also showed that some of the freshwater and marine
strains had additional cross-reactive bands that were not associated with any ARAF

activity detectable by zymogram analysis (Figure 9B; lanes 7-10).

Internal peptide sequences and glycosylation of Arl'I. We were unable to obtain an N-
terminal amino acid sequence of purified Arfl, as the protein appeared to be blocked.
However, treatment of Arfl with trypsin (a protease that cleaves after a lysine or arginine
residue) followed by reversed phase-high pressure liquid chromatography (RP-HPLC)
yielded three fragments with the following amino acid sequences:
EAAQWVEYITSNNPSPMTNLR, YFCIGNENWGCGGHMTPEYYADLYR, and
DALVAGINLNIFNNNADR (Fragments no. 1-3, respectively). Analysis of fi'agments l,
2, and 3 by using the program BLASTp (Altschul, et a1. 1990) revealed that fragment no.
1 most closely resembled ARAFs fi'om Bacteroides ovatus strain V975 (asdII gene
product; GenBank accession no. U15179), Clostridium stercorarium (aer gene product;
Genbank accession no. AF002664), and Bacillus subtilis (arabinosidase; Genbank
accession no. Z75208) in having % identity/% similarity ratios of 66/80, 47/66, and
38/71. By contrast, fragment no. 2 resembled the same three ARAFs as fragment no. 1,
but in the order of C. stercorarium, B. subtilis, and B. ovatus as having % identity/%
similarity ratios of 84/96, 84/92, and 76/84. Fragment no. 3 closely resembled only the
ARAF fi'om C. stercorarium, with a % identity/% similarity ratio of 77/94. No evidence
was obtained for glycosylation of ARAF (not shown).

Treatment of Arfl with Endoproteinase Lys C (a protease that cleaves after a
lysine residue) followed by blotting and sequencing of resulting fragments yielded three

peptides with identical N—terrninal sequence of GSSIVFDEQSxHQILRHxLR. Analysis

76

of the fragment sequence (designated fragment no. 4) using the program BLASTp
revealed similarity to the ARAF from C. stercorarium (above), with a % identity/%
similarity ratio of 35/50.

The four peptide fragments were compared to the cloned ARAFs from C.
xylanolytica (Appendix; Figures 10 and 11). The three tryptic peptide fragments matched
the deduced amino acid sequence of arfl (Appendix; Figure 12). Fragment four produced
by Endoproteinase Lys C digestion matched the deduced amino acid sequence of arfl
except for an Isoleucine (1) instead of Tyrosine (T) at position number 14 of the peptide

fi'agment. Two amino acids of fragment no. 4 were undetemtined (denoted by x).

77

Figure 9. Arfl-like proteins and enzyme activity of various freshwater and marine
isolates of C. xylanolytica after growth in fieshwater (FW) or marine (MN) medium.
A. MU-AF zymogram of native PAGE gel showing ARAF activity (viewed by UV
light). B. Western immunoblot of native PAGE gel with polyclonal anti-Arfl antiserum.
Lane: 1, purified Arfl; 2, strain XM3 (FW); 3, strain XMB (MN); 4, strain MAB (FW); 5,
strain EWl (FW); 6, strain SL1 (FW); 7, strain EPFW (FW); 8, strain EPFW (MN); 9,
strain EPA (1901); 10, strain EPB (MN); 11, strain OP2E (MN); 12, strain OPZF (MN);
13, strain PR2L (MN) (0.6, 91.0, 142.0, 138.0, 121.0, 166.0, 146.0, 68.0, 226.0, 201.0,
85.0, 130.0, and 59.0 pg of protein, respectively). Lanes 2-13 contained soluble cell-free
extract produced after sonification and centrifugation. Note: Panel B is of higher

magnification than A.

78

A

12 34 56 7 8910111213

.‘r‘ 4‘.

    

B
12345678910111213
“§¢ -

-

Figure 9. Arfl-like proteins and enzyme activity of various freshwater and marine

isolates of C. xylanolytica afier growth in freshwater (FW) or marine (MN) medium.

79

DISCUSSION

A cytoplasmic a-L-arabinofiiranosidase (Arfl) from C. xylanolytica was purified
85-fold by column chromatography and was judged to be a single protein species on the
basis of SDS-PAGE, native PAGE, and IEF analyses, all of which yielded a single
protein band (the latter two retaining ARAF activity in zymograms). No evidence was
obtained that the Arfl existed in multiple forms, as had been observed with some
endoxylanase and arabinofuranosidase enzymes produced by other organisms (Komae, et

al. 1982, Matte and Forsberg 1992, Van Laere, el al. 1997, Vincent, er al. 1997).

Arfl accounted for virtually all of the ARAF activity in cultures of
C. xylanolytica, and it appeared to be the only ARAF produced by this bacterium under
our grth conditions. A recent study (Van Laere, er al. 1997) suggested that some
ARAFs may go undetected owing to their inability to cleave pNP-AF, the substrate
commonly used to assay ARAF activity. However, MU-AF was also used as a substrate
in the present study, and again the only MU-AF-hydrolyzing activity observed was that
attributable to Arfl and seen as a single electrophoretically-separable band in various
fractions produced throughout the purification procedure (Figure 2B). Arfl also appeared
as single bands with identical p13 in IEF gels prepared previously from both xylan- and
xylose-grown cells (Haack and Breznak 1992). The pI of Art] reported in that
preliminary study [i.e., 5.85; (Haack and Breznak 1992)] was slightly lower than that
reported herein for the purified protein (p1 = 6.1), but the former was determined by IEF
zymogram analysis of crude Triton extracts and may have been underestimated. The trace

amount of extracellular ARAF activity occasionally observed in cell-free culture fluids of

80

xylan- and xylose-grown cells (Haack and Breznak 1992) is probably the result of lysis of
some cells in the population, since the protein responsible for that activity has properties
similar to that of Arfl. However, although ArfI was the only ARAF detected in this
present study, it is apparently not the only ARAF that cells are capable of producing.
Indeed, two different ARAF-encoding genes have been cloned from the genome of C.
xylanolytic: arfl, which encodes Arfl; and arfll, which encodes a putative ARAF (ArflI)
expressed by E. coli, but which has not yet been observed in cells or culture fluids of C.
xylanolytica (Appendix; Figures 10 and 11, respectively). In any case, the single protein
(Arfl) accounting for ARAF activity was in marked contrast to the multiplicity of
endoxylanase activities, which segregated into three major zones (ENDOX pools) during
native PAGE and were associated with ca. 15 individual bands. Such multiplicity of
endoxylanases is not uncommon among xylanolytic systems (Dekker 1985, Wong, et al.

1988)

Some of the properties of Arfl from C. xylanolytica were similar to those of
ARAFs from various other organisms, e.g., subunit molecular mass (which is usually
within 30-95 kDa), an acidic pH optimum (typically lying between pH 2.5 - 6.9), and the
ability to release arabinose from pNP- and MU-a-L-arabinofiiranosides, arabinoxylans,
and arabinan (but with an otherwise narrow substrate specificity). However, the apparent
native molecular mass of 160-210 kDa, which was consistent with that of a trimer or
tetramer composed of 56 kDa subunits (Table 3), was higher than that of many other
ARAFs (Table 1). Among purified ARAFS, Arfl bears perhaps the closest resemblance to
the ARAF of Butyn'vibrio fibrisolvens GSI 13 in terms of physical parameters (similar

pIs; identical subunits of 56 kDa vs. 31 kDa, respectively), location (both cytoplasmic,

81

see below), catalytic properties (similar Km values and temperature and pH optima), and
narrow substrate specificity (i. e., specific for the a-L-furanoside configuration and no

activity on arabinogalactan [Hespell and O'Bryan 1992]).

One curious property of the Arfl of C. xylanobltica was its ability to induce
Congo red non-binding to oat spelt arabinoxylan present in zymogram gels (Figure 2A,
arrow). However, we are reluctant to attribute true endoxylanase activity (hence bi-
functionality) to this enzyme because (i) little or no reducing sugar was liberated (above
that expected for arabinose alone) when Arfl acted on oat spelt arabinoxylan, (ii) neither
a significant amount of reducing sugar, nor spots on TLC plates corresponding to
xylooligomers, were liberated by Arfl from an essentially arabinose-free Lenzing
beechwood xylan (data not shown), and (iii) no apparent xylooligomers accompanied the
release of arabinose from rye or wheat arabinoxylan (Figure 5). Nevertheless, it is
conceivable that a small amount of xylan-depolymerizing activity might be associated
with Arfl, akin to that seen with certain endoxylanases of Clostridium thermocellum
(which liberate too little reducing sugar to be detected by conventional colorimetric
assays, but can effect enough depolymerization of the substrate to be detected in Congo
red-stained zymograms (MacKenzie, er al. 1989]) or with the ARAF of Streptomyces
Iividans (which hydrolyzed the xylan backbone after prolonged incubation (Vincent, et
a1. 1997]). Such activity might also be responsible for the small amount of saccharide
trailing the arabinose spot in TLC of Art] digests of corn cob- and oat spelt arabinoxylans
(Figure 5). However, the Arfl of C. xylanolytica bears little resemblance to the arabinose-
releasing endoxylanases that release arabinose side groups before attacking the xylan

backbone (Matte and Forsberg 1992). Such debranching endoxylanases do not act on

82

MU-AF and pNP-AF substrates, whereas the Arfl of C. xylanolytica has significant

activity on these and very little activity on oat spelt arabinoxylan.

An osmotic shock method, originally developed for the isolation of inner and
outer membrane components from several Cytophaga species (Godchaux and Leadbetter
1988), was employed as part of a cell fractionation procedure that inferred an almost
entirely cytoplasmic location for Art]. Even though no validated periplasmic enzyme
markers are known for cytophagas, the activity of an endoxylanase present in the shock
fraction suggested a possible periplasmic location (Figure 9, lane 5). Our assessment of a
periplasmic location for at least one ENDOX component is a first approximation,
however, since so little is known about complex cell-associated xylanolytic enzyme
systems. Other researchers have shown that the distribution of xylanase activities found
within xylan-degrading microorganisms can be affected by the amount and type of
grth substrate, the culture conditions employed, the phase of grth at cell harvest,
and/or some combination of these (as examples, see [Lee, et al. 1993, Lee and Zeikus
1993, McDermid, et al. 1990, Rodriguez, et al. 1985]). In addition, research has shown
that intrinsic differences in the substrates used for detection of xylanases, and the ability
or inability of enzymes to act on them, can make it difficult to determine the distribution
of xylanase activities (e.g., the different specificities of ARAFs for artificial and natural

substrates).

In light of the largely (if not entirely) cytoplasmic nature of Art], it seems
reasonable to assume that the major role of this enzyme in C. xylanolytica is to remove

or-L-arabinofuranosyl residues from arabinose-containing xylooligosaccharide fragments

83

that pass through the cytoplasmic membrane and whose presence might otherwise
impede the action of cytoplasmic ENDOXS and or B-xylosidases. This interpretation is
consistent with the ability of Arfl to release arabinose from various arabinoxylans (Figure
5) and to exhibit synergy with various ENDOX components (Table 4), including those
present in the cytoplasmic fiaction of cells (Figure 8, lanes 2 and 6). Such synergy is not
unusual and has been reported for the xylanolytic enzyme systems of other organisms
(Bachmann and McCarthy 1991, Matte and Forsberg 1992). Although one might assume
that xylooligosaccharides capable of passing through the cytoplasmic membrane are
restricted to short oligomers, preliminary evidence for the uptake of large
oligosaccharides (up to seven glucose units in length) has been obtained for Bacteroides
thetaiotaomicron, a bacterium closely related to cytophagas phylogenetically (Salyers, et

al. 1996).

The comparison of four peptide fragments from Art] to other proteins in the
database gave an unexpected result. Although the order of similarity of fragments no. 1
and 2 were different, the fact that fragment no. 3 and 4 had similarity only to an ARAF
from Clostridium stercorarium was unusual. Perhaps these fragments are from a less

conserved region of ARAFs than the regions containing fragments no. 1 and 2.

Further studies on the nature and cellular location of specific ENDOXs, as well as
on the mechanisms and limits of saccharide uptake by cells, would undoubtedly refine
our concept of arabinoxylan degradation by cells. Hence, we consider this study as but

one step in dissecting the complex xylanase system of this fascinating gliding bacterium.

APPENDIX

APPENDIX

a-L—ARABINOF URANOSIDASE-ENCODIN G GENES FROM Cytophaga xylanolytica

85

PREFACE

I specifically want to acknowledge the work of Kwi Kim (cloning and
sequencing) and the efforts of Dr. Tim Lilburn (sequence searches and PCR design) for
the information contained in this Appendix (Kim, K. S., T. G. Lilburn, M. J. Renner,
and J. A. Breznak. arfl and arfll: two genes encoding a—L—arabinofiiranosidases in

C ytophaga xylanolytica).

86

Figure 10. Nucleotide sequence of arfl and the deduced amino acid sequence of Art]
from C. xylanolytica. The sequence of both DNA strands was determined. The ORF
(residues 232 to 1761, encoding 509 amino acids) corresponds to arfl. Encoded amino
acids are indicated below the nucleotide sequence. Numbers to the left of the figure
designate the first nucleotide in each row. Plus symbols (+) designate every 10
nucleotides in sequence. Bold and underlined portions designate the amino acid sequence
of tryptic peptides used to design degenerate primers for PCR. Asterisk represents

proposed termination codon.

87

Figure 10. Nucleotide sequence of arfl and the deduced amino acid sequence of Arfl

from C. xylanolytica.

1 TGGTCGTTGGTGAAATACAGCGGTTTGTGTTTGGAGACTCAGCATTTCCC
+ + + + +

51 CGACTCGCCCAATAAGGCTAATTTTCCCTCGGTGATTCTTAATCCCGGCG
+ + + + +

101 AGAAATATACTTCGACTACCATTATGAAATTTGGGGTAAAGTAACCCAAT
+ + + + +

151 CTTTAATCAGGAGGCACCGCTCGTTGAATAATCAACTTGGGTGCCTCTTT
+ + + + +

201 TGTTTGGTCATTATTAACTTTTGATATACTGATGAATATGAAAAGAATTT
+ + + + +

M N M K R I S

251 CTATTGCCTTTTTGCCGACGCTTGCCTTATTGCCTTTGTCGGCGCAAACC
+ + + + +

I A F L P T L A L L P L S A Q T

301 AAAATTACTTTGCAGGCCGATCAGGCCAAGGAGCGCATTAATCCTGAAAT
+ + + + +

K I T L Q A D Q A K E R I N P E I

351 TTATGGTCATTTTGCCGAGCACCTTGGGCGATTGATTTATGGTGGAATTC
+ + + + +

Y G H F A E H L G R L I Y G G I L

401 TGGTCGAGCCGGGTTCTCAAATTGACAATATTCGGGGCTTCCGTACCGAT
+ + + + +

V E P G S Q I D N I R G F R T D

451 GTGATTGGAGCCCTCCGCGACATGAAAGTGCCCATTTTGCGTTGGCCCGG
+ + + + +

V I G A L R D M K V P I L R W P G

501 CGGTTGTTTTGCCGATACTTACAACTGGCGCGATGGCATTGGTCCCAAGC
+ + + + +

G C F A D T Y N W R D G I G P K Q

551 AAAATCGTCCCTCTATTGTGAATATCCACTGGGGTGGGGTCACCGAAGAT
+ + + + +

N R P S I V N I H W G G V T E D

88

Figure 10. (cont’d).

601

651

701

751

801

851

901

951

1001

1051

1101

AACAGTTTTGGAACGCATGAGTTTTTCGATTTTTGTGAACTGATTGGTGC
+ + + + +

N S F G T H E F F D F C E L I G A

CGAACCATACGTGAATCTGAACGTAGGCTCAGGAACTGTGCGCGAAGCTG

+ + + + +
E P Y V N L N V G S G T V R E A A

CTCAGTGGGTGGAGTACATTACTTCTAACAACCCCAGCCCGATGACCAAT
+ + + + +

Q fl 2 E z I g S N N P S 2 M I N

TTGCGCAAAGAAAACGGTCGTGATAAACCCTGGGATATCAAGTATTTTTG
+ + + + +

uxaucanxpworxvrc

CATTGGTAATGAAAACTGGGGATGTGGTGGACACATGACGCCTGAGTATT
+ + + + +

I G N E N W G C G G H M T P E Y Y

ATGCCGATTTGTATCGTAATTATGCAACCTATTGTGGTGGTGTGAAATAT
+ + + + +

A D L Y R N Y A T Y C G G V K Y

AAAATCGCCGGCGGTCCCAATGTGGATGATTATCGTTGGACGGAAGTGTT
+ + + + +

K I A G G P N V D D Y R W T E V L

GATGAATAAAACGCAGCATCACAAGTTCCTCATGCAGGGATTGTCACTTC
+ + + + +

M N K T Q H H K F L M Q G L S L H

ATTATTATACGTTCCCCGGTCGTTGGGAAGATAAGGGTTCTTCTATTGTT
+ + + + +

Y Y T F P G R W E D K G S S I V

TTTGATGAACAGTCGTGGCATCAAACCTTGCGTCATACCTTGCGCATGCA

+ + + + +

F D E Q S W H Q T L R H T L R M Q

GGAACTCATCAGCAAGCATGGCGAAATTATGGACAAATATGACCCCAAAA
+ + + + +

E L I S K H G E I M D K Y D P K K

89

Figure 10. (cont’d).

1151

1201

1251

1301

1351

1401

1451

1501

1551

1601

1651

AGCAAATCGGCTTGATTGTTGACGAATGGGGAACCTGGTACGATGTGATG
+ + + + +

Q I G L I V D E W G T W Y D V M

CCCGGTACGAACCCCGGCTTTTTGTATCAGCAAAATACGTTACGTGATGC
+ + + + +

PGTNPGFLYQQNTLRU

TCTGGTGGCGGGGATCAATCTTAATATCTTCAACAACAACGCTGATCGGG
+ + + + +

L V A G I N L N I F N N N A D R V

 

TGAAAATGGCTAATATTGCCCAAATGGTCAATGTGTTACAGGCTGTAATT
+ + + + +

K M A N I A Q M V N V L Q A V I

CTCACTCAGGGCGATGAAATGATTCTGACGCCCACTTACTATGTCTTTAA
+ + + + +

L T Q G D E M I L T P T Y Y V F K

AATGTACAATGTGCACCATGGTGCTACGTTGATTCCCATGAACATTCAGA
+ + + + +

M Y N V H H G A T L I P M N I Q T

CGCAAGATTATGTGATGGGAGATCAAAAAATTCCGATGGTCAATGCCTCT
+ + + + +

Q D Y V M G D Q K I P M V N A S

GCCTCCATCAAGGATAAAACGGTGAGTGTAACCTTGTGCAACCTTCACGC

+ + + + +

A S I K D K T V S V T L C N L H A

ATCCCAATCAACCAAAGTTGAAATTGATGTGACTGGTTTTGAAGGCAAAA
+ + + + +

S Q S T K V E I D V T G F E G K T

CGGCTACCGGTCAAATCATCACCGGCGAAAAAATTACCGATTACAACGAT
+ + + + +

A T G Q I I T G E K I T D Y N D

TTTGGTAAGCCTGAAATGGTTGGTCTCAAGGCTTTTAATGTGGCCAAGCC
+ + + + +

F G K P E M V G L K A F N V A K P

90

Figure 10. (cont’d).

1701 TAAAGGCAATAAACTGACTGTTGAAATCCCCGCGAAGTCAGTTGTGTTAG
+ + + + +

K G N K L T V E I P A K S V V L V

1751 TGCAAATGAAGTAATTTCAATTTCTTTTCCCTTATAAAGCCGTTGCCCTA
+ + + + +

Q M K *

1801 TAGCAGCGGCTTTTTTTGTGTTCGGTGGGAGCGATTTGTC
+ + + +

91

Figure 11. Nucleotide sequence of arflI and the deduced amino acid sequence of a
putative or-L-arabinofiiranosidase fi'om C. xylanolytica. The sequence of both DNA
strands was determined. The ORF (residues 294 to 1895, encoding 534 amino acids)
corresponds to will. Encoded amino acids are indicated below the nucleotide sequence.
Numbers to the left of the figure designate the first nucleotide in each row. Plus symbols
(+) designate every 10 nucleotides in sequence. Asterisk represents proposed termination

codon.

92

Figure 11. Nucleotide sequence of arfll and the deduced amino acid sequence of a

putative a-L-arabinofilranosidase from C. xylanolytica.

1 GAATTCATGGCCGGTGATGTACGTTTCGCAAGGACTCTTTTTTCTCTTAA

+ + + + +

51 GGAAACGATATGGATTGTTGATTCAATGTCGTTAAGTTAGTGCGATTCAC
+ + + + +

101 AAAAGAGACAAGAAGATGATGAGACTAAAGACAGATAGATTAGACTGAAG
+ + + + +

151 AGACGAAGTTCACGGATCTAAATATCTGTTTTTTCATCTCATCATCTGTC
+ + + + +

201 TCTTCGTCTGACATCTTTTTCTCTTAACTAAACGACATTGATTGTTGATT
+ + + + +

251 TGTCGTTTGATGATTGATTTGAAATAGTATAAATTGAAATAGTATGAAGA
+ + + + +

M K K

301 AACGAATGAGTATTCAAAAGCGAATTGCTTTCAATGGAGCCTTGGTGTTG
+ + + + +

R M S I Q K R I A F N G A L V L

351 GCATTTATGTTGACCATGTGCACCCCAAAACCTCCTCAGAATGAGGATGT
+ + + + +

A F M L T M C T P K P P Q N E D V

401 CGTAAAAATGTCCGTCGACGTCACGCAAAGTGGCCCTGAAATCAACCGTC
+ + + + +

V K M S V D V T Q S G P E I N R H

451 ACATCTTTGGCCAGTTTGCCGAACATTTGGGCACGGGCATTTACGGCGGC
+ + + + +

I F G Q P A E H L G T G I Y G. G

501 ATTTGGGTGGGACCCGAATCGGAAATCCCCAATACCCGCGGGATTCGTAA
+ + + + +

I W V G P E S E I P N T R G I R N

551 TGATGTGGTGAAAGCTTTAAAGGCCATCAAAGTGCCCAACGTGCGCTGGC

+ + + + +
D V V K A L K A I K V P N V R W P

601 CGGGCGGTTGTTTTGCCGATCAGTACCACTGGCGCGATGGCATCGGTGCA,
+ + + + +

G G C F A D Q Y H W R D G I G A

93

Figure 1 l. (cont’d).

651 CCCGAGGAACGCAAAAGTCGCATCAATGTCAGCTGGGGCGGTAGTCCCGA
+ + + + +

P E E R K S R I N V S W G G S P E

701 AGCCAACACCTTTGGCACGCATGAATATTTCGATTTCATTTCTCAAATTG

+ + + + +
A N T F G T H E Y F D F I S Q I G

751 GTTCCGAAGCCTTTATTTCGGCCAATGTGGGATCGGGTACTGTGCAGGAA
+ + + + +

S E A F I S A N V G S G T V Q E

801 TCGGCTGATTGGCTGGAGTATCTCACGGCTTCAGGGTCGACATTGGCTCT
+ + + + +

S A D W L E Y L T A S G S T L A L

851 CGAGCGTGCCCGAAACGGTCACCCCGACCCCTACGATGTTGCCTTTTGGG
+ + + + +

E R A R N G H P D P Y D V A F W G

901 GCGTGGGCAACGAAGTGTGGGGATGCGGCGGTCCTTTTACCCCGCAGGAA
+ + + + +

V G N E V W G C G G P F» T P Q E

951 TATATCACCGAGCTTAAAAAGTTTGCCACTTTTACCAATAACTACAATGC
+ + + + +

Y I T E L K K F A T F T N N Y N A

1001 CAACGTCAATACTCAGTTTGTTGCGGTGGGCCCCGACTCGTTTGATGAAT
+ + + + +

N V N T Q P V A V G P D S F D E Y

1051 ACAGCTTTGGCTATCCCGAAGCCATCATGGAGGCCTGGGCAAAGAAAACC
+ + + + +

S F G Y P E A I M E A W A K K T

1101 TGGACCTGGGATATTCAAGGGCTTTCGTTGCACATGTACACGCGTGGCGG
+ + + + +

W T W D I Q G L S L H M Y T R G G

1151 CTGGCCGGCAGTGATTCCTGCTACCGGTTTCAACGAATCGGGATACGCAG
+ + + + +

W P A V I P A T G F N E S G Y A A

94

Figure 11. (cont’d).

1201 CAGTCATTAAGGAAACTCTGGAAATGGACAAATTTATCAGCGACAATCGC
+ + + + +

V I K E T L E M D K F I S D N R

1251 GCCATCATGGATAAGTTCGACCCCGAACACAAAGTGTCGATCATGGTGGA
+ + + + +

A I M D K F D P E H K V S I M V D

1301 TGAGTGGGGCACCTGGTATGCACCCACCGAAGGGACCAATCCCGGTTTTC
+ + + + +

E W G T W Y A P T E G T N P G F L

1351 TGCAACAGCAAAACTCACAACGCGATGCTGTGTTGGCGGCTTTGAATTTC
+ + + + +

Q Q Q N S Q R D A V L A A L N F

1401 AACATCTTTATTCGTCATGCCGAGCGGGTGAAAGGGGCTAACATTGCCCA
+ + + + +

N I F I R H A E R V K G A N I A Q

1451 GATGATCAATGTGTTGCAAGCCATGATTCTGACCGAAGGTGAGAAGATGG
+ + + + +

M I N V L Q A M I L T E G E K M V

1501 TGCTCACACCCACTTATCACACTTTCCGCATGTACGTGCCTTTTCAGGAT

+ + + + +
L T P T Y H T F R M Y V P P Q D

1551 GCTACGCGTTTGCCAATCAATTTCAATAAAGGCTTTTATAAAGAAGGAAC
+ + + + +

A T R L P I N F N K G F Y K E G T

1601 CATTGAATTGCCGCGTGTGGATGCCATTGCTGCCAAAGGAAGAGATGGCA
+ + + + +

I E L P R V D A I A A K G R D G K

1651 AAATTTACGTGGCCATCACCAATATCGACCCCAAAAACAACGCTTCGTTG
+ + + + +

I Y V A I T N I D P K N N A S L

1701 GATCTCTCGTTTGATGAGCAAACCATCAAGGATGTGAAGGGGGAAACGCT
+ + + + +

D L S F D E Q T I K D V K G E T L

95

Figure 11. (cont’d).

1751 GTATGCTTCGGCCATTGATGCTGTTAACACATTTGACAACCCTAACAATG
+ + + + +

Y A S A I D A V N T F D N P N N V

1801 TTGCTCCTACATCCATTAACGCAAGTTTAACGGGCAGCAATGTGTCGGTA
+ + + + +

‘APTSINASLTGSNVSV

1851 ACGGTGCAGCCTCAGTCGGTCACGGTGCTCGAAATTGTATCGGAGTGATT
+ + + + +

T V Q P Q S V T V L E I V S E *

1901 GCGTTTGGAGGTTTTGGTTTATAGCATCCACCTGTTCTGTGAATTC
+ + + +

96

Figure 12. CLUSTAL W comparison of the deduced amino acid sequences of arfl and
arfll to similar ARAFs fi'om other organisms. A multiple alignment was performed with
the deduced amino acid sequences of ad] and arfll from C. xylanolytica to ARAFs from
Bacteroides ovatus strain V975 (accession no. U15179), Clostridium stercorarium
(accession no. AF002664), and Bacillus subtilis (accession no. Z75208). Boxes around
amino acid sequences designate regions of sequence identity. The four shaded regions
represent the amino acid sequences of three trypsin- and Endoproteinase Lys C-generated
peptides of Arfl. Dashes indicate gaps in the sequence. Numbers to the left designate the
number of the first amino acid in each row and numbers to the right designate the last
amino acid in each row. Designations: Arfl = purified arabinofuranosidase from
C. xylanolytica; Arl'II = putative arabinofuranosidase from C. xylanolytica; B. ovatus =
Bacteroides ovatus asdll gene product; C. sterc. = Clostridium stercorarium aer gene
product; and B. subtilis = Bacillus subtilis gene product. Dashes indicate gaps in the

sequence.

97

Figure 12. CLUSTAL W comparison of the deduced amino acid sequences of arfl and

arfll to similar ARAFs from other organisms.

 

 

 

Am I ............ MNMKRISIAFLPTLI4
Arlll u MKKRMS IQKRIAFNGALVLAFMLTMC 2.
B.ovatus u ----------- MKAKLLVSTAFLAAS Is
C.:terc. I -------------------------- o
8.:ubtms I -------------------------- o
Arll Is ALLPLSAQTKITLQA QAEE--RINP 39
MI 27 TPKPPQNEDVVKMSV vr SGPEINRsz
8.0mm: :6 vs LSAQKSATITVHA QGKE--IIPK39
Csterc. . ....... MAKYVINTLNRKS --KINR .7
exam: I ..... MSEHQAVIQTQIAKG--TINK .9
Arll 39 HFAEHLGRLIYGGILVEPG? I 64
Am: 53 QFAEHLGTGIYGGI VGPESEI n
8.0mm: 4o QFAEHLGSCIYGGL VGENSDI as
Csterc. Ia HFEEHLGRCIYGGFYVGENliPI 43
B.:ubtllls 20 XHWFAEHLGRGIYEGI VGTDSDI 4s

 

 

 

 

   
    

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Artl as
Arlll 79
B. ovum: as
C. sterc. 44
B. subtilis 46
Arll 9|
Arfll nos
8. ovatus 92
C. store. 70
B. subtilis 72
Arll :17
Art" 13:
B. ovatus Ila
C. store. 96
B. subtilis 95
Am :43 .VNLNVGSGTV
Art" 157 FISANVGSGTV

C.sterc. m YINGNIGSGTV

8.0mm: I44 IVSGNVGSGTV
8.3066": 121 YICGNVGSGTV

 

 

 

 

 

 

 

Arll I69
Arfll :92
B. ovatus 170
C. store. 147
B. subtilis 147

98

Figure 12. (cont’d).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Arfl I95 NYATYCGG 220
Arlll 209 E KFATFTN 233
B.ovatus I96 Y RYSTYCR 22I
Csterc. I73 F RYQTYVR I99
B.subtllis I73 Y RFQTYVR I99
Arll 221 KIAGE VDEJJYR-E 239
mm 234 YNA VNTQFVAVGPDSFDEYSFGYPE 259
9.6mm 222 YDG R-LFKIAS GAS ---DY YK TD 243
Csterc. I99 YGN K-lYKIACGPN---SDEYNEE 220
BJubtllls I99 YSG o-IYKI__A_G_G_AN-..volI=N T0220
Am 239 VL NKTQHI—IKF--L|E] G YYTFP 262
Arlll 266 Al EAWAKKTWTWDI [EYTRG 266
B.cvatus 244 VL NRVGHR ----- YYTVT 264
Csterc. 22I TVVKIAGRF ----- YYTVP 24I
amen: 221 VLEKKAAGL ----- YYTIP 241
Arfl 263 GRWED-K .__._*‘ “ = #297
Arm 286 G PAV--IPATGFNESGYAAVIKETL309
9.6mm 265 G SGS -KGSATdFNKDDYY TMGKCL 269
Csterc. 242 GTWHS -KGSSTKFT EEWYITLFKTL 266
B.subtms 242 GDFWKGKGS AlEFT DEWFITMKKAK 267
Arfl 289 EMQELmslzanE KKQIGIIV 3:3
Arfll 3m EMDKF SD_RA EHKVS IMV 33S
9.6mm 290 EVEDVLKKHCT EDKKIALLL 3:5
Csterc. 267 KMDEL TKHTA DNRIGLVV 292
B.subtllls 266 YlDEL ciflGT EQRVGL II 293

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Arll 3.4 oewcr YIEIV ":1 339
Art" 336 DEWGT YAP 36I
B. ovatus 3I6 D E WG T EE 34I
Csterc. 293 DEWGT Y V m
8.331136": 294 DEWGT F P ,3I9
Art! 340 ‘ '*_-'"_" .- ‘2 I ~ = 'VKMANIA M 365
Art" 362 AVLAAL FNIFI RVKGANIA M397
8.0mm: 342 AF VASL LDVFH RLKMANIA I 367
C.starc. 3:9 LVAGI LNIFNKHSDKVVMANIA T 344
8.3mm": 320 ALVAAS FHIFHQHCRRV MANIA T 345
Arfl 366 VNVLQ 1LT G YVFK 39I
Art" 399 INVLQ ILTEG HTFR 4I3
8.0mm: 369 VNVL ILTKD YVFK 393
Csterc. 34s INVLQA ILTEG HVFD 370
B.subtllis 346 VNVLQA ILTEG HVFN 37I

 

 

 

 

 

 

 

 

 

 

.99

Figure 12. (cont’d).

 

 

 

 

 

 

 

Arfl 392 YNV HGATLIPMNIQTQDYVMGD--Q 41$
Arlll 4.4 var DATRLPINFNKGFYKEGT--l 437
B.ovatus 394 YKVH DATYLPIDLTCEKMSVRDN-R4I9
C.sterc. 37I YKEH GAELVEKLLLKTEKKS AQTKY 396
9.60mi: 372 FKVH DASLLATETMS ADYEWNG-w-E 39s
Arfl 416 KI'P'MVNASAS-II<DI<TvserCNI.HA440
Arm 439 ELPRVDAIAAKGRDGKIYVAITNIDP 463
9.69m: 4:9 TVPMVSATAS KNKDGVIHISLSNVDA444
Csterc. 397 TVPNLHES AS VNADGKLHITLCNLSL 422
8.90136”: 396 TFLQISISAS KQAEGDINITICNIDH 42I
Arll 44. SQSTKVEIDVT-GFEGKTATGIIIIG 466
Art" 464 KNNASLDLSFD-EQTIKDVKGET YA466
B.ovatus 443 DEAQEITINLG-DTKAKKAIGE A469
C.sterc. 423 SESYRVETEll-GKKAENVTGR G447
8.:ubtills 422 QNKAEAEIELRGLHKAAoHsgv A447
Am 466 EIZIITDYNDFGKPEMVGLKAEINVAKPK49I
Arfll 489 SAIDAVNTFDNPNNVAPTSlNAS-LT 513
9.6mm 470 s LTDYNSFEKPNIVKPAP KEVKIN495
C.starc. 449 - MNDHNTFDAPETVKPQT DKISVR 472
9.3066706 446 E MNAHNTFDDPHHVKPES RQYTLS 473

 

 

 

 

C.sterc. 473 QNGLVIEI -- 49I

Arll 492 GNKLTVEIEIAK LVQM---- 509
Arlll Sl4 GS st VTVP TVLE vse- s34
9.6mm 496 KGTMKVKLEAK [I TLE Q--- 514
SIT 0.
9.3376906 474 KNKLKIKL LLT RADS 49s

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