FEMS Microbiology Reviews 23 (1999) 411^456

Molecular and biotechnological aspects of xylanases

Neeta Kulkarni, Abhay Shendye, Mala Rao *
Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008, India

Received 23 December 1998; accepted 22 February 1999

Abstract

Hemicellulolytic microorganisms play a significant role in nature by recycling hemicellulose, one of the main components of
plant polysaccharides. Xylanases (EC 3.2.1.8) catalyze the hydrolysis of xylan, the major constituent of hemicellulose. The use
of these enzymes could greatly improve the overall economics of processing lignocellulosic materials for the generation of liquid
fuels and chemicals. Recently cellulase-free xylanases have received great attention in the development of environmentally
friendly technologies in the paper and pulp industry. In microorganisms that produce xylanases low molecular mass fragments
of xylan and their positional isomers play a key role in regulating its biosynthesis. Xylanase and cellulase production appear to
be regulated separately, although the pleiotropy of mutations, which causes the elimination of both genes, suggests some
linkage in the synthesis of the two enzymes. Xylanases are found in a cornucopia of organisms and the genes encoding them
have been cloned in homologous and heterologous hosts with the objectives of overproducing the enzyme and altering its
properties to suit commercial applications. Sequence analyses of xylanases have revealed distinct catalytic and cellulose binding
domains, with a separate non-catalytic domain that has been reported to confer enhanced thermostability in some xylanases.
Analyses of three-dimensional structures and the properties of mutants have revealed the involvement of specific tyrosine and
tryptophan residues in the substrate binding site and of glutamate and aspartate residues in the catalytic mechanism. Many
lines of evidence suggest that xylanases operate via a double displacement mechanism in which the anomeric configuration is
retained, although some of the enzymes catalyze single displacement reactions with inversion of configuration. Based on a
dendrogram obtained from amino acid sequence similarities the evolutionary relationship between xylanases is assessed. In
addition the properties of xylanases from extremophilic organisms have been evaluated in terms of biotechnological
applications. 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights
reserved.

Keywords : Xylanase ; Regulation of biosynthesis ; Extremophile; Overexpression ; Protein engineering ; Domain structure

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
2. Scope of the present review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413
3. Structure of xylan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413
3.1. Chemical structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413

* Corresponding author. Tel.: +91 (212) 338 234; Fax: +91 (212) 338 234.

0168-6445 / 99 / $20.00 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 4 5 ( 9 9 ) 0 0 0 0 6 - 6

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3.2. Three-dimensional structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414

4. Xylanase production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415
4.1. Factors aecting xylanase yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416
5. Regulation of xylanase synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417
5.1. Induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417
5.2. Regulation at the molecular level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417
6. Biochemical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419
6.1. Carbohydrate content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419
6.2. Substrate specicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 420
7. Xylanases of extremophilic origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421
7.1. Xylanases from alkaliphilic microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421
7.2. Thermophilic xylanases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422
8. Cloning and expression of xylanase gene(s) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422
8.1. Heterologous cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422
8.2. Overexpression in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 426
8.3. Expression in plant system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 426
8.4. Homologous cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427
8.5. Cloning suitable for biotechnological application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427
9. Protein engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
9.1. Amino acid modication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
9.2. X-ray crystallography studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
10. Site-directed mutagenesis (SDM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430
10.1. SDM and structure-function analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430
10.2. SDM for altered desirable properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431
11. Mechanism of action of the xylanases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432
11.1. Nucleophile and acid-base catalyst . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432
12. Domain organization of xylanases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434
12.1. Hydrophobic cluster analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434
12.2. Domain structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434
13. Molecular evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438
13.1. Sequence homology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438
13.2. Evolutionary relationship of xylanases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440
14. Biotechnological potentials of xylan and xylanases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442
14.1. Applications of xylanases in paper and pulp technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442
14.2. Other applications of xylanases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443
15. Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446

1. Introduction degradation to useful end products. Hence the devel-

The naturally occurring lignocellulosic plant bio-
mass consists of 20^30% hemicellulosic materials
which are heterogeneous polysaccharides found in
association with cellulose [1]. Biomass is an alterna-
tive natural source for chemical feedstocks with a
replacement cycle short enough to meet the demand
in the world fuel market. Xylan is the major constit-
uent of hemicellulose and is the second most abun-
dant renewable resource with a high potential for
opment of inexpensive technologies based on hemi-
cellulose is called for. Eventually, if not in the near
future, xylan, in combination with cellulose, will sup-
ply most of the global demand for raw materials. It
is not unrealistic to foresee that coal and crude oil
are likely to be substituted by biomass in another 50
years [2]. Microbial xylanases (1,4-L-D-xylan xylano-
hydrolase, EC 3.2.1.8) are the preferred catalysts for
xylan hydrolysis due to their high specicity, mild
reaction conditions, negligible substrate loss and

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 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456
side product generation. However, the cost of enzy-
mic hydrolysis of biomass is one of the factors limit-
ing the economic feasibility of the process. The pro-
duction of xylanases must therefore be improved by
nding more potent fungal or bacterial strains, or by
inducing mutant strains to excrete greater amounts
of enzymes, or both. Xylanases are produced by a
plethora of organisms like bacteria, algae, fungi, pro-
tozoa, gastropods, and arthropods [3]. Most of the
bacteria and fungi secrete extracellular xylanases
which act on the hemicellulosic material to liberate
xylose as a directly assimilable end product allowing
the organisms to grow heterotrophically on xylan.
Ruminal microorganisms are known to be potent
xylanase producers, possibly due to the high dietary
hemicellulose content of the feed of ruminant ani-
mals. The use of hemicellulolytic enzymes as a sub-
stitute for chlorine chemicals in pulp bleaching has
recently attracted considerable interest because of
environmental concerns. The limited hydrolysis of
hemicellulose in pulps by hemicellulases, mainly xyl-
anases, increases the extractability of lignin from the
kraft pulp in the subsequent bleaching sequences,
reducing the chloro-organic discharges.
2. Scope of the present review
The various applications of xylanases have stimu-
lated research on the biochemical and molecular as-
pects of this important enzyme of the family of gly-
cosyl hydrolases. Considering the future prospects of
xylanases for biotechnological exploitations, espe-
cially in the eld of biopulping and bleaching, it is
very essential to analyze the properties of xylanases
with respect to regulation of biosynthesis and mech-
anism of action. An increasing number of publica-
tions in recent years describe numerous xylanases
from new sources, their cloning, sequencing, muta-
genesis and crystallographic analysis. Many of these
reports are about hemicellulase-aided bleaching of
kraft pulp used in the paper industry, which truly
exploits the unique specicity and safety of use of
biocatalysts. Since xylanases are industrially impor-
tant enzymes, many reviews have been published
covering the various aspects of the enzyme [4^8].
The present article is a comprehensive state-of-the-
art review describing the xylanases with special em-
FEMSRE 646 28-6-99
413
phasis on the latest developments in the area of mo-
lecular biology and biotechnology. The article also
covers the xylanases from extremophilic microorgan-
isms which have gained importance by virtue of their
stability and activity at extremes of pH and temper-
ature. Theoretical analysis of a few of the reported
thermostable xylanases has also been included; it
will be useful in application-oriented protein engi-
neering studies. The recent reviews on related topics
complement the present work [9,10].
3. Structure of xylan
The L-1,4-xylans are heteropolysaccharides with a
homopolymeric backbone chain of 1,4-linked L-D-xy-
lopyranose units. The backbone consists of O-acetyl,
K-L-arabinofuranosyl, K-1,2-linked glucuronic or 4-
O-methylglucuronic acid substituents [11]. However,
unsubstituted linear xylans have been isolated from
guar seed husk, esparto grass and tobacco stalks [12].
Wood xylans exist as O-acetyl-4-O-methylglucuro-
noxylans in hardwoods or as arabino-4-O-methylglu-
curonoxylans in softwoods. The degree of polymer-
ization of hardwood xylans (150^200) is higher than
that of softwoods (70^130) [13]. The cereal xylans
are made up of D-glucuronic acid and/or its 4-O-
methyl ether and arabinose [14]. Endospermic arabi-
noxylans of annual plants, also called pentosans, are
more soluble in water and dilute alkali than xylans
of lignocellulosic materials because of their branched
structures [15].
3.1. Chemical structure
Based on the common substituents found on the
backbone, xylans are categorized as linear homoxy-
lan, arabinoxylan, glucuronoxylan and glucuronoar-
abinoxylan. However, in each category there exists a
microheterogeneity with respect to the degree and
nature of branching. The basic backbone and the
possible substituent groups are shown in Fig. 1A.
Xylans are present in a partly acetylated form in
various plants. The O-acetyl groups present at C-2
and C-3 positions of xylosyl residues inhibit xyla-
nases from completely degrading acetylxylan, prob-
ably by steric hindrance. The synergistic action of
acetylxylan esterases and xylanases is therefore es-
414 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456

sential for the complete hydrolysis of acetylxylans.
The presence of small amounts of feruloyl and p-
coumaroyl acids linked via L-arabinose residues has
been shown on the structure of xylan in several stud-
ies. These hydroxycinnamic acids are bound to C-5
of the arabinose residue [16]. The presence of a co-
valent bond between lignin and hemicellulose, per-
haps through xylan substituents in many cases, has
been documented. Evidence for the existence of an
ether linkage between arabinose and lignin [13] and
an ester linkage between glucuronic acid and lignin
[17] has also been shown. Feruloyl groups may also
crosslink xylan and lignin [18]. The side chains de-
termine the solubility, physical conformation and re-
activity of the xylan molecule with the other hemi-
cellulosic components and hence greatly inuence the
mode and extent of enzymatic cleavage.
3.2. Three-dimensional structure
The three-dimensional structure of the xylan mol-
ecules has been elegantly described by Atkins [19].
The xylan backbone shows a threefold left-handed
conformation under crystallized conditions; the ge-
ometry of the glycosidic linkage is not aected by the
side chains (Fig. 1B). Studies of the polysaccharide
indicate that the backbone imposes certain minimum
constraints and the interactions between the chains
determine the nal conformation. Electron dirac-
tion patterns also conrm the threefold conforma-

Fig. 1. A: Chemical structure of xylan. B: Three-dimensional structure. Projections perpendicular (top) and parallel (bottom) of (a) xylan
backbone, (b) backbone and one L-arabinose side group: (c) backbone and two L-arabinose side groups. Hydrogen bonds are shown dot-
ted. In each case the backbone is a left-handed threefold helix. (Based on [19].)

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 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456 415

Fig. 1 (continued)

tion and show that the chains are organized in a
trigonal lattice with hexagonal morphology. The sin-
gle hydrogen substituent at position 5 on the xylose
ring has a dramatic eect on the intra- and inter-
chain hydrogen bonding interactions. Energy calcu-
lations suggest that the D-xylose ring exists in the
common 4 C1 chair conformation, which indicates
that in both the 1-4 and 1-3 glycosidic linked xylans
the bonding is diequatorial (1e-4e). The intra-chain
hydrogen bond O(2')HmO(6) reinforces the
O(3)mO(5') hydrogen bond to support a twofold
extended, helical structure, like a ribbon, and the
O(6)H group hydrogen bonds to O(3) atoms in ad-
jacent chains to form sheets [19]. The concept that
the glycosidic linkage geometry maintains the basic
conformation is evident from these studies. However,
three-dimensional structure data on xylan, in aque-
ous environment, would be extremely important in
understanding the xylan and xylanase interactions.
4. Xylanase production
The basic factors for ecient production of xyla-

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416 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456
nolytic enzymes are the choice of an appropriate in-
ducing substrate and an optimum medium composi-
tion. The importance of cellulase-free xylanase sys-
tems in the paper and pulp industry has initiated
research into the correlation between the production
of xylanases and cellulases by microorganisms. Fila-
mentous fungi are particularly interesting producers
of xylanases since they excrete the enzymes into the
medium and their enzyme levels are much higher
than those of yeast and bacteria. However, fungal
xylanases are generally associated with cellulases
[20]. Selective production of xylanase is possible in
the case of Trichoderma and Aspergillus species using
only xylan as the carbon source. On cellulose these
strains produce both cellulase and xylanase which
may be due to traces of hemicellulose present in
the cellulosic substrates [6]. The mechanisms that
govern the formation of extracellular enzymes with
reference to carbon sources present in the medium
are inuenced by the availability of precursors for
protein synthesis. Therefore, in some fungi, growing
the cells on xylan not contaminated by cellulose
under a lower nitrogen/carbon ratio in the medium
may be one of the strategies for producing xylano-
lytic systems free of cellulases [21]. However, cellulo-
sic substrates were also found to be essential in the
medium for maximum xylanase production by Clos-
tridium stercorarium [22], Thermomonospora curvata
[23] and Neurospora crassa [24]. Cheaper hemicellu-
losic substrates like corn cob, wheat bran, rice bran,
rice straw, corn stalk and bagasse have also been
found to be most suitable for the production of xyl-
anases in the case of certain microorganisms such as
Aspergillus awamori, Penicillium purpurogenum [25]
and alkaliphilic thermophilic Bacillus sp. NCIM 59
[26]. The xylanase activity is found to be higher in
fungi than in bacteria. Among fungi, the maximum
activity reported is 3350 IU ml31 in Trichoderma
reesei [27]. The maximum xylanase activity in solid-
state fermentation has been obtained from the fun-
gus Schizophyllum commune (22 700 IU g31 ) [28].
Trichoderma hamatum is reported to produce 7000
IU g31 dry wt using wheat straw as substrate [29].
The production of cellulase-free xylanase has been
reported in a few Bacillus sp. [26] and fungi [30,31].
Archana and Satyanarayana have reported xylanase
production by the bacterial strain, Bacillus lichenifor-
mis A99, in solid state fermentation (SSF) [32]. SSF
FEMSRE 646 28-6-99
systems resemble the natural habitats of microbes
and, therefore, may prove ecient in producing cer-
tain enzymes and metabolites. Although a plethora
of xylanase producing strains have been described,
their use for commercial production at present is
restricted mainly to Trichoderma sp. and Aspergillus
sp. [25]. However, the future scenario may be dier-
ent since several promising strains that produce xyl-
anases in higher yields, with increased stability at
extreme conditions of pH and temperature, have
been recently identied.
Actinomycetes and bacteria exhibit near-neutral
pH optima for growth and enzyme production [33],
in contrast to the generally acidic pH requirements
of fungi. However, certain alkaliphilic Bacilli are
known which have pH optima for growth and en-
zyme production at 9^10 [34]. In Streptomyces avo-
griseus [35] and in the Bacillus sp. NCIM 59 [36]
simultaneous production of glucose (xylose) isomer-
ase, in addition to cellulases and xylanases, has been
described.
4.1. Factors aecting xylanase yield
The yield of xylanases in a fermentation process is
governed by a few key factors in addition to the
standard parameters. When xylanase fermentation
is carried out on complex heterogeneous substrates,
various factors have a combined eect on the level of
xylanase expression. They include substrate accessi-
bility, rate and amount of release of the xylooligo-
saccharides and their chemical nature and quantity
of xylose released ^ which acts as the carbon source
and as an inhibitor of xylanase synthesis in most of
the cases. Generally, the slow release of the inducer
molecules and the possibility of the culture ltrate
converting the inducer to its non-metabolizable de-
rivative are believed to boost the level of xylanase
activity.
The xylanases bind tightly to the substrate. A part
of the enzyme produced during the fermentation is
often lost and discarded, as bound enzyme, along
with the insoluble substrate. The metabolic enzymes
of the xylanase producer such as proteases [37] and
transglycosidases also aect the actual yield of the
enzyme [38]. These enzymes are optimally expressed
at the end of the exponential phase; and the harvest-
ing time of the xylanases must be correlated to the
 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456
production of these enzymes on the medium under
consideration. Other bioprocess parameters that can
aect the activity and productivity of xylanase at-
tained in a fermentation process include the pH, tem-
perature and agitation.
5. Regulation of xylanase synthesis
Despite the increase in knowledge of microbial
xylanolytic systems in the past few years further
studies on induction and secretion of xylanases are
necessary to develop ecient xylanase producers for
possible commercial applications. Xylanase produc-
tion by various bacteria and fungi has been shown to
be inducible. But rare examples of constitutive xyla-
nase expression have also been reported [39]. In gen-
eral the xylanase induction is a complex phenomen-
on and the level of response to an individual inducer
varies with the organisms. An inducer producing
maximum xylanase activity in one species may be
the inhibitor of activity in an other species [40].
The substrate derivatives and the enzymatic end
products may often play a key positive role in the
induction of xylanases; they can also act as the end-
product inhibitors, possibly at much higher concen-
trations.
5.1. Induction
Xylan, being a high molecular mass polymer, can-
not penetrate the cell wall. The low molecular mass
fragments of xylan play a key role in the regulation
of xylanase biosynthesis. These fragments include
xylose, xylobiose, xylooligosaccharides, heterodisac-
charides of xylose and glucose and their positional
isomers. These molecules are liberated from xylan by
the action of small amounts of constitutively pro-
duced enzyme. Cellulose has also been shown to
act as an inducer of the xylanase in a few cases,
but its not clear whether the inducing eect lies
with cellulose or the contaminating xylan fraction.
In Streptomyces sp. xylanase activity appears to in-
crease with the crystallinity of the cellulosic substrate
[41]. Sugarcane bagasse is found to be the best in-
ducer of xylanase and L-xylosidase in Cellulomonas
avigena [42]. A synergistic eect on the synthesis of
both the enzymes was observed when cellulose and
FEMSRE 646 28-6-99
417
hemicellulose were used together as the carbon
source.
The xylanase from Aspergillus sp. (2MI), induced
by pine xylan, exhibited higher stability than that
induced by other xylans [43]. In the presence of xyl-
ose higher enzyme yields were obtained from Bacil-
lus pumilus [44], Streptomyces lividans 66 [45] and
Aureobasidium pullulans [46]. However, in Cryptococ-
cus albidus [8] xylose repressed xylanase production
while in thermophilic actinomycetes [47] it had no
inuence on the regulation of xylanase expression.
Xylobiose was found to be a specic inducer of xyl-
anase in T. reesei [48] and Aspergillus terreus [38].
Xylanolytic systems of yeasts and fungi were also
induced by positional isomers of xylobiose. In Cryp-
tococcus albidus the positional isomers of xylobiose
behave dierentially from xylobiose [49] in that the
response of the cells to them is slower, however, the
enzyme yields are higher in the presence of isomeric
xylobioses indicating that they are not direct in-
ducers. D-Xylan fragments, as well as methyl-L-D-xy-
lopyranoside, induce xylanase in Streptomyces sp.
[50,51] and in the yeasts of the genera Cryptococcus
and Trichosporon [52,53]. In Trichosporon cutaneum
[54] and Trichoderma lignonum [55] thioxylobiose
was found to induce xylanase activity. It is also re-
ported that not all L-xylopyranosides serve as in-
ducers of xylanase since their induction capacity is
also dependent on the structure of aglycon.
The role of transglycosylating enzymes in the syn-
thesis of positional isomers of these heterodisaccha-
rides is indicated in Aspergillus terreus [38] and T.
reesei [48]. The low molecular mass substances that
have been identied as xylanase inducers need trans-
ferase enzymes for their translocation into the cyto-
plasm. Hence the level of inducers and/or the re-
quired enzymes in the culture ltrate also aect the
xylanase synthesis. The possible factors aecting xyl-
anase induction have been diagrammatically repre-
sented (Fig. 2), largely based on the gure by Thom-
son [56].
5.2. Regulation at the molecular level
Xylanase biosynthesis and the phenomenon of en-
zyme induction at the molecular level are compara-
tively less investigated. This may be due to the lack
of cell-free systems which are necessary for the eval-
418 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456

Fig. 2. Regulation of xylanase biosynthesis (hypothetical model). Constitutive xylanases degrade xylan to xylooligosaccharides and xylo-
biose, which are taken up by the cell and induce other xylanase genes. The inducible xylanases degrade xylan further to xylooligosaccha-
rides and xylobiose. The L-xylosidases, which may be produced constitutively and/or inducibly, convert xylobiose to xylose and may sub-
sequently transglycosylate it to XylB1-2Xyl and GlcB1-2Xyl. These compounds are taken up by the cell and act as additional inducers of
genes encoding xylanolytic enzymes. (Based on [56].)

uation of inducers under experimental conditions
which prevent their transport into the cells [6]. A
separate regulation for the formation of xylanase
and cellulase has been reported in a few microorgan-
isms [38,57]. In spite of this evidence, an additional
linkage governing the synthesis of the two enzyme
systems seems to be present, as evident from the
pleiotropy of mutations, causing the elimination of
both cellulase and xylanase genes [58].
Esteben et al. [59] have reported that xylanase was
undetected in glucose grown cultures of Bacillus cir-
culans WL-12 but xylose, mannose and cellobiose
supported xylanase production. The xylanase and
xylosidase in Butyrivibrio brisolvens GS 113 are
shown to be under coordinate control, induced by
xylan and repressed by glucose [60]. An analysis of
DNA fragments containing L-xylanase genes from B.
pumilus [61,62] indicated that the xylanase and xylo-
sidase genes are closely associated and are linked in a
14.4-kb DNA fragment. However, they did not ap-
pear to be controlled by the same operon.
5.2.1. Catabolite repression
Catabolite repression by glucose is a common phe-
nomenon observed in xylanase biosynthesis. Rela-
tively few reports on the relation of cAMP to xyla-
nase induction are available. In the case of yeast C.
albidus, when xylan or methyl-L-xylopyranoside were
used as inducers, cAMP caused a twofold increase in
xylanase production [63]. However, cAMP had no
eect on the repression caused by D-xylose. It has
been suggested that a 15-bp nucleotide sequence, up-
stream from the L-xylanase gene, may be a part of
the cyclic AMP regulatory sequence. In Aspergillus
tubigensis, a 158-bp region, 5' upstream of the xyla-
nase gene, was shown to be involved in xylan-specic
induction [64]. The authors have also reported that
catabolite repression of xylanase gene appeared to be

FEMSRE 646 28-6-99

 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456
controlled at two levels, directly by repression of
gene transcription and indirectly by repression of
transcriptional activator. The same pattern of regu-
lation was observed in A. niger and A. nidulans.
Mach et al. [65] have studied the carbon catabolite
repression of xylanase gene expression and have an-
alyzed the molecular basis for the absence of xyla-
nase I formation by the lamentous fungus T. reesei.
They have concluded on the basis of Northern blot
analysis that the repression of basal xyn 1 transcrip-
tion was mediated by the carbon catabolite repressor
protein Cre 1. Cre 1 in vivo binds to two of four
consensus sites (5P-SYG-GRG-3P) in the xyn 1 pro-
moter, which occurred in the form of an inverted
repeat.
Deletion and functional analysis of the xylanase
genes from A. niger and A. tubigensis [66] led to
the discovery of a triplicated sequence that appears
to control the enzyme induction. Gat et al. [67] have
demonstrated that, in the case of B. stearothermophi-
lus T-6, induction of xylanase synthesis by xylose
was controlled at the transcriptional level, indicating
the presence of a repressor protein mediating the
regulation. Enzyme synthesis was also found to be
repressed when easily metabolizable carbon sources
were present in the growth medium, suggesting that
the synthesis of the enzyme is controlled by transi-
tion state regulators and catabolite repression. The
sequence analysis of T-6 xylanase has revealed the
presence of an AT-rich region preceding the pro-
moter that has been identied as the binding site
for a protein that regulates induction [68]. It has
been suggested that these regions interact or facili-
tate interaction with regulators. A putative catabolite
repression sequence was found 230 bp inside the xyl-
anase structural gene. The catabolite repression con-
sensus sequence was TGT/AAANCMGNTNA/TCA,
where the underlined letters represent the most crit-
ical bases, N is any base, and the vertical line denotes
an axis of symmetry. In B. subtilis, this consensus
sequence was found inside the structural genes in
several operons, including the xyl operon [69].
The biosynthesis of xylanase occurs several hours
after the depletion of the inducer added to the me-
dium, in contrast to the synthesis of L-xyloside per-
mease and L-xylosidase which have very short induc-
tion periods. This is in agreement with the suggestion
that mRNAs of secreted proteins do not undergo a
FEMSRE 646 28-6-99
419
rapid turnover as that of intracellular proteins [70].
Characterization of mRNA transcripts of xylanases
and studies on in vivo genetics will considerably help
a better understanding of the regulation of xylanase
biosynthesis.
6. Biochemical properties
The available information about the properties of
xylanases stems mostly from studies on bacterial and
fungal enzymes. Microbial xylanases are single sub-
unit proteins with molecular masses in the range of
8^145 kDa [10].
The optimum temperature for endoxylanase from
bacterial and fungal sources varies between 40 and
60C. Fungal xylanases are generally less thermo-
stable than bacterial xylanases. Fungi which are mes-
ophilic in origin but produce thermostable xylanases
include Ceratocystis paradoxa, the xylanase of which
is stable at 80C for l h [71]. A low temperature
active enzyme having both carboxymethyl cellulase
and xylanase activities from Acremoniun alcalophilum
JCM 7366 has been reported recently. The xylanase
and cellulase activities at 0C were 25 and 48.8%,
respectively, of their activities at the optimum tem-
perature of 40C [72]. D-Xylanases from dierent or-
ganisms are usually stable over a wide pH range (3^
10) and show optimum pH in the range of 4^7. The
xylanases from fungi such as Aspergillus kawachii
[73] and Penicillium herque [74] exhibit optimum
pH towards the acidic side (pH 2^6). The isoelectric
points for endoxylanases from various sources
ranged from 3 to 10. Generally, bacteria are known
to produce two xylanases ^ high molecular mass
acidic xylanase and low molecular mass basic xyla-
nase. However, this type of relationship is not ob-
served in fungi; but low molecular mass basic xyla-
nases are common. The amino acid compositions of
xylanases reported from various sources indicate pre-
dominantly aspartic acid, glutamic acid, glycine, ser-
ine and threonine.
6.1. Carbohydrate content
The occurrence of glycosylated enzymes is a com-
mon phenomenon among many eukaryotic xylanases
[74]. The xylanases from prokaryotic sources, like
420 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456
Clostridium stercorarium [22], Streptomyces sp. [75]
and an alkaliphilic, thermophilic Bacillus sp. [26],
were found to be glycoproteins. Carbohydrate
groups are covalently linked with protein or are
present as dissociable complexes with xylanases. Gly-
cosylation has been implicated in the stabilization of
glycanases against extreme environments [76]. The
recombinant xylanases expressed in Escherichia coli
from an alkaliphilic thermophilic Bacillus sp. [77]
showed lower stability at higher temperature and
reduced ability to bind xylan compared to xylanases
from the parent strain which is attributed to degly-
cosylation. In the case of xylanases from Talaromy-
ces byssochlamydoides YH-50 the carbohydrate resi-
dues are found to be mannose, glucose and fucose
[78]. It has been suggested that both dierential gly-
cosylation and proteolysis may contribute to the
multiplicity of xylanases [7].
6.2. Substrate specicity
Knowledge of the mechanism of action of xylan-
degrading enzymes has been gained from studies on
substrate specicity, the role of side chain substitu-
ents on activity, the specicity of bonds cleaved and
the end products. The xylanases of fungal origin are
well characterized; they are mainly of two types ^
non-debranching, which do not liberate arabinose,
or debranching, which liberate arabinose from the
side chain substituents, in addition to cleaving
main chain linkages [79]. Many xylanases of fungal
origin, such as Neurospora crassa [80] and Aspergillus
niger [81], were found to release arabinose from ara-
binoxylan. An endoxylanase from Streptomyces rose-
iscleorticus has also been shown to be a debranching
type of enzyme. However, xylanases from Trichoder-
ma harzianum [82] and another strain of A. niger
were found not to release free arabinose from arabi-
noxylan. The presence of debranching as well as
non-debranching xylanases have been reported
from T. koningii and Ceratocystis paradoxa, and var-
ious other sources. A novel enzyme has been recently
isolated from A. awamori which cleaves arabinose
substituents from cereal arabinoxylans, is specic to
the substituent on the relevant xylose, and does not
cleave the xylan main linkage [83].
It is commonly observed that substituents in the
highly branched polysaccharides interfere with xyla-
FEMSRE 646 28-6-99
nase activity. However, enzymes having more anity
for main chain linkages, near branch points, were
reported from A. niger, T. viride and other sources
[3]. The xylanases also vary in their activity against
various cellulosic substrates. A few of them act only
on xylan whereas the non-specic xylanases from
Myrothecium verrucaria, Penicillium capsulatum and
P. funiculosum act against carboxymethyl cellulose
and xylan. The relaxed specicity of some xylanases
as against the more restricted specicity of others
could be due to dierences between residues involved
in the catalytic groups. Generally, xylanases appear
to be specic toward the intersugar linkage [3]. In C.
thermocellum, endoglucanase was reported to hydro-
lyze L-1,3 linkages in barley, L-glucan as well as L-
1,4 linkages in other substrates. Transglycosylation
by endoglucanases was believed to lead to products
with the same linkage as the substrate because they
contain stereospecic binding sites on either side of
the catalytic site [84]. The xylanase from Cryptococ-
cus albidus was reported to form a 1,3- L-D-linkage
due to transglycosylation reactions [85].
6.2.1. Subsite mapping
Subsite mapping and determination of end prod-
uct analysis are signicantly useful in understanding
the mode of action of xylanases. Based on the kinetic
and end product analysis techniques the subsite
maps of the endoxylanase from Aspergillus niger
have been determined. The enzyme did not show
any appreciable hydrolysis of xylobiose and had
higher anity towards xylooligosaccharides sub-
strates with increasing chain length. Since the bind-
ing site of the endoxylanase is shown to consist of
eight subsites with the catalytic site in the middle,
xylobiose did not form a productive enzyme com-
plex. The xylooligosaccharides of larger length
showed hydrolysis of substrate with the formation
of a productive enzyme complex. The presence of
ve main subsites and the localization of a catalytic
site between the third and fourth subsites has been
demonstrated in the case of xylanase from A. niger
[86]. Simon et al. [87] have recently shown the occur-
rence of an additional subsite (subsite F, i.e. the sixth
xylose binding site) in the substrate binding cleft of
xylanase A from Pseudomonas uorescens. It is found
that the primary role of F subsite of xylanase A is to
prevent small oligosaccharides from forming non-
 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456
productive enzyme-substrate complexes. The endox-
ylanase from a dierent strain of A. niger [88] was
found to have seven subsites whereas that from
Cryptococcus albidus has four with the catalytic
group in the middle [89]. Debeire et al. [90] mapped
the subsites of a xylanase from Clostridium thermo-
lacticum and also determined the structures of end
products by nuclear magnetic resonance spectros-
copy and methylation analysis. They concluded
that xylanase from Clostridium thermolacticum was
composed of ve subsites, a^e, binding ve xylosyl
rings A^E. The catalytic site was located between
xylosyl rings B and C and subsites d and e were
able to bind substituted xylosyl residues unlike sub-
sites a, b and c. The dierences in the subsite num-
bers of the xylanases may be due to the dierent
mode of action of the individual xylanases and the
length of the end products released by them. The
heterogeneous nature of xylan may be one of the
reasons for the multiplicity of xylanases. The diver-
gent specicities of these enzymes could play a sig-
nicant role in their synergistic action towards the
complex substrate.
7. Xylanases of extremophilic origin
Considerable progress has been made in the iso-
lation of extremophilic microorganisms and their
successful cultivation in the laboratory. A plethora
of enzymes from diverse sources, and techniques to
modify interesting candidates for basic or applied
research, have laid the basis for the expansion of
biocatalysts in ways not previously envisioned [91].
Commercial applications of the xylanases demand
identication of highly stable enzymes active under
routine handling conditions. Many advantages such
as reduced contamination risk and faster reaction
rates have been proposed for the use of thermophiles
in biotechnology processes. In general, parameters
such as temperature, pH, and chemical and enzy-
matic stability are important for the industrial ap-
plicability of any enzyme. Recently a simple model,
based on the thermodynamic and kinetic parameters
for inactivation of the enzyme, has been presented
for predicting (1) thermostabilities in the presence of
substrate, and (2) residual activities of enzymes in
harsh processing environments. The authors have
FEMSRE 646 28-6-99
421
proposed that the model could be applied to a large
number of industrially relevant enzymes and process-
ing conditions where the reversible denaturation of
protein is fast as compared to the rate of the subse-
quent irreversible inactivation step [92]. One of the
ways to identify the industrially suitable xylanase
preparations is to look for the enzymes from extrem-
ophilic microorganisms. In the words of M.W.W.
Adams, University of Georgia, a pioneer in the study
of extremophiles, ``If you want highly stable en-
zymes, you must go to the extreme environments.
Nature's bounty, it seems, abides in its black smok-
ers, steam vents and salt lakes'' [93]. The use of bio-
catalysts has been constrained due to their labile na-
ture under extreme temperature and pH conditions.
The study of extremophiles and their enzymes can
extend the present understanding of protein chemis-
try in addition to expanding the potential applica-
tions of biocatalysts.
7.1. Xylanases from alkaliphilic microorganisms
Studies of alkaliphiles have led to the discovery of
a variety of enzymes which exhibit some unique
properties. Biological detergents contain enzymes
such as alkaline proteases and/or alkaline cellulases
from alkaliphiles. One signicant application is the
use of the enzyme, cyclodextrin glycosyl transferase
(CGT), to catalyze the degradation of starch to cy-
clodextrins. Commercial production became eco-
nomical only after the discovery of alkaliphilic Ba-
cillus producing CGT with enhanced pH stability.
Alkaline xylanases have gained importance due to
their application for the development of eco-friendly
technologies used in paper and pulp industries. The
enzymes are able to hydrolyze xylan which is soluble
in alkaline solutions [94]. The rst report on xylanase
from alkaliphilic bacteria was published in 1973 by
Horikoshi and Atsukawa [95]. The puried enzyme
of Bacillus sp. C-59-2 exhibited a broad pH optimum
ranging from 6.0 to 8.0. Many of the xylanases pro-
duced by alkaliphilic microorganisms such as Bacil-
lus sp. [34] and Aeromonas sp. 212 [96] with optimum
growth at pH 10.0 showed remarkable stability at
pH 9^10 but were not highly active above pH 8.0.
The enzymes from Bacillus sp. TAR-1 [97], C-125
[98] and alkaliphilic Bacillus sp. (NCL-86-6-10) [99]
were optimally active at pH 9^10. Recently an alkali-
422 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456
tolerant xylanase from Aspergillus scheri [100] was
reported to exhibit remarkable (pH 9.0) stability.
The xylanase from Cephalosporium was the only
one reported from an alkaliphilic fungus having ac-
tivity at broad pH range of 6.5^9.0 [101]. Xylanases
from four alkaliphilic, thermophilic Bacillus strains
(W1 JCM 2888), W2 JCM 2889, W3 and W4 with
optimum pH of 6.0^7.0 and temperature of 65^70C)
are reported [102]. An alkaliphilic thermophilic Ba-
cillus sp. (NCIM 59) producing two types of cellu-
lase-free xylanases at pH 10 and 50C has been
documented [26].
7.2. Thermophilic xylanases
The xylanases from thermophilic bacteria such as
Thermonospora fusca [47], thermophilic Bacillus sp.
[103] and Bacillus stearothermophilus [104] show an
optimum temperature in the range of 65^80C. The
thermostable xylanase produced by a thermotolerant
Aspergillus strain at 37C showed maximum activity
at 80C [105]. Xylanase A from the thermophilic
anaerobe Clostridium stercorarium has a temperature
optimum of 70C and a half-life of 90 min at 80C,
whereas the xylanase from Thermotoga sp. has been
shown to have a temperature optimum of 105C at
pH 5.5 with a half-life of 90 min at 95C [106]. Xyl-
anase from Dictyoglomus sp.exhibited a half-life of
80 min at 90C [107]. Among the thermophilic fungi,
xylanase from Thermoascus aurantiacus has been re-
ported to be stable at 70C for 24 h with a half-life
of 54 min at 80C [108]. The other thermophilic fun-
gi producing thermostable xylanases include Paecilo-
myces variota [109] and T. byssochlamydoides [110]
which show an optimum temperature of 65^75C
at pH 5^6.5. Recently endoxylanases from the ther-
mophilic actinomycete Microtetraspora exuosa SIIX
were reported to have an optimum temperature of
80C, at pH 6.0 [111]. Despite the prevalence of xy-
lan-degrading enzymes in actinomycetes, compara-
tively little information is available about xylanases
from other groups of thermophilic actinomycetes
such as Microbispora, Saccharomonospora, etc. Table
1 describes a few of the extremophilic organisms
producing xylanase.
As pointed out by Hodgson [112], dramatic
changes are predicted in the future scenario of the
industrial enzyme business which is mostly based on
FEMSRE 646 28-6-99
high technology. Multinational companies like
Novo, Genencor and Diversa plan to mobilize bio-
diversity by establishing a large culture collection of
unique microorganisms and to screen for enzyme
activities. Diversa (formerly Recombinant Biocataly-
sis) is specically harvesting DNA from entire ex-
tremophilic communities and is using probes, based
on known enzyme sequences to perform biopanning
of expression libraries derived from the DNA. That
the extremozymes have signicant nancial impact
for the companies that exploit them is evident from
the example of Taq polymerase which has sales of
$80 million per annum [113]. The unique framework
of enzymes isolated from extreme environments will
be the ideal choice to engineer novel proteins with
the desired functions suitable for a particular appli-
cation. In spite of having interesting and novel prop-
erties, the extremophilic enzymes have not yet been
commercialized. Recently, Genencor International
(Rochester, NY) introduced cellulase 103, isolated
from an alkaline bacterium, for application in the
detergent and fabric industries. This is the rst
large-scale commercial application of an extremo-
molecule. With the advent of recombinant DNA
technology and protein engineering one can foresee
a tremendous future for such enzymes.
8. Cloning and expression of xylanase gene(s)
For the commercial realization and economic via-
bility of xylanase production, it is necessary to iden-
tify organisms which can hyperproduce the enzymes.
Recombinant DNA techniques oer the means to
enhance protein production. Xylanase genes have
been cloned from dierent microbial genera into var-
ious suitable hosts. The bacterial genes encoding xy-
lan-degrading enzymes have been found to be adja-
cent on the genome or in close proximity to other
genes encoding cellulase-related functions. In P. u-
orescens subsp. cellulosa, xylanase and arabinofura-
nosidase genes were transcribed in the same direction
and were separated by only 148 bp [114], while the
second xylanase gene mapped to within 125 bp of the
endoglucanase gene. In the case of B. polymyxa, xyl-
anase and endoglucanase genes were separated by
155 bp [115]. Similarly the close linkage between xyl-
anase and L-xylosidase genes has been reported in
 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456 423

Table 1
Xylanases from extremophilic organisms
Source Xylanase Optimum conditions Reference
Growth Activity
pH Temp. (C) pH Temp. (C)
Thermophilic bacteria
1 Bacillus acidocaldarius 3.5^4.0 65 4.0 80 [284]
2 Bacillus licheniformis A 99 7.0 60 7.0 50 [32]
3 Bacillus stearothermophilus T-6 T-6 7.0^7.3 60 9.0 65 [104]
4 Bacillus stearothermophilus No. 21 A 7.0 55 7.0 60 [285]
5 Bacillus thermoalkalophilus 90 60 6.0^7.0 60, 70 [286]
6 Clostridium acetobutylicum ATCC A 6.0 37 5.0 50 [287]
824
B 37 5.5^6.0 60
7 Clostridium stercorarium HX-1 D 6.0^7.0 60 6.5 75 [288]
8 Clostridium stercorarium F-9 A 6.0^7.0 65 6.5 75 [22]
9 Clostridium thermolacticum (TC 21) 6.0^7.0 65 6.5 80 [289]
10 Dictyoglomus thermophilum strain B1 7.0 68 7.0 80 [290]
11 Microtetraspora exuosa S II X 6.0 80 9.0 52 [111]
12 Thermophilic Bacillus Strain XE 7.0 55 6.0 75 [291]
13 Thermophilic Bacillus sp 7.0 65 6.5^7.0 78 [103]
14 Thermophilic bacteria ITI 283, ITI 7.5 65 8.0 80 [292]
236
15 Thermoanaerobacterium sp. JW/SL- 6.0 60 6,2 80 [293]
YS485
16 Thermotoga sp. (Fjss3-B.1) A 6.8^7.0 80 5.3 105^110 [106]
17 Thermotoga maritima (MSB 8) A 7.0 80 6.2 92 [294]
B 80 5.4 105
18 Thermotoga thermarum 1 6.0^7.0 77 6.0 80 [295]
2 77 7.0 90^100
19 Thermomonospora curvata 1 6.0^7.0 55 7.8 75 [23]
2 7.2 75
3 6.8 75
20 Thermomonospora chromogena 8.0 50 5.0^8.0 75 [47]
MT814
21 Thermomonospora fusca BD 21 8.0 50 6.0^8.0 65 [296]
23 Thermomonospora fusca YX 8.0 50 6.0^8.0 70 [239]
24 Streptomyces thermoviolaceus OPC- I 7.0 50 7.0 70 [297]
520
II 50 7.0 60
Thermophilic fungi
1 Gloephyllum trabeum ^ 4.0 80 [298]
2 Talaromyces byssochlamydoides YH- 6.2 50 5.0 70 [78]
50
3 Thermoascus aurantiacus 6.0 45 5.0 75 [108]
4 Thermomyces lanuginosus DSM5826 6.5 50 6.5 50 [299]
5 Talaromyces emersonii CBS 814.7 II 4.5 45 4.2 78 [300]
III 4.5 45 3.5 67
Alkaliphilic bacteria
1 Alkalophilic Bacillus 41M-1 10.3 37 9.0 50 [97]
2 Bacillus sp. TAR-1 10.5 50 9.0 70 [97]
3 Bacillus sp. C-59-2 8.0 37 5.5^9.0 60 [301]
4 Bacillus sp. C-125 10.5 ^ 6.0^10.0 70 [98]
5 Bacillus sp. NCIM 59 10.0 50 6.0 50^60 [26]
6 Aeromonas sp. 212 10.0 37 7.0^8.0 50 [118]

FEMSRE 646 28-6-99

424 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456

Table 1 (continued)
Xylanases from extremophilic organisms
Source Xylanase Optimum conditions Reference
Growth Activity
pH Temp. (C) pH Temp. (C)
7 Bacillus sp. NG-27 9.0^10.0 27 7.0, 8.4 70 [302]
8 Bacillus sp 9.0^10.0 45^50 [102]
W1 I 6.0 65
II 7.0^9.0 70
W2 I 6.0 65
II 7.0^9.5 70
W3 6.0 65
W4 6.0^7.0 70
9 Bacillus NCL-87-6-10 9.5 28 8.0 60 [99]

B. pumilus [61] and C. saccharolyticum [116]. In spite
of this frequent clustering of the genes encoding xy-
lan-degrading enzymes, there is no evidence for poly-
cistronic transcription [58]. The nucleotide sequence
data of the 4.2-kb chromosomal segment of the ge-
nome of Bacillus stearothermophilus 21 indicated that
L-xylosidase gene was located upstream of the xyla-
nase gene. Further analysis indicated that the two
genes were separated by only 2 bp and were tran-
scribed independently [117].
8.1. Heterologous cloning
The xylanase genes have been isolated from dier-
ent microbial genera and expressed in E. coli. The
expression in E. coli is generally found to be lower
than the parent organism, and conned to the cyto-
plasmic or the periplasmic fractions. The absence of
post-translational modications such as glycosyla-
tion in E. coli and the intracellular accumulation of
the recombinant xylanases have been suggested to be
the key reasons for the low levels of activity. Extra-
cellular activity has been reported in recombinant E.
coli for the xylanases from alkaliphilic Aeromonas
[118], alkaliphilic Bacillus [119], alkaliphilic, thermo-
philic Bacillus sp. [120] and Cellulomonas sp. [121].
The xylanase gene cloned from Bacillus stearother-
mophilus T-6 was of particular interest because the
enzyme was optimally active at pH 9.0 and 65C
[67]. The sequence data for the gene is also available,
which can be used in further studies on site-directed
mutagenesis. Cloning and expression of a xylanase
gene from the extreme thermophile Dictyglomus ther-
mophilum Rt46B.1 in E. coli has been reported [122];
the recombinant enzyme was found to have an opti-
mum temperature of 85C. The xylanase cloned from
Thermotoga maritima showed an optimum temper-
ature of 90C at pH 5.5 and was stable up to
100C [123]. The recombinant xylanase from Ther-
motoga neapolitana was found to be stable at 90C
for 4 h with a half-life of 2 h at 100C [124]. The
latter had an optimum temperature of 102C at pH
5.5. Studies on the cloned xylanase from Clostridium
thermocellum F1 revealed that it was optimally active
at 80C and was stable up to 70C at neutral pH and
over the pH range of 4^11 at 25C [125]. Keen et al.
[126] have reported the cloning of a xylanase gene
from corn strains of Erwinia chrysanthemi. Sequence
analysis revealed that the leader peptide of the pro-
tein was unusual and long. The protein was found to
be distinct from xylanases belonging to glycohydro-
lase families 10 and 11 and appeared to be intermedi-
ate between families 5 and 30. The XYN 2 gene from
T. reesei QM6a [127] was expressed in yeast Saccha-
romyces cerevisiae under the control of alcohol de-
hydrogenase (ADH 2) and phosphoglycerate kinase
(PGK 1) gene promoters and terminators, respec-
tively. The recombinant strains produced 1200 and
160 nkat of xylanase activity per ml, respectively,
under the control of ADH 2 and PGK 1 promoters.
The recombinant xylanase had an optimum temper-
ature of 60C at pH 6 and retained more than 90%
of its activity after 60 min at 50C. Recently Graessle
et al. [128] reported a system for regulated heterolo-
gous gene expression in the lamentous fungus Peni-
cillium chrysogenum. The heterologous expression

FEMSRE 646 28-6-99

 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456 425

system was developed by placing the encoding se-
quences under the control of the repressible acid
phosphatase gene promoter of P. chrysogenum. The
cloning and expression of xylanases from various
organisms is summarized in Table 2.
Most of the xylanolytic organisms produce multi-
ple isomeric forms which may result from post-trans-
lational processing of a single gene product or, more
often, as the products of multiple genes. The hetero-
geneity of xylanases was attributed to post- transla-
tional modications such as dierential glycosylation
and/or proteolysis [58]. The two xylanases from
Aeromonas sp. appeared to be dierentially modied
products from the same gene due to their similar
hydrolytic, immunological and physicochemical
properties [7]. However, the presence of multiple xyl-
anase genes has been demonstrated in the case of
Bacillus circulans [129], Caldocellum saccharolyticum
[116], Clostridium thermocellum [130], Pseudomonas
uorescens subsp. cellulosa [131] and Ruminococcus
avefaciens 17 [132]. Extensive cloning work has
been undertaken on Clostridium sp. to track down
the enzymatic specicities of the individual proteins
that form the complex cellulosome and xylanosome
structures. These studies have already been reviewed
extensively [56].
As against the wealth of information available on
the xylanase gene cloning of bacterial systems, very
few reports are to be found on fungal systems. Only
recently some detailed reports on the cloning and

Table 2
Cloning and expression of xylanase genes in E. coli
Source organism Vector MW (kDa) Expression of cloned xylanase Reference
31 31
P R U ml U mg
Aeromonas sp. 212 (ATCC 31085) pBR 322 145 135 1.63 ^ [118]
Bacillus sp. C125 pBR 322 43 43 0.47 ^ [119]
B. circulans NRC 9024/USDA 729 pUC 19 59 59 0.04 ^ [129]
22 22
B. polymyxa NRC 2822/NRRL 8505 pBR 322 51 48 0.037 ^ [304]
pUC 13 22 ^ ^
B. polymyxa NCIB 8158/ATCC 842 pBR 322 ^ ^ 10.0 0.1 [305]
B. pumilus IPO pBR 322 ^ ^ ^ 0.002 [62]
B. subtilis PAP 115 ^ 22 0.5 ^ [303]
Bacillus sp. NCIM 59 pUC 8 35 35 2.0 ^
15.8 14.5 [120]
B. ruminicola #23 pUC 18 ^ ^ ^ 1.1 [138]
Butyrivibrio brosolvens #49 pUC 19 45 46.6 ^ 0.01 [306]
Caldocellum saccharolyticum V1059 pBR 322 ^ 42 ^ ^ [116]
Cellulomonas sp. NCIM 2353 pUC 18 ^ 45 ^ 0.056 [121]
Chainia sp. NCL 82-5-1 Vgt 10 pUC 8 6 ^ 0.003 0.018 [307]
Clostridium stercorarium F9 pBR 322 ^ ^ ^ 8.16 [308]
C. acetobutylicum #P262 pEcoR 251 28 28 64 4.0 [309]
C. thermocellum NRCC pUC 8 90 41, 39 ^ 1.5 [130]
F. succinogenes VgtWESVB ^ ^ 0.57 28.5 [139]
Neocallimastix patriciarum VZAP II 93 73, 42 ^ 193.75 [135]
Pseudomonas uorescens subsp. cellulosa V 47.1 ^ ^ ^ 0.061 [131]
^ ^ ^ 0.061
^ ^ ^ 0.039
Ruminococcus albus #SY3 pBR 322 ^ 56 ^ 0.013 [310]
R. avefaciens #17 VEMBL3 ^ ^ 1.6 ^ [132]
^ 20^30 0.9 ^
Thermomonospora fusca YX VgtWESVB ^ ^ 0.79 ^ [311]
Trichoderma reesei C30 pGEM5Z(+) 19 19 ^ ^ [134]
21 20.7 ^ ^
P and R correspond to parent and recombinant protein. MW corresponds to the molecular mass in kDa.

FEMSRE 646 28-6-99

426 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456
expression of xylanases from the fungi Aspergillus
kawachii [133], Trichoderma reesei [134], Neocalli-
mastix patriciarum [135] and Orpinomyces PC-2
[136] have appeared. The expression of fungal xyla-
nase in E. coli and the rumen bacterium Butyrivibrio
brisolvens OB 156 using the putative xylanase pro-
moter from B. brisolvens strain 49 has been re-
ported [137].
8.2. Overexpression in E. coli
Enhanced xylanase production in E. coli has been
achieved in the cases of alkaline Aeromonas sp. [118],
Bacteroides ruminicola [138] and Fibrobacter succino-
genes 135 [139]. A thermostable xylanase from C.
saccharolyticum has also been cloned and overex-
pressed in E. coli. The enzyme expressed by the re-
combinant had a thermal half-life of 2^3 min at 80C
[140]. Although this enzyme cannot t into the ideal
criteria for industrial application, the isolated gene
itself could prove a starting material for mutagenesis
to further improve the properties of the engineered
enzyme. Recently Koo et al. [141] have reported
overexpression of xylanase from Clostridium thermo-
cellum in E. coli. The recombinant enzyme had an
optimum temperature of 60C at pH 5.4. Overex-
pression of B. subtilis and B. circulans xylanases in
E. coli has also been described. A gene encoding
mature B. circulans xylanase has been designed to
imitate the frequency of degenerate codons used in
E. coli. Synthetic B. circulans gene is then converted
to B. subtilis xylanase gene via single codon substi-
tution (Thr147 Ser). The plasmids containing both
synthetic genes were further modied for direct ex-
pression in E. coli. Under the control of the lac pro-
moter, recombinant xylanase has been produced at
levels as high as 300 mg l31 in solution form in
cytoplasm. Characterization of the products indi-
cated that the puried recombinant protein was cor-
rectly processed and enzymatically active [142]. B.
subtilis xylanase gene, fused to the 5' end of C. mi
cenA, has been overexpressed in E. coli [143]. The
fusion protein exhibited strong anity for both mi-
crocrystalline cellulose and regenerated cellulose.
High level expression in E. coli has also been ob-
tained with the modied domain II construct of xy-
lanase cDNA from the anaerobic fungus Neocalli-
mastix patriciarum. The modied domain II
FEMSRE 646 28-6-99
xylanase produced in E. coli had a specic activity
of 1229 U mg31 protein. The high level expression
was largely attributed to the presence of a favorable
N-terminal coding sequence [144]. Xue et al. [145]
have reported temperature-regulated expression of
recombinant xylanase from N. patriciarum in an E.
coli strain containing natural lacI gene under the
control of the tac promoter. The specic activites
of recombinant xylanase and cellulase were 4.5 times
higher at 42C than those obtained at 23C. The
temperature-modulated Ptac system has also been
used for the overproduction of xylanase in 10-l fer-
mentation studies using a fed-batch process. Re-
cently Lapidot et al. [146] have reported overexpres-
sion and single-step purication of a thermostable
xylanase from B. stearothermophilus T-6. The xyla-
nase gene was cloned into T-7 polymerase expression
vectors. The enzyme was found to constitute over
70% of the cell protein and was eciently puried
from the host proteins by a single heating step. Over
2 g soluble and active enzyme per liter culture was
achieved. Xylanase A from the extremely thermo-
philic eubacterial strain Rt8B.4 was overexpressed
in E. coli. The xylanase activity from domain 2 was
associated with a 36-kDa protein, which was stable
at 70C for at least 12 h at pH 7.0 [147].
8.3. Expression in plant system
In a recent report Helbers et al. [148] have shown
the high level expression of the thermostable xyla-
nase from Clostridium thermocellum (xylanase Z-
truncated protein) into transgenic tobacco plants.
The xylanase gene was introduced into the tobacco
plant by an integration system with Agrobacterium
tumefaciens. The protein molecules were correctly
targeted into the intracellular space with the help
of the signal peptide from the proteinase II.
Although the active enzyme was synthesized inside
the tobacco cells, it did not harm the host cells due
to either the high temperature optimum of the en-
zyme or the relative scarcity of the substrate mole-
cules in the plant cell wall. The response of the trans-
genic plant to pathogenic stimuli has been shown to
be mediated through the xylanase and other related
enzymes secreted by the pathogen and hence the
transgenic tobacco plants producing high levels of
the xylanase from C. thermocellum assume a special
 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456
signicance. Xylanase B (Xyn B) from C. stercora-
rium is also expressed in tobacco suspension cells
(Nicotiana tabacum L. cv BY2 cell) under the control
of the CaMV 35S promoter and noparin synthetase
terminator [149]. The plasmid constructed using
pUC 118 was introduced into BY2 protoplasts by
electroporation and the transformed cells were incu-
bated in the medium containing kanamycin, in agar-
ose bead-type culture. After a 4^6-week cultivation,
the calli depicting clear halos on the xylan containing
agar plates were selected. The amount of expressed
Xyn B protein was also around 4^5% of total pro-
teins in the soluble extracts of tobacco suspension
cells. These studies have shown that the bacterial
xylanases were stably expressed in the tobacco plant,
as high as around 4% of total proteins, without any
inhibitory eect on plant growth. In addition, the
thermostable enzymes are easily isolated from other
tobacco proteins by heating, which has opened an
avenue of creating the bioreactors for hydrolytic en-
zymes.
8.4. Homologous cloning
Several heterologous proteins cannot be eciently
expressed in E. coli due to any one of several rea-
sons, such as the relatively abundant occurrence of
rare codons in the cloned gene, the need for specic
post-synthetic modication(s), structural complexity
of the protein, toxicity of the coded protein to the
host cells, and susceptibility of the foreign protein to
proteases coded by E. coli. The xylanase genes
cloned in E. coli may be underexpressed due to
poor or complete lack of an induction mechanism.
It is well known that higher expression levels are
obtained in the homologous host system. Hence the
cloning of xylanase genes in the homologous host
systems is important, although studies on the homol-
ogous expression of cloned xylanase genes are scarce.
The level of expression of the xylanases from Bacillus
sp. is relatively higher in a homologous host system
than that in E. coli. In the case of B. pumilus IPO,
the extracellular secretion of the xylanase was
achieved using the homologous host B. subtilis
[150]. The homologous expression of the xylanase
gene from an alkaliphilic, thermophilic Bacillus sp.
has been demonstrated in a xyn mutant B. subtilis
A8 [151], and B. subtilis MI 111 [152]. The low level
FEMSRE 646 28-6-99
427
of expression observed has been attributed by the
authors to the weak recognition of the signals from
the host strain which is an unusual extremophilic
species. Enhanced production of the xylanases has
also been reported by chromosomal gene integration
in an alkaliphilic, thermophilic Bacillus sp. [153].
This is of signicance because the use of recombi-
nants in the large-scale fermentation process has
been restricted due to plasmid instability and con-
stant requirement of antibiotic-selective pressure.
An Aspergillus nidulans multicopy tranformant for
the gene xylanase B encoding minor xylanase has
been constructed recently [154]. The transformant
was reported to secrete 114 U of xylanase per mg
protein.
In the case of Streptomyces, the promoter elements
may not be recognized by the E. coli sigma factor, in
turn leading to the failure of the gene expression.
Hence the construction of a genomic library and
screening for the xylanase gene have been carried
out from Streptomyces sp. #36a and S. lividans
#1326 using a homologous host system. The xyla-
nase gene of S. lividans #1326 was cloned by func-
tional complementation of the xyn3 mutant of S.
lividans using multicopy plasmid PIJ 702; a maxi-
mum enzyme production of 380 U ml31 was re-
ported [155]. Thus the cloning of a xylanase gene
into a homologous system not only allowed excellent
secretion but also yielded 60 times higher enzyme
activity than that of the wild-type. Iwasaki et al.
[156] have also reported the molecular cloning of a
xylanase gene from another strain of Streptomyces;
the recombinant showed maximum activity of 2830
U ml31 of culture broth. The eects of signal peptide
alterations and replacement on export of xylanase
have been reported in Streptomyces [157]. The au-
thors have suggested that the changes in the signal
peptide aect the level of xylanase production. How-
ever, further experiments are necessary that will al-
low the combination of a suitable signal peptide with
the enzyme to achieve the overproduction. The re-
ports on homologous cloning and expression of the
xylanases are summarized in Table 3.
8.5. Cloning suitable for biotechnological application
The cloning and expression of xylanases in non-
xylanolytic organisms has been largely restricted to
428 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456

Table 3
Homologous expression of xylanase genes
Parent strain Host Vector Xylanase activity (U ml31 ) Reference
P R
Bacillus pumilus IPO B. subtilis MI 111 PUB110 0.6 1.8 [62]
Bacillus sp. (NCIM 59) Bacillus sp. NCIM-59 PUC8 66 128 [153]
Streptomyces sp S. lividans TK21 PIJ-702 38.9 2839 [156]
Strain #36a S. kasugansis G3 PSK2 ^ 1841
S. lividans 1326 S. lividans PIJ-702 5.8 380 [155]
Streptomyces EC3 S. lividans PIJ-702 600 ^ [312]
S. parvulus PIJ-702 ^ ^
Streptomyces halstedii S. parvulus PIJ-702 4.12 ^ [313]
P and R correspond to parent and recombinant protein.

microorganisms such as Saccharomyces, Kluyveromy-
ces, Lactobacillus and Bacillus subtilis. These organ-
isms have been well characterized as far as their in-
dustrial applicability is concerned. Lactobacillus
plantarum can actively produce and secrete heterolo-
gous enzymes from a variety of Gram-positive bac-
teria and is comparable with Bacillus subtilis as a
host for recombinant protein production [158]. The
xylanase produced by the recombinant L. plantarum
was able to release the fermentable carbohydrates
from ensiled crops and thereby improve the silage
quality. The cloned xylanase gene has also been
shown to oer fermentative advantages to both these
host organisms in the sense that the organism can
directly utilize an additional substrate ^ hemicellu-
lose, a cheap, renewable, abundant, fermentable,
raw resource material. Saccharomyces has been con-
sidered a suitable host for the cloning and expression
of the genes from eukaryotes [159]. The yeast cells do
not produce endotoxins and are considered to be
safe in medicine and food products. Also large-scale
fermentation and downstream processing are estab-
lished. Saccharomyces has been used to express the
xylanases from Aspergillus and Cryptococcus. Re-
cently cloning and expression of xylanase genes
from Meripilus giganteus, Myceloipthora thermophi-
lum and Thielavia terrestris in Aspergillus oryzae
have been reported [160^162], illustrating the exam-
ples of heterologous cloning of xylanase genes. The
application of recombinant xylanase in the food and
paper industries has also been described. Walsh and
Bergquist have reported the expression of Xyn A
from an extremely thermophilic anaerobe Dictyoglo-
mus thermophilum Rt 46 B.1 in the yeast Kluyvero-
myces lactis [163]. The gene was fused in frame with
the secretion signal of the K. lactis killer toxin in
episomal expression vectors. Xyn A was secreted pre-
dominantly as an unglycosylated protein comprising
of 90% of the total extracellular proteins. Also xyla-
nase from thermophilic bacterium Caldicellulosirup-
tor saccharolyticus was secreted to a level of 10Wg
ml31 in the same yeast. The reports on the expres-
sion of the cloned xylanase genes in heterologous
host systems with relevant application potential are
summarized in Table 4.

Table 4
Cloning of xylanase genes into hosts suitable for biotechnological application
Parent strain Host Vector Reference
Aspergillus kawachii S. cerevisiae pVT 100 [133]
A. pullulans S. cerevisiae pYES 2 [314]
Clostridium acetobutylicum L. plantarum pWP 37 [158]
C. thermocellum L. plantarum pWP 37 [158]
Cryptococcus albidus S. cerevisiae pVT 100 [315]
Pichia stipitis pJHS
C. saccharolyticum S. cerevisiae pFLAGU 2 [316]

FEMSRE 646 28-6-99

 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456
9. Protein engineering
Biotechnological applications of the xylanases re-
quire thermostable enzyme preparation with a wide
pH and temperature range. Since the availability of
the ideal enzyme preparation is limited, the applica-
tion of protein engineering studies to xylanases has
gained importance. Protein engineering is also one of
the principal means of examining the active site of an
enzyme to identify the roles of specic residues in
catalysis; site- directed mutagenesis provides the
technology required to redesign the protein. The
identication of active site residues by chemical mod-
ication, X-ray crystallographic data and site-di-
rected mutagenesis has provided basic information
regarding the structure-function correlation of the
xylanases. These studies have formed the basis for
the protein engineering of xylanases for specic ma-
nipulation of the gene for desired enzymatic proper-
ties.
9.1. Amino acid modication
Amino acid modication is carried out usually
chemically or by site-directed mutagenesis, and the
residues essential for substrate binding or catalysis
are identied. Chemical modication, using group-
specic reagents, may suggest the type of residue
involved in catalysis or in substrate binding, how-
ever, it does not identify the specic residue involved.
Substrates and competitive inhibitors which can bind
to the active site frequently protect the enzyme
against inactivation. The participation of tryptophan
in the active site of xylanases from Chainia [164] and
Streptomyces [165] has been reported. The uoro-
metric analysis of the xylanases from Chainia [166]
and alkaliphilic thermophilic Bacillus [167] revealed
that the tryptophan microenvironment was electro-
negative. Chemical modication of xylanases from
the fungus Schizophyllum commune [168] and an al-
kaliphilic thermophilic Bacillus sp. [169] indicated
the involvement of carboxyl groups in the catalysis.
Evidence for the specic interaction of guanidine
hydrochloride with the essential carboxyl group of
xylanase from an alkaliphilic, thermophilic Bacillus
sp. NCIM 59 has also been presented [170]. The
presence of cysteine in the active site of a few bacte-
rial xylanases has been reported [164,165]. However,
FEMSRE 646 28-6-99
429
a possible role for such residues has not so far been
addressed. Cysteines may be critical for the proper
folding of the enzyme and modication of the resi-
due may result in loss in activity. It is also possible
that they may participate in the formation of cova-
lent glycosyl enzyme intermediates.
9.1.1. Active site peptide
In the case of xylanases, inhibitors can be used to
identify the active site residues. The characterization
and sequencing of the cysteine containing active site
peptide of the xylanase from Streptomyces T-7 [171]
and Chainia [172] have been reported. The peptides
showed the presence of a conserved aspartic acid
residue consistent with the catalytic regions of other
glucanases. The possibility of the cysteine residue
directly participating in catalysis by forming a cova-
lent link with the incipient reducing sugar has been
postulated in the case of the variant T4 lysozyme.
9.2. X-ray crystallography studies
The three-dimensional structures of low molecular
mass xylanases (family 11, Mr 20) from B. pumilus
[173], B. circulans, T. harzianum [174], thermophillic
Bacillus sp. [175] and B. stearothermophilus T-6 [176]
have been reported. These studies have helped to
determine the overall structure of xylanases, in pos-
sible identication of specic residues involved in
substrate binding and catalysis.
Crystallization and diraction analysis of xyla-
nases have been carried out at 1.5^3.0 A . In B. pumi-
lus IPO [177], the enzyme molecule is of ellipsoidal
shape (40U35U35 A ) with a well-dened cleft down
one side of the molecule. However, the crystals of
two major xylanases from the fungus T. reesei [178]
are reported to be monoclinic and those of T. har-
zianum to be orthorhombic [179]. The crystallogra-
phy studies of xylanase I from A. niger indicated a
characteristic fold which is unique for family 11 xyl-
anases. It consists of a single domain composed pre-
dominantly of L-strands. Two L-sheets are twisted
around a deep, long cleft which is lined with many
aromatic residues and is large enough to accommo-
date at least four xylose residues. Two conserved
glutamate residues Glu79 and Glu170 reach into the
cleft from opposite sides [180]. The enzymes of fam-
ily 11 are single domain proteins composed of three
430 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456
antiparallel L-sheets and one K-helix, with the active
site lying between the second and third sheets. How-
ever, the three-dimensional structure of xylanase II
from T. reesei [181] has revealed that the L-sheet
structure is twisted, forming a large cleft on one
side of the molecule. In the case of T. harzianum
the xylanase contains two extra strands at the begin-
ning of sheets I and II and a few insertions and
deletions. The observed crystal structure of xylanase
from B. circulans is in close agreement with the
NMR-derived secondary structure of the protein
[182]. In addition to conserved residues in the active
site cleft of xylanases from B. circulans [183] there
are a number of other residues conserved on either
side of the cleft. The three-dimensional structures of
the xylanases from T. harzianum and B. circulans
were found to be very similar. Many of the con-
served amino acids of xylanases are believed to be
structurally important for conrming the correct
folding and packing. The putative catalytic residues
Glu86 and Glu177 of Xyn II from T. reesei are con-
served. Also, a clear cluster of conserved residues
consisting of Gln136 , Tyr77 and Tyr88 is observed
around Glu86 . The hydrogen bond between Tyr171
and Tyr77 exists in other xylanases although the ty-
rosine residue is substituted by histidine in a few
cases. However, the residues around Glu177 are
much less conserved. In addition to these amino
acids, three residues, Pro98 , Asn124 and Thr133 , are
conserved although their role is not clear. It is specu-
lated that they may take part in substrate binding.
The at Ser/Thr face of L-sheet A is also conserved
in family 11 whose functional role may, to a certain
extent, be similar to that of cellulose binding do-
mains present in many cellulases [181].
The three-dimensional structures of the catalytic
domain of a few family 10 enzymes have been
solved. These are xylanase/exoglucanse (Cex) from
C. mi, xylanase A from P. uorescens, xylanase Z
from C. thermocellum and xylanase from S. lividans
[184]. They all have an eight-fold K/L-barrel structure
in which conserved glutamates function as catalytic
nucleophile and acid/base catalytic residues. The
presence of active site residues, Glu127 on strand 4
and Glu246 on strand 7, have been demonstrated in
xylanase A from P. uorescens subsp. cellulosa. The
three bulge-type distortions occurring on L-strands 3,
4, and 7 seem to be functionally signicant as they
FEMSRE 646 28-6-99
serve to orient important active site residues
[185,186]. Xylopentose binds across the carboxy-ter-
minal end of the K/L-barrel in an active site cleft
containing the two catalytic glutamates. Recent crys-
tal structure analysis revealed the presence of the Ca
binding site (located in loop 7) in xylanase A [184].
This is the only xylanase reported to contain a Ca
binding site. The authors have suggested that the
occupation of Ca binding loop with its ligand pro-
tected the enzyme from thermal inactivation, thermal
unfolding and proteolysis. Mutational analysis indi-
cated that Asp256 , Asn261 and Asp262 present within
the Ca binding domain play a pivotal role in the
anity of Xyl A to the divalent cation. A mutant
of XYLA in which the key residues of the calcium
binding domain were replaced by alanine exhibited
thermal stability similar to that of XYLA complexed
with Ca2 ions; however, the xylanase variant was
susceptible to cleavage by chymotrypsin.
10. Site-directed mutagenesis (SDM)
Previously the identication of enzyme active site
residues relied on the chemical modication of pro-
teins. The essential reactive groups were identied by
selective chemical modication. Rapid developments
in molecular biology techniques have made it possi-
ble for the individual amino acids to be substituted
by site-specic mutagenesis. The knowledge derived
from chemical modication and crystallographic
studies of the active site facilitates the understanding
of the structure-function relationship of the protein.
Protein engineering is also applied to alter the sub-
strate specicity, pH optima and to increase the ther-
mal stability of the enzymes.
10.1. SDM and structure-function analysis
The highly conserved amino acid residues located
at specic positions in xylanases are important in
structure-function analysis and hence are targeted
for SDM. Based on the sequence similarity of xyla-
nase from Bacillus pumilus with other xylanases of
known origin and the knowledge of its three-dimen-
sional structure, the authors have proposed the res-
idues Glu93 and Glu182 to be the most suitable can-
didates for the essential catalytic activity of xylanase
 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456
[187]. Mutation of these residues resulted in a de-
crease in specic activity, suggesting that they are
essential catalytical residues. No change in protein
conformation was observed, as conrmed by circular
dichroism (CD) spectra of the mutant enzyme. The
conserved glutamate residues (Glu87 and Glu184 )
have also been shown to be present in the active
site of xylanase A from S. commune [188]. Recently
mutagenesis and analysis of 3D structure of xylanase
A from S. lividans revealed Asn127 to be the impor-
tant residue in maintaining the ionization states of
two catalytic residues and in the stabilization of cat-
alytic intermediates [189]. The three-dimensional
structures of two bacterial xylanases from B. pumilus
and B. circulans and a fungal xylanase from T. har-
zianum have also revealed the presence of two com-
pletely conserved glutamic acid residues correspond-
ing to Glu87 and Glu184 of S. commune xylanase A.
The mutational and crystallographic analysis of the
active site residues of the B. circulans xylanase indi-
cate the presence of six tyrosine and three trypto-
phan residues [183]. However, in the case of the B.
circulans xylanase, a Tyr residue rather than Trp
may play a signicant role in substrate binding, as
demonstrated by kinetic analysis of mutant enzymes.
Tyrosine residues have also been implicated in the
catalytic mechanism for E. coli L-galactosidase
[190]. The authors have demonstrated that two glu-
tamic acid residues (Glu78 and Glu172 ) are involved
in catalysis in the case of the xylanase from B. cir-
culans. Arg112 has been implicated to play a signi-
cant role in catalysis, and Tyr69 and Tyr80 in sub-
strate binding. Recently, NMR studies revealed
His149 to be an important residue in establishing
the conformation of the xylanase. His, Ser and Tyr
residues are known to be completely conserved in all
family 11 xylanases. Mutagenesis of H149 to Phe
and Gln did not alter the active site, but the stability
of the folded protein was found to decrease in irre-
versible thermal denaturation studies [191].
Moreau et al. [192] have identied two acidic res-
idues, Glu128 and Glu236 , as essential residues in the
active site of xylanase A belonging to family 10 from
Streptomyces lividans. The carboxyl group of Asp124
also contributed to the catalytic mechanism. The
mutant enzymes obtained by SDM were 10^50%
more active than the wild-type xylanase A. Con-
versely, none of the aspartic acid residues of xyla-
FEMSRE 646 28-6-99
431
nases of family 11 were completely conserved; it ap-
pears that they are not essential for activity [188].
Roberge et al. [193] showed that two histidine resi-
dues (H81 and H207) out of three were present in the
active site of xylanase A from S. lividans and were
found to be completely conserved in family 10 gly-
canases. The structural analysis revealed that they
were part of an important hydrogen bond network
in the vicinity of two catalytic residues (E128 and
E236). SDM studies showed that they were also es-
sential for protein stability and were probably in-
volved in the folding of native and denatured states
of protein. The mutational analysis of xylanase J
from alkaliphilic Bacillus sp. strain 41M-1 indicated
that Glu93 , Glu183 , Trp18 , Trp86 , Tyr84 and Tyr95
play an important role in the catalytic activity [194].
10.2. SDM for altered desirable properties
The potential of hemicellulases in the biobleaching
of kraft pulp has been recognized in the last decade.
However, the commercial application of this technol-
ogy requires highly active and thermostable enzymes.
Site-directed mutagenesis of the cloned gene oers
interesting research opportunities to change the
properties of a protein suitable for its application.
In the case of xylanase from S. lividans 1326 [195]
the thermostability is increased by replacing Arg156
by glutamic acid. The modied enzyme has shown a
temperature optimum 5C higher than that of the
wild-type. The half-life of Arg156 Glu was 6 min lon-
ger than that of the wild-type enzyme, suggesting
that although the engineered xylanase had a higher
optimal temperature, the stability was not signi-
cantly aected. Previous reports suggested that the
same substitution occurs naturally in the xylanases
produced by Bacillus sp. C-125 and C. saccharolyti-
cum [196]. Both xylanases have an optimum temper-
ature of 70C, which is the same for the modied
enzyme (Arg156 Glu). The studies on cellulases from
T. reesei TD L-6 and Thielavia terrestris NRRL 8126
[197] have revealed that if two favorable mutations
are combined, such as Arg156 Glu and Asn173 Asp, the
resulting enzyme is twice as stable as the wild-type,
with a half-life of 220 min, at 60C. The introduction
of disulde crosslinks into proteins, to protect them
from unfolding, requires the creation of cysteine res-
idues that form disulde bonds spontaneously in sol-
432 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456
ution and do not obstruct functional domains. In B.
circulans mutant xylanase proteins [198], disulde
bridges conferred thermoprotection as observed by
15C increase in thermostability.
Site-directed mutagenesis of a xylanase gene from
C. saccharolyticum has yielded six mutant xylanases
with altered temperature stability and temperature
optimum [196], whereas no change was observed in
the pH optimum. The stabilization of xylanases by
random mutagenesis of the cloned gene fragment
from B. pumilus IPO has been described [199].
Four mutants, each showing a single amino acid
substitution, have been selected on the basis of ac-
tivity at 60C. Based on computer graphic simulation
it was conrmed that these substitutions do not
change the wild-type conformation. Kinetic analysis
has revealed that the mutants are stabilized by a
decrease in activation entropy, except for Asn104
which was stabilized by an increase in activation en-
thalpy.
Even though protein engineering is the most
powerful tool to redesign the protein, the reports
on xylanases to date suggest that it has not been
fully exploited to improve the properties of xyla-
nases. The future scope includes alteration of the
residues to improve the pH stability and shift in
pH optimum of the enzyme for its commercial ap-
plication at alkaline pH. SDM has also been applied
to alter the substrate specicity of proteases [200]
and glucose-xylose isomerases [201]. Designing a xyl-
anase with multiple substrate specicities will also
lead to ecient utilization of hemicellulosic sub-
strates.
11. Mechanism of action of the xylanases
It has frequently been suggested that the catalytic
mechanism of glycosidases resembles that of lyso-
zyme. The hydrolysis reaction catalyzed by xylanases
as well as cellulases proceeds through an acid-base
mechanism involving two residues. The rst residue
acts as a general catalyst and protonates the oxygen
of the osidic bond. The second residue acts as a
nucleophile which, in the case of retaining enzymes,
interacts with the oxocarbonium intermediate or
promotes the formation of an OH3 ion from a water
molecule, as observed for inverting enzymes. Reac-
FEMSRE 646 28-6-99
tion with retention of conguration involves a two-
step mechanism in which proton transfer occurs to
and from an oxygen atom in an equatorial position
at the anomeric center [202] (Fig. 3). This reaction
mechanism is similar to that of lysozyme [203]. Re-
actions leading to inversion of conguration proceed
through a single substitution, as observed in the case
of L-amylase [204]. It is, therefore, likely that a single
amino acid residue (acid/base catalyst) is responsible
in both proton transfer steps. Xylanases mainly ex-
hibit a double displacement mechanism involving a
glycosyl enzyme intermediate which is formed and
hydrolyzed via oxocarbonium ion like transition
state.
11.1. Nucleophile and acid-base catalyst
Wakarchuk et al. [183] have proposed Glu78 and
Glu172 to be the nucleophile and acid-base catalyst,
respectively, based on crystallographic studies of the
enzyme-substrate complex of xylanase from B. circu-
lans. The critical distance between two catalytic car-
boxylic acids (approx. 5.5 A in B. circulans xylanase)
is less in retaining enzymes as compared to that in
inverting glycosidases (approx. 10^11A ). It was also
observed that the precise placement of the acid/base
catalyst is not critical, since both shortening and
lengthening this carboxyl side chain resulted in ap-
proximately the same modest decrease in kcat /Km val-
ues [205]. This is in sharp contrast to the positional
requirements of the catalytic nucleophile Glu78 .
Thus, as expected, the positional requirements for
proton transfer are less demanding than those for
carbon-oxygen bond formation. The important
property of glutamic acid residue, to undergo con-
formational change on a rise of the pH (e.g. at pH
6.5) for xylanase II from T. reesei, could be useful in
catalysis since it will initiate the reaction and conse-
quently may assist a water molecule to attack the
substrate [181]. Further studies suggested that the
primary function of the distal sugar moiety in the
disaccharide substrate is to increase the rate of for-
mation of the glycosyl enzyme intermediate through
improved acid catalysis and greater nucleophilic pre-
association, without aecting its rate of decomposi-
tion [84]. Hence, the residues acting as nucleophiles
have been identied on the basis of structural anal-
ysis and mutagenesis data. Tull et al. [206] have
 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456 433

Fig. 3. Reaction mechanism of the xylanases. (1) Single displacement reaction. Involvement of a general acid (Glu), a general base (Asp
or Glu) and attack by a nucleophilic water molecule is shown. (2) Double displacement reaction (a) involving stabilization of an oxocar-
bonium ion by electrostatic interaction with the carboxylate of an Asp (or Glu) at the active site or (b) involving formation of a covalent
intermediate by nucleophilic attack of the Asp (or Glu) on C-1 of the incipient sugar. (Based on [80].)

FEMSRE 646 28-6-99

434 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456
mapped the nucleophile (Glu274 ) of exoglucanase/
xylanase Cex from C. mi and Glu127 has been con-
rmed as the acid-base catalyst based on kinetic
studies [207].
Thus, from various reports it appears that xyla-
nases belonging to family 11 operate via a double
displacement mechanism in which the anomeric con-
guration is retained. However, endoxylanase from
A. niger [84] was found to be an inverting enzyme
following the single displacement reaction mecha-
nism (Fig. 3). Extensive NMR assignments of xyla-
nase from B. circulans revealed the presence of po-
tential amide-aromatic hydrogen bond between HN
of Ile118 and the indole ring of Trp71 . This interac-
tion, which is conserved in all low molecular mass
xylanases of known structure, may play an impor-
tant role in establishing the active site conformation
of these enzymes [182]. The X-ray crystallography
data on xylanases from P. uorescens [184] and ki-
netic studies on exoglucanase/xylanase from C. mi
[208] belonging to family 10 have also shown that
the substrates are hydrolyzed with net retention of
anomeric conguration.
12. Domain organization of xylanases
The understanding of the basic structure and its
correlation with function has become a key step in
studies dealing with the molecular aspects of the en-
zyme. However, detailed structural data available on
the xylanases is limited. A considerable sequence var-
iability exists among the hemicellulases making the
detection of homologies dicult. Hence various
means of theoretical analysis are used to complement
the data. Of these, hydrophobic cluster analysis and
thermostability analysis are of particular importance
from the point of view of basic and industrial appli-
cations.
12.1. Hydrophobic cluster analysis
Hydrophobic cluster analysis (HCA) is a sensitive
method for the comparison of amino acid sequences
to derive structural, functional and evolutionary in-
formation. HCA detects homologies between similar
three-dimensional structures of proteins having low
sequence identity. These homologies imply a struc-
FEMSRE 646 28-6-99
tural as well as functional correlation, and hence are
indispensable tools in classifying the related enzymes
into families. HCA also provides information on
conserved amino acids which are likely to be in-
volved in catalysis.
On the basis of amino acid sequence homology
and hydrophobic cluster analysis catalytic domains
of cellulases and xylanases were rst classied into
six families (A^F) [209]. In a subsequent study, the
families were upgraded to 11 [210] and in a third
update to 45, including 482 glycosyl hydrolase amino
acid sequences [211]. Currently Henrissat and Bair-
och have classied all the available sequences of gly-
cosyl hydrolases into 58 families [212]. Based on
HCA the xylanases are subdivided into two families,
F and G, which are also shared by other glycanases
such as endoglucanase, exoglucanase, and cellobio-
hydrolase [211]. The families F and G, which are
analogous to glycohydrolase families 10 and 11,
comprise high and low molecular mass xylanases,
respectively. Generally no signicant homology was
found between the xylanases from the two families,
including the region around the catalytic residues,
and they have an altogether dierent pattern of pro-
tein folding.
12.2. Domain structure
At the molecular level the xylanase protein com-
prises functional or non-functional domains and
linker regions. The functional domains are further
dissected into the catalytic and the substrate binding
domains. Classically, the domains are clusters of
amino acids that represent structural homology mo-
tifs which can be well distinguished from each other
on the basis of the structural and spatial identity.
The domains are linked together by sequences,
highly enriched in hydroxy amino acids, which are
often extensively O-glycosylated. It is suggested that
the serine-rich linker sequences in xylanases and cel-
lulases may play a role analogous to that of introns
(non-coding sequences) in eukaryotes. On the basis
of structural similarities, it is also believed that the
cellulases and the xylanases have evolved by domain
shuing, with subsequent modication of the do-
mains [210]. Hence, it was assumed that the catalytic
domains and the substrate binding domains of the
xylanases, too, should be spatially separable or dis-
 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456
tinguishable. However, an analysis of the a family 10
xylanase from P. uorescens subsp. cellulosa (Xyl A)
has revealed that the spatial separation of protein
domains is not necessary for substrate binding or
catalytic activity [213]. The substrate binding domain
was found to be a cellulose binding domain with no
anity towards xylan and therefore did not contrib-
ute to the catalytic activity. Truncated derivatives of
xylanase A, lacking either the serine-rich linker se-
quence or cellulose binding domain, were found to
be less active against xylan contained in a cellulose-
hemicellulose complex as compared with the full
length xylanase [214]. A 374-residue linker sequence
rich in asparagine and glutamine has been reported
in the xylanase from R. avefaciens [215]. Xylanase
D from C. mi, in addition, contains two glycine-rich
linker sequences that provide spatial separation of
domains, imparting structural exibility within the
protein structure [216]. In the case of the xylanase
from Neocallimastix patriciarum, a non-catalytic
linker sequence of 455 residues has been reported
[217]. The sequence unusually comprised 57 repeats
of an octapeptide XSKTLPGG where X could be S,
K, or N. The O-glycosylation of hydroxy amino
acids present in most of the linker sequences is sug-
gested to confer protection from proteolysis. The
short linker sequence between the catalytic domain
and the C-terminus of xylanase C from Clostridium
thermocellum has been found to contain a docking
domain which is responsible for cellulosome assem-
bly [125].
12.2.1. Catalytic domain
In general xylanases consist of a single catalytic
domain. However, analysis of truncated forms of
xylanase 3 from Neocallimastix frontalis has indi-
cated that the full length protein contained two cat-
alytic domains displaying similar substrate specicity
[218]. In the case of Ruminococcus avefaciens two
catalytic domains have been reported. Catalytic ac-
tivity measurements and dierential scanning calo-
rimetry of exoglucanase/xylanase Cex from C. mi
suggested that binding and catalytic domains of the
protein fold independently [92]. The amino acid se-
quence of the endoxylanase from Cryptococcus albi-
dus has some homology with the catalytic region of
the egg white lysozyme [219]. Based on sequence ho-
mology it has been interpreted that the glycosidic
FEMSRE 646 28-6-99
435
bond cleavage in xylan is similar to that of lysozyme.
However, West et al. [84] have shown that the cata-
lytic domain of this enzyme is homologus to that of
cellobiohydrolase from C. mi and xylanase B from
B. subtilis C 125. A xylanase from C. saccharolyticum
[116] is found to be homologous with the cellobiohy-
drolase domain of the Caldocellum bifunctional exo-
cellulase-endocellulase (celB) catalytic domain of cel-
lobiohydrolase from C. mi [220], xylanases from B.
subtilis C 125, C. thermocellum [221], and Cryptococ-
cus albidus [222]. These homology studies suggest
that these enzymes may have evolved by shuing
the two catalytic domains with several substrate
binding domains. When the xylanases and cellulases
from such diverse groups of microbes share extensive
homology in their catalytic domains, it is expected
that their mechanisms of action also would be
closely related. At the same time, the striking ab-
sence of homology between the two xylanases from
B. circulans [129] must be noted; this could be an
indication of a distinct phylogenetic route and/or
catalytic mechanism. The full length xylanase C
from Fibrobacter succinogenes, expressed in E. coli,
is reported to be less active than the two truncated
xylanase C proteins, each possessing an intact cata-
lytic domain. It may be that the tertiary structure of
the enzyme is such that the catalytic sites are parti-
ally buried, as a consequence of improper folding in
E. coli. Comparison of protein sequence by HCA
showed a clear similarity in the secondary structure
elements for the two catalytic domains of Xyn C and
the B. pumilus xylanase, indicating a common ances-
try as well as related three-dimensional organization.
Two tryptophan residues in each domain are found
to be completely conserved [223].
12.2.2. Cellulose binding domain (CBD)
Catalytic domains and CBDs are grouped into
dierent families based on sequence similarities.
Structural studies using NMR have revealed the
presence of very few charged amino acids and an
unusually large number of conserved aromatic resi-
dues in CBD of CbhI and Cex. The CBD of the
mixed function glucanase-xylanase Cex from C.
mi contains ve tryptophans, two of which are lo-
cated within the K/L-barrel structure and three are
exposed on the surface. NMR analysis and chemical
modication studies conrmed that the exposed ar-
436 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456
omatic residues play a direct role in binding to cel-
lulose; their interaction with cellulose appeared to be
reversible [224]. The cellulose binding domains have
also been detected in xylanases from Pseudomonas
uorescens, Cellulomonas mi and Clostridium ther-
mocellum [210]. These domains seem to play two
possible roles, i.e. they either open the structure of
the plant cell wall, making it more accessible to en-
zymic hydrolysis, or provide a general mechanism by
which a consortium of hydrolases accumulate on the
surface of the plant cell wall, resulting in synergistic
action between the enzymes. The full length and
truncated forms of xylanase D from C. mi were
found to be equally eective in hydrolyzing pulp
xylans; enzyme derivatives containing a polysaccha-
ride binding domain were marginally more ecient
in decreasing kappa number [225]. Black et al. [226]
have studied cellulose and xylan binding domains
from xylanase D of C. mi. The deletion of the cel-
lulose binding domain abolished the cellulose bind-
ing capacity of the enzyme without aecting the xy-
lan binding properties. However, the Km values of
the truncated xylanase for the insoluble xylan were
higher, indicating that the internal cellulose binding
homologue in xylanase D constitutes a discrete xylan
binding domain which inuences the anity of the
enzyme for the insoluble substrate, but does not di-
rectly aect the xylanase activity. The existence of a
truly functional internal CBD of the type found in C.
mi enzymes has not yet been demonstrated unam-
biguously [143]. Recently, domain organization and
stability of xylanase A from Thermotoga maritima
and its C-terminal CBD were studied by expressing
the two separate proteins in E. coli. CBD was ex-
pressed as a glutathione S-transferase fusion protein.
Denaturation/renaturation studies have shown that
the domain folds independently [227]. As observed
for all Thermotoga proteins investigated so far
[228], CBD of Xyn A exhibits extremely high intrin-
sic stability in the case of CBD. The apparent Tm
value of both proteins exceeds 100C. CBD was
found to retain residual secondary structure even at
a temperature beyond Tm . CBDs encoded by xyla-
nase 1 from Rhodothermus marinus are shown to be
repeated in tandem at the N-terminus exhibiting sim-
ilarity with CBD family IV [229]. It appears to be the
rst example of xylanase gene encoding a CBD fam-
ily IV in combination with the catalytic domain of
FEMSRE 646 28-6-99
glycosyl hydrolase family 10. The molecular architec-
ture of four new xylanases, from the aerobic soil
bacteria P. uorescens subsp. cellulosa and C. mixtus,
has been studied [230]. Each of the enzymes is mod-
ular and contains a novel cellulose binding domain
that is separate from the catalytic domain. Two of
the enzymes, xylanase E and F from P. uorescens
subsp. cellulosa, contain a domain that is homolo-
gous with NodB from nitrogen xing rhizobia. The
authors therefore suggest that there has been a
strong selective pressure for the retention of CBDs
in xylanases from saprophytic soil bacteria. Recently,
functional analysis of the NodB domain of xylanase
D from C. mi has shown that like other members of
the family it is a deacetylase, whose function is to
remove acetyl groups from acetylated xylan [231].
Characterization of the xylanase A gene from Ther-
moanaerobacterium thermosulfurigenes EM 1 has re-
vealed the presence of two CBDs and a triplicated
sequence at its C-terminus. The latter was found to
be identical to S-layer-like domains of previously
characterized pullulanase from the same organism
[232]. In xylanase producing Streptomyces strains
the proteolytic activity for the removal of CBD
seems to be conserved [233] since a similar xylanase
pattern was obtained for all of them. It is suggested
that autoprocessing of protein and protease activity
are the two reasons that cause the loss of CBD that
might help in the selective hydrolysis of xylan.
12.2.3. Thermostabilizing domain
Repeated domains unrelated to the catalytic do-
main are relatively common in bacterial xylanases
and glucanases; however, in most cases, little is
known about their functions. Recent work on the
thermostable xylanase from Thermoanaerobacterium
saccharolyticum [234] has correlated the intrinsic
thermostability to the N-terminal domains of the
enzymes. Evidence has also been presented that Xyl
Y from Clostridium thermocellum [235] contained a
homologue of this domain that also appeared to
confer thermostability. The thermostabilizing do-
mains have been closely associated with the xylanase
catalytic domain. The 180-residue domain of xyla-
nase Y from C. thermocellum has been shown to
have 28% sequence identity with the thermostabiliz-
ing domain of xylanase A (residues 200^353) from T.
saccharolyticum. Recently sequence analysis of the
 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456 437

xylanase C gene from C. thermocellum F1 revealed
the presence of a 165-amino acid region which was
found to be homologous to the thermostabilizing
domain [125]. We compared the sequence of Xyn
A from T. saccharolyticum with the other reported
xylanases from thermophilic organisms. The analysis
revealed that the xylanases from B. stearothermophi-
lus, thermophilic bacterium RT8.B4, T. maritima,
Anaerocellum thermophilum and Caldicellulosiruptor
saccharolyticus have a region similar to the thermo-
stabilizing domain of Xyn A from T. saccharolyticum
(Fig. 4). Multiple sequence alignment of these xyla-
nases with Xyn A from T. saccharolyticum indicated
conserved Gly and Tyr residues in the region corre-
sponding to the thermostabilizing domain of Xyn A
from T. saccharolyticum. The identity of xylanases
from other thermophilic organisms with the thermo-
stabilizing domain ranged from 5 to 32%. Xylanase
C from C. thermocellum showed a maximum similar-
ity of 32%, while xylanase from B. stearothermophi-
lus showed very little identity. It may be possible that
these domains confer thermostability to the corre-
sponding xylanases (Fig. 4). However, more experi-
mental evidence is necessary to validate the concept

Fig. 4. Homology of the thermostabilizing domain of xylanase Y from Clostridium thermocellum with other thermophilic xylanases. Acces-
sion numbers are in parentheses. (1) T. saccharolyticum B6A-RI (A48490) xylanase; (2) C. thermocellum F1 xylanase C (D84188); (3) B.
stearothermophilus 21 xylanase (D28122) ; (4) thermophilic bacterium RT8.B4 (S12745); (5) T. maritima xylanase A (Z462664) ; (6) Anaero-
cellum thermophilum xylanase A (Z69782); (7) Caldicellulosiruptor saccharolyticus xylanase I (AF005382) ; (8) C. thermocellum YS xylanase
Y (X83269). The conserved amino acid residues are indicated by asterisks. Homology to the xylanase from T. saccharolyticum B6A-RI is
shown by bold letters.

FEMSRE 646 28-6-99

438 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456
that thermostability is conferred by a specic do-
main. Examination of the conserved residues in the
thermostabilizing domain reveals the absence of cys-
teine. A predominance of bulkier aliphatic residues is
observed, which may increase thermostability by in-
creasing the compactness of the folded molecule
[236,237]. The occurrence of a large number of gly-
cine residues supports the proposition that they may
be located in the loop regions of thermophilic pro-
teins [238]. However, comparatively few asparagine
and glutamine residues are observed which can cause
thermoinactivation, as they are more susceptible to
denaturation at elevated temperatures.
It was observed that xylanases from thermophilic
organisms exhibited more homology in their catalytic
domains. The conserved residues in the catalytic do-
mains could suggest the close evolutionary relation-
ship between the thermophilic bacteria that might
have arisen through the lateral transfer of a single
ancestral gene between them. The thermophilic xyl-
anase A of Thermomonospora fusca is the one
reported to date belonging to family 11 [239]. How-
ever, non-catalytic domains conferring thermo-
stability have not yet been detected in family 11 xyl-
anases. One of the reasons may be that these
enzymes are inherently more thermolabile, unlike
family 10 enzymes in which folding is such that it
has been relatively easy for the enzyme to evolve into
a thermophilic enzyme [235]. The data imply that
domains which confer increased thermostability
may be a common phenomenon among family 10
xylanases from thermophilic organisms. Such do-
mains are located only in xylanases and have not
been observed in a number of thermophilic endoglu-
canases characterized to date.
13. Molecular evolution
Recent developments in the molecular genetics of
xylanases have added a new dimension to the studies
of evolution. Sequence alignment is one of the ap-
proaches for obtaining useful information about
functional and evolutionary relationships. Residues
occupying equivalent positions are believed to share
common ancestors and/or to have equivalent biolog-
ical roles. HCA analysis of amino acid sequences of
xylanases and cellulases has revealed that isoenzyme
FEMSRE 646 28-6-99
forms of these enzymes, observed in several organ-
isms, are a consequence of large multigene families
and are not solely the result of processing of a single
gene product. Cellulase and xylanase genes from dif-
ferent families are often present in a single organism
suggesting that microorganisms have acquired multi-
ple plant cell wall hydrolase genes not exclusively
through gene duplication but via extensive horizon-
tal gene transfer [5]. The occurrence of both fungal
and bacterial enzymes in the families 5, 6, 10 and 12
and the presence of prokaryotic and plant enzymes
in family 9 led to the proposition that lateral transfer
of cellulase and xylanase genes has also occurred.
13.1. Sequence homology
A number of reports on sequence homology of
xylanases are documented. In general, sequences
from the same family (10 or 11) were closely related
and exhibited more homology. This criterion has
been used to assign particular sequences to particular
families. The amino acid sequence of Xyn II (family
11) from T. reesei showed signicant homology to
alkaline low molecular mass xylanases from the
same family, but resembled more bacterial xylanase
in several aspects [134]. Schizophyllum commune xyl-
anase (family 11) was found to be the most similar
to Trichoderma xylanases and distantly related to
Bacillus xylanases [188]. Xylanase B from the fungus
Penicillium purpurogenum showed signicant similar-
ity with 38 other fungal and bacterial xylanases be-
longing to family 11. As expected, xylanase B was
more closely related to other fungal endoxylanases
than to bacterial enzymes with signicant homology
to xylanase A from A. awamori (73%) [240]. Xyla-
nase A from B. stearothermophilus 21 was 45^50%
identical to xylanases from other thermophilic or-
ganisms such as C. saccharolyticum and C. thermo-
cellum [117]. Xylanase C (acid xylanase) from A.
kawachii showed 40^50% homology with xylanases
from B. pumilus, B. circulans and C. acetobutylicum.
However, Xyn C from A. kawachii showed no sig-
nicant homology with xylanase A from the same
organism and glucanases from other lamentous
fungi [241]. The catalytic domain of STX-II (35.2
kDa) from S. thermoviolaceus OPC-520 showed ex-
tensive homology with family 11 xylanases. The two
glutamic acid residues were found to be essential for
 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456
catalysis in the xylanase (STX-II) [242]. Irwin et al.
[239] reported that the catalytic domain of xylanase
(TfxA) from T. fusca showed 40^72% identity with
other xylanases. The xylanases from thermophilic
organisms such as xylanase A from C. saccharolyti-
cum [116], xylanase B from C. stercorarium [243] and
xylanase T-6 from B. stearothermophilus T-6 [67]
showed high homology to xylanase A from alkali-
philic Bacillus C-125. B. steararothermophilus xyla-
nase T-6 showed comparatively less homology to
other xylanases from thermophilic organisms, such
as Clostidium saccharolyticum, T. saccharolyticum
B6A-RI. Xylanase A from C. stercorarium had an
Mr of 56 kDa; however, it consisted of a catalytic
domain belonging to family 11, and showed high
homology to other xylanases from family 11 such
as xylanase B from C. acetobutylicum and xylanase
A from B. pumilus, suggesting that these genes orig-
inate from a common ancestral gene, i.e. these genes
were acquired by each bacterium through interspe-
cies gene transfer. Xylanase A (Mr 56 kDa) was an
exception as it belonged to family 11, although it had
a high molecular mass. Xylanase B from C. stercora-
rium showed the highest homology to xylanase A
irrespective of the dierences in their thermal stabil-
ities [244]. The amino-terminal domain of xylanase
D (Mr 90 kDa) from R. avefaciens showed signi-
cant sequence similarity with xylanases beloning to
family 11. The C-terminal domain of xylanase D, on
the other hand, showed identity with glucanases
from Bacillus sp. and C. thermocellum. Surprisingly,
alignment with the L(1,3-1,4)-glucanase from the ru-
men species F. succinogenes revealed much lower
identity [132]. Xylanase C from the ruminant organ-
ism F. succinogenes S85 belonged to family 10. How-
ever, domains A and B of xylanase C shared homol-
ogy with family 11 xylanases from B. circulans, B.
subtilis and B. pumilus. Domain A of xylanase C
from F. succinogenes showed more homology with
fungal ruminal enzyme, whereas domain B exhibited
similarities with the bacterial enzymes. It was sug-
gested that these organisms were found in environ-
mental niches totally dierent from the ruminal or-
ganisms, suggesting that their catalytic domains
might have had a common origin but they had di-
verged a long time ago. On the other hand, the en-
zymes from the three ruminal organisms (R. avefa-
ciens, N. patriciarum and F. succinogenes) shared a
FEMSRE 646 28-6-99
439
similar multiple catalytic domain structure, which
might stem from environmental selection [245]. Xyl-
anases from two dierent strains of the rumen anae-
robic bacterium, Prevotella ruminicola, showed sim-
ilarities with the N- and C-terminal regions of other
family 10 xylanases. The two glutamic acid residues
that had been shown to be involved in the active site
of family 10 xylanases were situated in the conserved
motifs present in the carboxy-terminal regions of
both enzymes, distal to the point of insertion of un-
related sequences. The structures of xylanases from
the two strains of P. ruminicola represent a departure
from the recognized evolutionary route for xylanases
and other glycoside hydrolases, which is thought to
have involved reassortment of integral domain
blocks associated with catalytic, substrate binding
and other functions. The relationship between the
two enzymes in their N- and C-terminal family 10
sequences suggests that a common ancestral gene
must have undergone two independent insertional
events during evolution. A possible role for these
insertions in the P. ruminicola enzymes might be in
anchoring enzymes to other cell wall components,
but because of their position adjacent to the catalytic
center, it seems likely that they alter the substrate
preferences and kinetic properties of the enzymes.
It remains to be established whether these insertions
are a feature unique to xylanases from the Bacte-
roides group of bacteria, which may have diverged
early in the evolution from other eubacterial phyla
[246].
The modular pattern found in the sequence of Xyn
Z from C. thermocellum was similar to the structural
organization of several cellulases in which similar
domains are shued at dierent locations within
the sequences [221,247]. Xyn Y from C. thermocel-
lum, in common with xylanases from several thermo-
philic bacteria, contained a family 10 catalytic do-
main. This observation could reect the close
evolutionary relationship between thermophilic bac-
teria. Thus the evolution of xylanase activity might
have arisen through the lateral transfer of a single
ancestral gene between thermophilic bacteria. This
scenario appeared unlikely in view of the fact that
endoglucanases from C. thermocellum were derived
from several distinct glycosyl hydrolase families, sug-
gesting that the cellulase system of this bacterium
arose through extensive lateral gene transfer [235].
440 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456

13.2. Evolutionary relationship of xylanases
Many xylanases and cellulases are known to pos-
sess a modular structure comprising a catalytic do-
main linked to a non-catalytic cellulose binding do-
main (CBD). CBDs present in cellulases are distinct
from catalytic domains, and cellulases are known to
bind crystalline and/or amorphous cellulose via
CBDs. CBDs are also found in xylanases [216,244]
and other plant cell wall hydrolases such as K-L-ara-
binofuranosidase and acetylxylan esterase [248] and
L-mannanase [249] in addition to endoglucanases
and exoglucanases. It is necessary for xylanases
and other hemicellulases to act cooperatively on
plant cell walls which consist of cellulose, hemicellu-
lose and lignin. Hence the binding of hemicellulases
to cellulose via CBDs should be advantageous in the
hydrolysis of hemicellulose in plant cell walls. This
may be the reason for the evolution of xylanases
containing CBDs. Although xylan binding domains
have also been reported in xylanases, such domains
need to be specically evolved for each xylan due to
the heterogeneous nature of the substrate. The inte-
gration of CBD may be the ecient way to save the
gene capacity exclusively for xylan.
Spurway et al. [184] have reported the presence of
a Ca binding site (located in loop 7) in xylanase A
from P. uorescens subsp. cellulosa. The literature
survey indicated that only ve of the 28 family 10
xylanases contain an extended loop 7. The authors
have suggested that the ancestral protein gave rise to
family 10 xylanases containing loop 7 which,
through natural selection deletions within the loop,
resulted in the evolution of stable xylanases. This
seems logical since loop 7 does not confer any cata-
lytic properties on the enzyme. Phylogenetic analysis
has revealed a relationship between xylanases con-
taining loop 7 and a common ancestral sequence
containing DNA insertion in the region encoding
loop 7. Since similar sequences occur in taxonomi-
cally diverse groups of organisms, a considerable
horizontal gene transfer between family 10 xylanases
seems to have occurred.
We have compiled the amino acid sequences of
xylanases from the PIR databank (Protein Interna-
tional Resource, Release 46.0) (Fig. 5). The sequen-
ces were aligned using Clustal V multiple sequence
alignment [250]. A minimum mutation distance ma-
trix was then constructed from the pairwise compar-
isons of 54 sequences. The distance matrix was then
used to obtain an evolutionary tree using TAXAN
(Release 2.0, Information Resources Group, Univer-
sity of Maryland, USA). The evolutionary tree im-
plies that the xylanases of fungal and bacterial origin
appear to be related to each other forming seven
dierent groups. Although xylanases produced by
the same organism share maximum homology as ob-
served in the case of T. aurantiacus, T. viride and S.
commune, xylanases from two dierent fungi, i.e. N.
frontalis and F. oriforme, are also identical. Fungal
xylanases from T. viride, T. aurantiacus and A. niger
with bacterial xylanases from C. stercorarium and R.
avefaciens form one group. Thermophilic xylanases
from C. acetobutylicum and C. thermocellum seem to
be related. However, xylanases produced by two
strains of P. uorescens were found to be distantly
related to each other.
Thus the xylanases that make up a family/group
appear to have diverged from a common evolution-
ary ancestor. Such enzymes are apt to retain similar
secondary and tertiary structure and have the same
amino acid residues at 20^50% of the corresponding
positions in their primary sequences. The folding of
the polypeptide chain is essentially the same in fam-
ily 11 enzymes with substantial variations occurring
only in external loops, e.g. xylanases from T. reesei
and T. harzianum. These enzymes are classical illus-
trations of diverging evolution from a common an-
cestor. Recently xylanase from Streptomyces virido-
sporus T7A has been found to be dierent from the
reported xylanases from family 10 or 11. It does not
fall into any of the two families. The large size of this
protein may be explained by gene duplication of the
original ancestral gene, a common occurrence in
Streptomyces [251].

C
Fig. 5. Dendrogram showing possible evolutionary relationships among xylanases. The amino acid sequences of xylanases compiled from
PIR (Release 46.0) were aligned using Clustal V multiple sequence alignment. Minimum mutation distance matrix then constructed was
used to obtain the dendrogram using TAXAN (Release 2.0, Information Resources Group, University of Maryland, USA).

FEMSRE 646 28-6-99

N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456 441

FEMSRE 646 28-6-99

442 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456
14. Biotechnological potentials of xylan and xylanases
During the last decade, the potential biotechnolog-
ical applications of xylan and xylanases have been of
particular interest to researchers. At present, the ma-
jor end products of xylan, which are of considerable
importance, are furfural and xylitol. Furfural pro-
duction is derived mainly from agricultural residues
whereas xylitol is obtained from wood residues. The
hydrolysis products of xylan (xylose and oligosac-
charides) have possible applications in the food in-
dustry as thickeners or as fat substitutes and as an
antifreeze food additive. In the pharmaceutical in-
dustry xylan is found suitable as an agent for `direct
tableting' and, in combination with other compo-
nents, it can be used for delayed release tablet con-
struction. The xylan hydrolysis products can be sub-
sequently converted to liquid fuel, single cell
proteins, solvents and articial low calorie sweet-
eners [252].
14.1. Applications of xylanases in paper and pulp
technology
Environmental regulations have put a restriction
on the usage of chlorine in the bleaching process in
the paper and pulp industry, especially in Western
European countries and in North America [253]. Xyl-
anases play an important role in debarking, deink-
ing of recycled bers, and in the purication of cel-
lulose for the preparation of dissolving pulp [254].
Xylanase pretreatment has been reported to lower
bleaching chemical consumption and to result in
greater nal brightness. Enzymatic bleaching results
from the cleavage of bonds between lignin and car-
bohydrate and the the opening of the pulp structure
[255]. Viikari et al. [256] rst showed that treating
pulps with hemicellulases can reduce subsequent
chlorine bleaching requirements and other investiga-
tors [255,257,258] have subsequently conrmed these
studies. Xylan and glucomannan form the basic pol-
ymers of the wood hemicellulose backbone. Xyla-
nases and mannanases are the two main glycanases
that depolymerize the hemicellulose backbone. The
eects of puried or partially puried endo-acting L-
mannanases from B. subtilis, A. niger and T. reesei
on pulp delignication have been studied. Treatment
of pine pulp with xylanase enriched with endo-L-
FEMSRE 646 28-6-99
mannanase of B. subtilis resulted in only a slight
increase in delignication compared with xylanase
alone [259]. Mannanase interacts synergisticaly with
xylanases to improve the bleaching of kraft pulps
specically from softwoods. Xylanases promote
bleaching by the hydrolysis of relocated, reprecipi-
tated xylan on the surface of the pulp bers, allowing
for better chemical penetration and thus improving
lignin extractibility [260]. The reprecipitated xylan
forms a barrier for the extraction of lignin, in both
hardwood and softwood pulps. Thus treatment with
xylanase makes the pulp more permeable for the
subsequent chemical extraction of residual brown
lignin and lignin-carbohydrate molecules from the
bers [261]. Scanning electron microscope studies
of xylanase-preteated pulps revealed an increase in
the porosity of pulp ber aiding in pulp accessibility
to bleaching chemicals [262]. However, no transverse
cracks were observed on the bers that could de-
crease their mechanical strength. Alkaline-stable lig-
nin-carbohydrate complexes present in the wood
seem to be the major obstacles to solubilization of
the residual lignin. During conventional bleaching,
these linkages are cleaved by acidic bleaching stages,
e.g. chlorine or chlorine dioxide. However, the deg-
radation products adversely contribute to the eu-
ent. In contrast to this, hemicellulose-degrading en-
zymes selectively hydrolyze polysaccharide chains
attached to lignin, thereby decreasing the amounts
of chemicals required for pulp bleaching. The xyla-
nases assume special importance in the paper and
pulp industry as they replace toxic chemicals such
as elemental chlorine and chlorine dioxide for devel-
oping eco-friendly processes. At the same time the
high molecular mass lignin molecules, which are cur-
rently gaining more importance as a valuable by-
product of the paper and pulp industry, can be re-
covered using enzymatic pretreatments [263].
14.1.1. Enzyme-aided bleaching
Xylanases from dierent organisms have been
evaluated for their interaction with various kinds
of pulps. On the laboratory scale, xylanases from
Streptomyces roseiscleroticus [264], actinomycetes
[265], T. harzianum [266], and Humiola sp. [267]
have been used for enzymatic pulp treatment to
test their bleach boosting abilities. The biotreatment
of bagasse pulp using xylanase from an alkaliphilic,
 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456
thermophilic Bacillus sp. and subsequent peroxide
bleaching has resulted in a decrease in kappa number
by 10 units and an increase in brightness by 2.5%
[268]. Recently, the thermostable xylanase from
Thermotoga maritima was compared with commer-
cial pulpzyme HC and was found to be ecient in
releasing lignin from kraft pulp [123]. The cloned
xylanases expressed in Bacillus cereus [269] and E.
coli [255] have also been reported to improve the
delignication of unbleached kraft pulps, thus reduc-
ing the chlorine required to achieve a certain degree
of brightness. In the process of pulp bleaching en-
zymes with a high pH and temperature optima are of
utmost importance. Many alkali-tolerant strains of
Bacillus produce xylanases with pH optima of
around 9 [270] and have been used for biobleaching.
The thermostable xylanase produced by the thermo-
philic anaerobic bacterium Dictyoglomus sp. [271]
has been evaluated for its suitability in pulp bleach-
ing. Xylanase pretreatment at 80C and pH 6^8 re-
sulted in an increase in brightness by 2 ISO units in
one-stage peroxide delignication. Thermostable xyl-
anase from B. sterarothermophilus T-6 bleached the
pulp eectively at 65C and pH 9.0, and has been
used successfully in an industrial-scale mill trial
[146]. The rst commercial xylanase preparation
available for pulp bleaching was marketed by
Novo Nordisk A/s, under the name `Pulpzyme
HA', which was produced by a strain of T. reesei.
Subsequently Pulpzyme HB and HC obtained from
bacterial sources were marketed. `Cartazyme HS' is
another xylanase preparation marketed by Sandoz
Chemicals. Irgazyme 40, a commercial preparation
containing xylanases from Trichoderma longibrachia-
tum and T. harzianum E 58, had been tested for
peroxide bleaching of Douglas r kraft pulp [272].
Recently three commercially available xylanases, viz.
Ecopulp (from Alko-ICI), Cartazyme-NS-10 (from
Clariant) and Pulpzyme HC (from Novo Nordisk),
were tested in the bleaching of Eucalyptus kraft
pulps. The results indicated a signicant decrease in
consumption of ClO2 and H2 O2 [273].
Detailed laboratory studies carried out to adapt
the enzymatic treatment to existing mill conditions
showed that no expensive investment is necessary for
full-scale runs, except for the pH adjustment facili-
ties. Thus, enzymatic pretreatment has been shown
to be fully compatible with existing industrial equip-
FEMSRE 646 28-6-99
443
ment, which is an added advantage. Addition of an
enzymatic step to any conventional bleaching se-
quence results in a higher nal brightness value of
the pulp. As world pioneers, the Finnish forest com-
panies started mill-scale trials in 1988. Since 1991,
this method has been continuously used on an indus-
trial scale in Finland together with other low-chlo-
rine or chlorine-free bleaching methods. The chlorine
requirement in prebleaching has been shown to be
reduced by 20^30%. As a result, the AOX (adsorb-
able organic halogen) load of the prebleaching eu-
ent has been reduced by 15^20%. The greatest num-
ber of mill trials have been performed in Europe,
mainly in Scandinavia, where most of the kraft
pulp is produced.
Recently total chlorine-free (TCF) bleaching meth-
ods are being developed in which enzymes have been
combined with O2 , O3 and/or hydrogen peroxide
[274]. In TCF bleaching sequences, the addition of
enzymes increases the nal brightness value, which is
a key parameter in marketing the chlorine-free pulps.
In addition to this, savings in the TCF bleaching
chemicals are important with respect to both costs
and the strength properties of the pulp. The more
ecient xylanase-yielding strains and technologies
will oer a low investment xylanase-aided bleaching
that is both environmentally and economically ad-
vantageous.
14.2. Other applications of xylanases
Xylanases also play a key role in the maceration of
vegetable matter [275], protoplastation of plant cells,
clarication of juices and wine [8], liquefaction of
coee mucilage for making liquid coee, recovery
of oil from subterranian mines, extraction of avors
and pigments, plant oils and starch [276] and to im-
prove the eciency of agricultural silage production
[252]. The food processing industry is already using
the commercial enzyme preparations manufactured
by Novo Nordisk. The fungal L-glucanase prepara-
tion from A. niger, marketed under the tradename
`Finizyme', is used in the fermentation of beer to
avoid the diculties encountered in ltration and
the haze caused by L-glucans. Xylanase is also one
of the components of the commercial enzyme prep-
aration `Ultrao L' produced by a selective strain of
Humicola insolens. It is a heat-stable multi-active L-
444 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456
glucanase used in the mashing process in beer brew-
ing to secure an ecient breakdown of L-glucans,
pentosans and other gums.
The puried xylanase from Trichoderma viride was
found to induce the biosynthesis of ethylene and two
other pathogen-related proteins in tobacco, suggest-
ing that xylanases may also play a role in induction
of plant defence mechanisms [277]. The xylanases
from the germinating plant seed primarily convert
the reserve food to the assimilable end product. It
is also proposed that they play a role in cell elonga-
tion, seem to be involved in fruit softening, and are
believed to have yet undiscovered important physio-
logical functions.
The xylanases nd application in the bakery and
the fodder industries due to the presence of substan-
tial amounts of residual hemicellulose in the raw
material. In bakeries the xylanases act on the gluten
fraction of the dough and help in the even redistrib-
ution of the water content of the bread [278], thereby
signicantly improving the desirable texture, loaf
volume and shelf life of the bread. A xylanase (No-
vozyme 867) has shown excellent performance in the
wheat separation process [279], since it has high ac-
tivity towards soluble arabinoxylan and eects a rap-
id decrease in the viscosity of wheat our slurries.
The dietary hemicelluloses have little nutritional sig-
nicance for non- ruminant organisms as they lack
the appropriate digestive enzymes. These undigested
bers increase the viscosity of the food in the gut,
which interferes with penetration of digestive en-
zymes, absorption of the digested food and may sup-
port pathogenic conditions, especially in broiler
chicks. The use of xylanases together with other
hemicellulases corrects the problems and also in-
creases the nutritive value of the feed. These biotech-
nological potentials of xylanases have prompted the
search for suitable enzymes and technologies for
large-scale economic production.
15. Future prospects
Several applications of xylanases are being devel-
oped for the food and paper industries which are
based on the partial hydrolysis of xylan. The long-
term application of xylanases such as conversion of
renewable biomass into liquid fuels, where xylanases
FEMSRE 646 28-6-99
play a crucial role in conjunction with the cellulases,
is not yet economically feasible. However, stringent
environmental regulations and awareness to reduce
the emission of greenhouse gases have added an in-
centive for future research developments in the study
of xylanases. In order to make the application of
xylanases realistic the improvement in enzyme yields
is of utmost importance. Considerable progress has
been made in the last few years in identifying the
process parameters which are important for obtain-
ing high xylanase yields and productivities, and thus
inuencing the economics of xylanase production.
The production of xylanolytic enzymes is higher
with increasing substrate concentration. However,
the high concentration of solid substrate gives rise
to mass transfer limitations in batch cultivations. A
fed-batch mode of cultivation, where much higher
substrate concentrations can be used, is an attractive
alternative process. This strategy has been success-
fully employed for the enhanced production of cellu-
lases by Trichoderma on both insoluble and soluble
substrates. It can be assumed that similar investiga-
tions on fermentation processes for the production
of xylanases will result in substantial increases in
xylanase activity and productivity. In addition to
the mode of operation, alternative designs and con-
gurations of the bioreactor oer opportunities for
improvement in the fermentation process. More re-
search eorts are necessary to obtain constitutive
mutants which will also eliminate the hindrance of
using insoluble carbon sources for the production of
xylanase in fermentors.
Developments in the eld of enzyme production
require strain developments as well as enzyme recov-
ery and downstream processing [280]. Although the
enzyme recovery methods are outside the scope of
the present review article, their importance in the
economics of enzyme production is beyond doubt
and greater attention needs to be focused on this
aspect. Large-scale enzyme recovery and purication
methods based on two-phase separation and anity
purication may be applied to the xylanases. The
laboratory-scale anity purication method already
described [272] seems to be promising. The complete
bioconversion of xylans to the sugar monomers has
so far not been achieved. This is the main hurdle in
the commercial success of bioconversion processes
from the technical as well as the economic point of
 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456
view. The enzymatic cleavage of xylan to smaller
oligosaccharides is itself a reversible reaction and
most of the enzymes show transglycosylating activity
and synthesis of higher oligosaccharides under cer-
tain reaction conditions. Hence a special process for
the enzymatic cleavage of xylan needs to be designed
^ such as the two-phase reactor in which the product
can be continuously separated from the reactants.
The specic activity of the xylanase preparations is
much lower than that of commercial preparations
such as amylases, proteases and glucose isomerases.
There is an urgent need for identifying, otherwise
developing, the strain capable of producing a high
specic activity xylanase, but the task may not be
simple, mainly because of the heterogeneous nature
of the substrate ^ xylan. In addition to the chemical
heterogeneity, the substrate is also divided into solu-
ble and insoluble physical states which makes the
actual catalysis an extremely complex phenomenon.
This catalytic complexity may have resulted in multi-
plicity amongst the xylanases. Hence the xylanase
multiplicity must be analyzed taking into account
the microheterogeneity of the substrate. Thus it
may be possible to design a mixture of xylanases
that has a better specic activity than the individual
components.
Cleaner biobleaching technology for the paper and
pulp industry is currently concentrated in the devel-
oped countries, whereas renewable energy generation
from agricultural waste has more relevance for the
developing nations. A lot of work needs to be done
to bring these research ideas to reality. The xylanases
that are commercially available today for possible
application in the paper and pulp industry, e.g. pulp-
zyme HA, HB, and HC from NOVO, do not meet
the ideal criteria idntied for enzymatic activity, i.e.
optimum activity at pH 10 and temperature s 90C.
Hence it is necessary to identify the potent xylanase
producer by screening for extremophiles or to design
a tailor-made enzyme by the application of protein
engineering. Although the creation of disulde cross-
links may prove useful for increasing the thermo-
stability of xylanases so as to make them suitable
in biotechnological processes, its use is restricted
where disulde bonds are stable. Interestingly, xyla-
nases without S-S cross-links are known to be more
thermostable than those with disulde bonds [188].
Also, in nature, thermostability of the enzymes from
FEMSRE 646 28-6-99
445
hyperthermophiles appears to be the result of the
reduction of water-accessible hydrophobic surfaces
rather than the disulde cross-link strategy [281].
Thus, even though protein engineering can be used
to increase conformational stability and enzyme ac-
tivity, much remains to be learned from natural ther-
mophilic enzymes before precise molecular predic-
tions can be guaranteed. Attempts have been made
to recover xylanase DNA from complex soil samples
by PCR amplication using degenerate primers. Re-
covered DNA, which is dierent from the known
xylanases, can be used in several ways to facilitate
the production of novel xylanases for industrial ap-
plications [282].
The transfer of xylanase genes to selected fermen-
tative microbes in which the enzyme can be produced
and secreted in sucient quantities will enable the
fermentative microbes to convert the xylan residue
directly into liquid fuels which will have a direct
implication in renewable energy conservation. Few
microbes possess the metabolic pathways required
for single-step conversion of xylan to ethanol [8].
Taguchi et al. [283] have reported for the rst time
an organism capable of producing hydrogen from
xylan. The hydrogen produced from xylan was
equivalent to about 73% of the xylose consumed.
Thus the strain might be useful to further our under-
standing the basic technology involved in the biolog-
ical process of hydrogen production from xylan in
plant wastes. Research eorts should be focused on
the improvement of such strains for the ecient uti-
lization of biomass. Xylan being an abundant agri-
cultural residue, the chemical energy of the molecule
can be meaningfully utilized if the nitrogen xation
activity can be linked to xylan degradation by cou-
pling the xylanase gene and the nif gene cluster.
Acknowledgements
The authors thank Urmila, Prashant and Sunita
from the Department of Bioinformatics, University
of Pune, Pune, India, for their help in the computer
analysis of xylanase sequences. The authors also
thank Drs. M.C. Srinivasan, V.V. Deshpande, A.
Ahmad and Ms. D. Nath for valuable discussions
and providing some of the literature information.
We thank Mr. H.B. Sing for the critical reading of
446 N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456
the nal text. A Senior Research Fellowship to N.
Kulkarni from the Council of Scientic and Indus-
trial Research is gratefully acknowledged.
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