Xilanasas

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.
2.
3.
Introduction . . . . . . . . . . . .
Scope of the present review
Structure of xylan . . . . . . .
3.1. Chemical structure . .
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412
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* 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
FEMSRE 646 28-6-99
412
N. Kulkarni et al. / FEMS Microbiology Reviews 23 (1999) 411^456
3.2. Three-dimensional structure . . . . . . . . . . . . . . . . . . . . . .

Xylanase production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Factors aecting xylanase yield . . . . . . . . . . . . . . . . . . .
5. Regulation of xylanase synthesis . . . . . . . . . . . . . . . . . . . . . . .
5.1. Induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Regulation at the molecular level . . . . . . . . . . . . . . . . .
6. Biochemical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1. Carbohydrate content . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2. Substrate specicity . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7. Xylanases of extremophilic origin . . . . . . . . . . . . . . . . . . . . . .
7.1. Xylanases from alkaliphilic microorganisms . . . . . . . . . .
7.2. Thermophilic xylanases . . . . . . . . . . . . . . . . . . . . . . . . .
8. Cloning and expression of xylanase gene(s) . . . . . . . . . . . . . . .
8.1. Heterologous cloning . . . . . . . . . . . . . . . . . . . . . . . . . .
8.2. Overexpression in E. coli . . . . . . . . . . . . . . . . . . . . . . . .
8.3. Expression in plant system . . . . . . . . . . . . . . . . . . . . . .
8.4. Homologous cloning . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.5. Cloning suitable for biotechnological application . . . . . .
9. Protein engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.1. Amino acid modication . . . . . . . . . . . . . . . . . . . . . . . .
9.2. X-ray crystallography studies . . . . . . . . . . . . . . . . . . . .
10. Site-directed mutagenesis (SDM) . . . . . . . . . . . . . . . . . . . . . . .
10.1. SDM and structure-function analysis . . . . . . . . . . . . . . .
10.2. SDM for altered desirable properties . . . . . . . . . . . . . . .
11. Mechanism of action of the xylanases . . . . . . . . . . . . . . . . . . .
11.1. Nucleophile and acid-base catalyst . . . . . . . . . . . . . . . .
12. Domain organization of xylanases . . . . . . . . . . . . . . . . . . . . . .
12.1. Hydrophobic cluster analysis . . . . . . . . . . . . . . . . . . . . .
12.2. Domain structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13. Molecular evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.1. Sequence homology . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.2. Evolutionary relationship of xylanases . . . . . . . . . . . . . .
14. Biotechnological potentials of xylan and xylanases . . . . . . . . . .
14.1. Applications of xylanases in paper and pulp technology .
14.2. Other applications of xylanases . . . . . . . . . . . . . . . . . . .
15. Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.
1. Introduction
The naturally occurring lignocellulosic plant biomass consists of 20^30% hemicellulosic materials
which are heterogeneous polysaccharides found in
association with cellulose [1]. Biomass is an alternative natural source for chemical feedstocks with a
replacement cycle short enough to meet the demand
in the world fuel market. Xylan is the major constituent of hemicellulose and is the second most abundant renewable resource with a high potential for
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degradation to useful end products. Hence the development of inexpensive technologies based on hemicellulose is called for. Eventually, if not in the near
future, xylan, in combination with cellulose, will supply 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 xylanohydrolase, EC 3.2.1.8) are the preferred catalysts for
xylan hydrolysis due to their high specicity, mild
reaction conditions, negligible substrate loss and
FEMSRE 646 28-6-99
side product generation. However, the cost of enzymic hydrolysis of biomass is one of the factors limiting the economic feasibility of the process. The production 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, protozoa, 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 animals. The use of hemicellulolytic enzymes as a substitute 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 xylanases, 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 stimulated research on the biochemical and molecular aspects of this important enzyme of the family of glycosyl hydrolases. Considering the future prospects of
xylanases for biotechnological exploitations, especially in the eld of biopulping and bleaching, it is
very essential to analyze the properties of xylanases
with respect to regulation of biosynthesis and mechanism of action. An increasing number of publications in recent years describe numerous xylanases
from new sources, their cloning, sequencing, mutagenesis 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 important enzymes, many reviews have been published
covering the various aspects of the enzyme [4^8].
The present article is a comprehensive state-of-theart review describing the xylanases with special em-
413
phasis on the latest developments in the area of molecular biology and biotechnology. The article also
covers the xylanases from extremophilic microorganisms which have gained importance by virtue of their
stability and activity at extremes of pH and temperature. Theoretical analysis of a few of the reported
thermostable xylanases has also been included; it
will be useful in application-oriented protein engineering 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-xylopyranose units. The backbone consists of O-acetyl,
K-L-arabinofuranosyl, K-1,2-linked glucuronic or 4O-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-methylglucuronoxylans in hardwoods or as arabino-4-O-methylglucuronoxylans in softwoods. The degree of polymerization 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-Omethyl ether and arabinose [14]. Endospermic arabinoxylans 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 homoxylan, arabinoxylan, glucuronoxylan and glucuronoarabinoxylan. 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 xylanases from completely degrading acetylxylan, probably by steric hindrance. The synergistic action of
acetylxylan esterases and xylanases is therefore es-
FEMSRE 646 28-6-99
414
sential for the complete hydrolysis of acetylxylans.

The presence of small amounts of feruloyl and pcoumaroyl acids linked via L-arabinose residues has
been shown on the structure of xylan in several studies. These hydroxycinnamic acids are bound to C-5
of the arabinose residue [16]. The presence of a covalent bond between lignin and hemicellulose, perhaps 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 determine the solubility, physical conformation and reactivity 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 molecules has been elegantly described by Atkins [19].
The xylan backbone shows a threefold left-handed
conformation under crystallized conditions; the geometry 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 diraction 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 dotted. In each case the backbone is a left-handed threefold helix. (Based on [19].)
FEMSRE 646 28-6-99
415
Fig. 1 (continued)
tion and show that the chains are organized in a

trigonal lattice with hexagonal morphology. The single hydrogen substituent at position 5 on the xylose
ring has a dramatic eect on the intra- and interchain hydrogen bonding interactions. Energy calculations 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 adjacent 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 aqueous environment, would be extremely important in
understanding the xylan and xylanase interactions.
4. Xylanase production
The basic factors for ecient production of xyla-
FEMSRE 646 28-6-99
416
nolytic enzymes are the choice of an appropriate inducing substrate and an optimum medium composition. The importance of cellulase-free xylanase systems in the paper and pulp industry has initiated
research into the correlation between the production
of xylanases and cellulases by microorganisms. Filamentous 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 xylanolytic systems free of cellulases [21]. However, cellulosic substrates were also found to be essential in the
medium for maximum xylanase production by Clostridium stercorarium [22], Thermomonospora curvata
[23] and Neurospora crassa [24]. Cheaper hemicellulosic 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 xylanases 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 solidstate fermentation has been obtained from the fungus 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 licheniformis A99, in solid state fermentation (SSF) [32]. SSF
systems resemble the natural habitats of microbes

and, therefore, may prove ecient in producing certain 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 dierent since several promising strains that produce xylanases 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 enzyme production at 9^10 [34]. In Streptomyces avogriseus [35] and in the Bacillus sp. NCIM 59 [36]
simultaneous production of glucose (xylose) isomerase, 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 accessibility, rate and amount of release of the xylooligosaccharides 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 derivative 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 harvesting time of the xylanases must be correlated to the
FEMSRE 646 28-6-99
production of these enzymes on the medium under

consideration. Other bioprocess parameters that can
aect the activity and productivity of xylanase attained in a fermentation process include the pH, temperature 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 production by various bacteria and fungi has been shown to
be inducible. But rare examples of constitutive xylanase expression have also been reported [39]. In general the xylanase induction is a complex phenomenon 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 endproduct inhibitors, possibly at much higher concentrations.
5.1. Induction
Xylan, being a high molecular mass polymer, cannot 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, heterodisaccharides of xylose and glucose and their positional
isomers. These molecules are liberated from xylan by
the action of small amounts of constitutively produced 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 increase with the crystallinity of the cellulosic substrate
[41]. Sugarcane bagasse is found to be the best inducer of xylanase and L-xylosidase in Cellulomonas
avigena [42]. A synergistic eect on the synthesis of
both the enzymes was observed when cellulose and
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 xylose higher enzyme yields were obtained from Bacillus pumilus [44], Streptomyces lividans 66 [45] and
Aureobasidium pullulans [46]. However, in Cryptococcus 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 xylanase in T. reesei [48] and Aspergillus terreus [38].
Xylanolytic systems of yeasts and fungi were also
induced by positional isomers of xylobiose. In Cryptococcus 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 inducers. D-Xylan fragments, as well as methyl-L-D-xylopyranoside, 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 reported that not all L-xylopyranosides serve as inducers of xylanase since their induction capacity is
also dependent on the structure of aglycon.
The role of transglycosylating enzymes in the synthesis of positional isomers of these heterodisaccharides is indicated in Aspergillus terreus [38] and T.
reesei [48]. The low molecular mass substances that
have been identied as xylanase inducers need transferase enzymes for their translocation into the cytoplasm. Hence the level of inducers and/or the required enzymes in the culture ltrate also aect the
xylanase synthesis. The possible factors aecting xylanase induction have been diagrammatically represented (Fig. 2), largely based on the gure by Thomson [56].
5.2. Regulation at the molecular level
Xylanase biosynthesis and the phenomenon of enzyme induction at the molecular level are comparatively less investigated. This may be due to the lack
of cell-free systems which are necessary for the eval-
FEMSRE 646 28-6-99
418
Fig. 2. Regulation of xylanase biosynthesis (hypothetical model). Constitutive xylanases degrade xylan to xylooligosaccharides and xylobiose, which are taken up by the cell and induce other xylanase genes. The inducible xylanases degrade xylan further to xylooligosaccharides and xylobiose. The L-xylosidases, which may be produced constitutively and/or inducibly, convert xylobiose to xylose and may subsequently 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 microorganisms [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 circulans 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 xylosidase genes are closely associated and are linked in a
14.4-kb DNA fragment. However, they did not appear to be controlled by the same operon.
5.2.1. Catabolite repression
Catabolite repression by glucose is a common phenomenon observed in xylanase biosynthesis. Relatively few reports on the relation of cAMP to xylanase 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, upstream 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 xylanase 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
controlled at two levels, directly by repression of

gene transcription and indirectly by repression of
transcriptional activator. The same pattern of regulation was observed in A. niger and A. nidulans.
Mach et al. [65] have studied the carbon catabolite
repression of xylanase gene expression and have analyzed the molecular basis for the absence of xylanase 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 transcription 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 promoter, 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. stearothermophilus 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 transition state regulators and catabolite repression. The
sequence analysis of T-6 xylanase has revealed the
presence of an AT-rich region preceding the promoter that has been identied as the binding site
for a protein that regulates induction [68]. It has
been suggested that these regions interact or facilitate interaction with regulators. A putative catabolite
repression sequence was found 230 bp inside the xylanase structural gene. The catabolite repression consensus sequence was TGT/AAANCMGNTNA/TCA,
where the underlined letters represent the most critical 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 medium, in contrast to the synthesis of L-xyloside permease and L-xylosidase which have very short induction periods. This is in agreement with the suggestion
that mRNAs of secreted proteins do not undergo a
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 subunit 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 thermostable than bacterial xylanases. Fungi which are mesophilic 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 temperature of 40C [72]. D-Xylanases from dierent organisms 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 xylanase. However, this type of relationship is not observed in fungi; but low molecular mass basic xylanases are common. The amino acid compositions of
xylanases reported from various sources indicate predominantly aspartic acid, glutamic acid, glycine, serine and threonine.
6.1. Carbohydrate content
The occurrence of glycosylated enzymes is a common phenomenon among many eukaryotic xylanases
[74]. The xylanases from prokaryotic sources, like
FEMSRE 646 28-6-99
420
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. Glycosylation 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 deglycosylation. In the case of xylanases from Talaromyces byssochlamydoides YH-50 the carbohydrate residues are found to be mannose, glucose and fucose
[78]. It has been suggested that both dierential glycosylation and proteolysis may contribute to the
multiplicity of xylanases [7].
6.2. Substrate specicity
Knowledge of the mechanism of action of xylandegrading enzymes has been gained from studies on
substrate specicity, the role of side chain substituents 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 arabinoxylan. An endoxylanase from Streptomyces roseiscleorticus has also been shown to be a debranching
type of enzyme. However, xylanases from Trichoderma harzianum [82] and another strain of A. niger
were found not to release free arabinose from arabinoxylan. The presence of debranching as well as
non-debranching xylanases have been reported
from T. koningii and Ceratocystis paradoxa, and various 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-
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 hydrolyze L-1,3 linkages in barley, L-glucan as well as L1,4 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 Cryptococcus 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 product 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 substrates with increasing chain length. Since the binding 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 complex. 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 occurrence 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-
FEMSRE 646 28-6-99
productive enzyme-substrate complexes. The endoxylanase 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 thermolacticum and also determined the structures of end
products by nuclear magnetic resonance spectroscopy and methylation analysis. They concluded
that xylanase from Clostridium thermolacticum was
composed of ve subsites, aê, binding ve xylosyl
rings AÊ. The catalytic site was located between
xylosyl rings B and C and subsites d and e were
able to bind substituted xylosyl residues unlike subsites a, b and c. The dierences in the subsite numbers 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 divergent specicities of these enzymes could play a signicant role in their synergistic action towards the
complex substrate.
7. Xylanases of extremophilic origin

Considerable progress has been made in the isolation 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 enzymatic stability are important for the industrial applicability 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
421
proposed that the model could be applied to a large

number of industrially relevant enzymes and processing conditions where the reversible denaturation of
protein is fast as compared to the rate of the subsequent irreversible inactivation step [92]. One of the
ways to identify the industrially suitable xylanase
preparations is to look for the enzymes from extremophilic microorganisms. In the words of M.W.W.
Adams, University of Georgia, a pioneer in the study
of extremophiles, `Ìf you want highly stable enzymes, you must go to the extreme environments.
Nature's bounty, it seems, abides in its black smokers, steam vents and salt lakes'' [93]. The use of biocatalysts has been constrained due to their labile nature under extreme temperature and pH conditions.
The study of extremophiles and their enzymes can
extend the present understanding of protein chemistry in addition to expanding the potential applications 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 cyclodextrins. Commercial production became economical only after the discovery of alkaliphilic Bacillus 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 produced by alkaliphilic microorganisms such as Bacillus 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-
FEMSRE 646 28-6-99
422
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 activity 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 Bacillus sp. (NCIM 59) producing two types of cellulase-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]. Xylanase from Dictyoglomus sp.exhibited a half-life of
80 min at 90C [107]. Among the thermophilic fungi,
xylanase from Thermoascus aurantiacus has been reported to be stable at 70C for 24 h with a half-life
of 54 min at 80C [108]. The other thermophilic fungi producing thermostable xylanases include Paecilomyces variota [109] and T. byssochlamydoides [110]
which show an optimum temperature of 65^75C
at pH 5^6.5. Recently endoxylanases from the thermophilic actinomycete Microtetraspora exuosa SIIX
were reported to have an optimum temperature of
80C, at pH 6.0 [111]. Despite the prevalence of xylan-degrading enzymes in actinomycetes, comparatively 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
high technology. Multinational companies like

Novo, Genencor and Diversa plan to mobilize biodiversity by establishing a large culture collection of
unique microorganisms and to screen for enzyme
activities. Diversa (formerly Recombinant Biocatalysis) is specically harvesting DNA from entire extremophilic 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 application. In spite of having interesting and novel properties, 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 extremomolecule. 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 viability of xylanase production, it is necessary to identify 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 various suitable hosts. The bacterial genes encoding xylan-degrading enzymes have been found to be adjacent on the genome or in close proximity to other
genes encoding cellulase-related functions. In P. uorescens subsp. cellulosa, xylanase and arabinofuranosidase 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, xylanase and endoglucanase genes were separated by
155 bp [115]. Similarly the close linkage between xylanase and L-xylosidase genes has been reported in
FEMSRE 646 28-6-99
423
Table 1
Xylanases from extremophilic organisms
Source
Xylanase
Optimum conditions
Growth
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
23
24
1
2
3
4
5
1
2
3
4
5
6
Thermophilic bacteria
Bacillus acidocaldarius
Bacillus licheniformis A 99
Bacillus stearothermophilus T-6
Bacillus stearothermophilus No. 21
Bacillus thermoalkalophilus
Clostridium acetobutylicum ATCC
824
T-6
A
A
B
Clostridium stercorarium HX-1
D
Clostridium stercorarium F-9
A
Clostridium thermolacticum (TC 21)
Dictyoglomus thermophilum strain B1
Microtetraspora exuosa S II X
Thermophilic Bacillus Strain XE
Thermophilic Bacillus sp
Thermophilic bacteria ITI 283, ITI
236
Thermoanaerobacterium sp. JW/SLYS485
Thermotoga sp. (Fjss3-B.1)
A
Thermotoga maritima (MSB 8)
A
B
Thermotoga thermarum
1
2
Thermomonospora curvata
1
2
3
Thermomonospora chromogena
MT814
Thermomonospora fusca BD 21
Thermomonospora fusca YX
Streptomyces thermoviolaceus OPC- I
520
II
Thermophilic fungi
Gloephyllum trabeum
Talaromyces byssochlamydoides YH50
Thermoascus aurantiacus
Thermomyces lanuginosus DSM5826
Talaromyces emersonii CBS 814.7
II
III
Alkaliphilic bacteria
Alkalophilic Bacillus 41M-1
Bacillus sp. TAR-1
Bacillus sp. C-59-2
Bacillus sp. C-125
Bacillus sp. NCIM 59
Aeromonas sp. 212
Reference
Activity
pH
Temp. (C)
pH
Temp. (C)
3.5^4.0
7.0
7.0^7.3
7.0
90
6.0
65
60
60
55
60
37
4.0
7.0
9.0
7.0
6.0^7.0
5.0
80
50
65
60
60, 70
50
[284]
[32]
[104]
[285]
[286]
[287]
6.0^7.0
6.0^7.0
6.0^7.0
7.0
6.0
7.0
7.0
7.5
37
60
65
65
68
80
55
65
65
5.5^6.0
6.5
6.5
6.5
7.0
9.0
6.0
6.5^7.0
8.0
60
75
75
80
80
52
75
78
80
[288]
[22]
[289]
[290]
[111]
[291]
[103]
[292]
6.0
60
6,2
80
[293]
6.8^7.0
7.0
8.0
50
5.3
6.2
5.4
6.0
7.0
7.8
7.2
6.8
5.0^8.0
105^110
92
105
80
90^100
75
75
75
75
[106]
[294]
6.0^7.0
80
80
80
77
77
55
[47]
8.0
8.0
7.0
50
50
50
6.0^8.0
6.0^8.0
7.0
65
70
70
[296]
[239]
[297]
50
7.0
60
6.2
^
50
4.0
5.0
80
70
[298]
[78]
6.0
6.5
4.5
4.5
45
50
45
45
5.0
6.5
4.2
3.5
75
50
78
67
[108]
[299]
[300]
10.3
10.5
8.0
10.5
10.0
10.0
37
50
37
^
50
37
9.0
9.0
5.5^9.0
6.0^10.0
6.0
7.0^8.0
50
70
60
70
50^60
50
[97]
[97]
[301]
[98]
[26]
[118]
6.0^7.0
FEMSRE 646 28-6-99
[295]
[23]
424
Table 1 (continued)
Xylanases from extremophilic organisms
Source
Xylanase
Optimum conditions
Growth
7
8
Bacillus sp. NG-27

Bacillus sp
W1
W2
W3
W4
Bacillus NCL-87-6-10
Reference
Activity
pH
Temp. (C)
pH
Temp. (C)
9.0^10.0
9.0^10.0
27
45^50
7.0, 8.4
70
6.0
7.0^9.0
6.0
7.0^9.5
6.0
6.0^7.0
8.0
65
70
65
70
65
70
60
I
II
I
II
9.5
B. pumilus [61] and C. saccharolyticum [116]. In spite

of this frequent clustering of the genes encoding xylan-degrading enzymes, there is no evidence for polycistronic transcription [58]. The nucleotide sequence
data of the 4.2-kb chromosomal segment of the genome of Bacillus stearothermophilus 21 indicated that
L-xylosidase gene was located upstream of the xylanase gene. Further analysis indicated that the two
genes were separated by only 2 bp and were transcribed independently [117].
8.1. Heterologous cloning
The xylanase genes have been isolated from dierent 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 cytoplasmic or the periplasmic fractions. The absence of
post-translational modications such as glycosylation 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. Extracellular activity has been reported in recombinant E.
coli for the xylanases from alkaliphilic Aeromonas
[118], alkaliphilic Bacillus [119], alkaliphilic, thermophilic Bacillus sp. [120] and Cellulomonas sp. [121].
The xylanase gene cloned from Bacillus stearothermophilus 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-
28
[302]
[102]
[99]
mophilum Rt46B.1 in E. coli has been reported [122];

the recombinant enzyme was found to have an optimum temperature of 85C. The xylanase cloned from
Thermotoga maritima showed an optimum temperature of 90C at pH 5.5 and was stable up to
100C [123]. The recombinant xylanase from Thermotoga 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 protein was unusual and long. The protein was found to
be distinct from xylanases belonging to glycohydrolase families 10 and 11 and appeared to be intermediate between families 5 and 30. The XYN 2 gene from
T. reesei QM6a [127] was expressed in yeast Saccharomyces cerevisiae under the control of alcohol dehydrogenase (ADH 2) and phosphoglycerate kinase
(PGK 1) gene promoters and terminators, respectively. 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 temperature 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 heterologous gene expression in the lamentous fungus Penicillium chrysogenum. The heterologous expression
FEMSRE 646 28-6-99
425
properties [7]. However, the presence of multiple xylanase 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
system was developed by placing the encoding sequences 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 multiple isomeric forms which may result from post-translational processing of a single gene product or, more
often, as the products of multiple genes. The heterogeneity of xylanases was attributed to post- translational 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
Table 2
Cloning and expression of xylanase genes in E. coli
Source organism
Vector
MW (kDa)
Aeromonas sp. 212 (ATCC 31085)

Bacillus sp. C125
B. circulans NRC 9024/USDA 729
pBR 322
pBR 322
pUC 19
B. polymyxa NRC 2822/NRRL 8505

B. polymyxa NCIB 8158/ATCC 842
B. pumilus IPO
B. subtilis
Bacillus sp. NCIM 59
pBR 322
pUC 13
pBR 322
pBR 322
PAP 115
pUC 8
B. ruminicola #23
Butyrivibrio brosolvens #49
Caldocellum saccharolyticum
Cellulomonas sp. NCIM 2353
Chainia sp. NCL 82-5-1
Clostridium stercorarium F9
C. acetobutylicum #P262
C. thermocellum NRCC
F. succinogenes
Neocallimastix patriciarum
Pseudomonas uorescens subsp. cellulosa
pUC 18
pUC 19
V1059 pBR 322
pUC 18
Vgt 10 pUC 8
pBR 322
pEcoR 251
pUC 8
VgtWESVB
VZAP II
V 47.1
Ruminococcus albus #SY3

R. avefaciens #17
pBR 322
VEMBL3
Thermomonospora fusca YX
Trichoderma reesei C30
VgtWESVB
pGEM5Z(+)
Expression of cloned xylanase
145
43
59
22
51
135
43
59
22
48
22
^
^
22
35
14.5
^
46.6
42
45
^
^
28
41, 39
^
73, 42
^
^
^
56
^
20^30
^
19
20.7
^
^
^
35
15.8
^
45
^
^
6
^
28
90
^
93
^
^
^
^
^
^
^
19
21
31
U ml
U mg
1.63
0.47
0.04
^
^
^
[118]
[119]
[129]
0.037
^
10.0
^
0.5
2.0
^
^
0.1
0.002
^
^
[304]
^
^
^
^
0.003
^
64
^
0.57
^
^
^
^
^
1.6
0.9
0.79
^
^
1.1
0.01
^
0.056
0.018
8.16
4.0
1.5
28.5
193.75
0.061
0.061
0.039
0.013
^
^
^
^
^
P and R correspond to parent and recombinant protein. MW corresponds to the molecular mass in kDa.
FEMSRE 646 28-6-99
Reference
31
[305]
[62]
[303]
[120]
[138]
[306]
[116]
[121]
[307]
[308]
[309]
[130]
[139]
[135]
[131]
[310]
[132]
[311]
[134]
426
expression of xylanases from the fungi Aspergillus

kawachii [133], Trichoderma reesei [134], Neocallimastix patriciarum [135] and Orpinomyces PC-2
[136] have appeared. The expression of fungal xylanase in E. coli and the rumen bacterium Butyrivibrio
brisolvens OB 156 using the putative xylanase promoter from B. brisolvens strain 49 has been reported [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 succinogenes 135 [139]. A thermostable xylanase from C.
saccharolyticum has also been cloned and overexpressed in E. coli. The enzyme expressed by the recombinant 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 thermocellum in E. coli. The recombinant enzyme had an
optimum temperature of 60C at pH 5.4. Overexpression 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 substitution (Thr147 Ser). The plasmids containing both
synthetic genes were further modied for direct expression in E. coli. Under the control of the lac promoter, recombinant xylanase has been produced at
levels as high as 300 mg l31 in solution form in
cytoplasm. Characterization of the products indicated that the puried recombinant protein was correctly 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 microcrystalline cellulose and regenerated cellulose.
High level expression in E. coli has also been obtained with the modied domain II construct of xylanase cDNA from the anaerobic fungus Neocallimastix patriciarum. The modied domain II
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 fermentation studies using a fed-batch process. Recently Lapidot et al. [146] have reported overexpression and single-step purication of a thermostable
xylanase from B. stearothermophilus T-6. The xylanase 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 thermophilic 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 xylanase from Clostridium thermocellum (xylanase Ztruncated 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 enzyme or the relative scarcity of the substrate molecules in the plant cell wall. The response of the transgenic 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
FEMSRE 646 28-6-99
signicance. Xylanase B (Xyn B) from C. stercorarium 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 incubated in the medium containing kanamycin, in agarose 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 proteins 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 enzymes.
8.4. Homologous cloning
Several heterologous proteins cannot be eciently
expressed in E. coli due to any one of several reasons, 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 homologous 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
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 recombinants in the large-scale fermentation process has
been restricted due to plasmid instability and constant 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 xylanase gene of S. lividans #1326 was cloned by functional complementation of the xyn3 mutant of S.
lividans using multicopy plasmid PIJ 702; a maximum enzyme production of 380 U ml31 was reported [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 authors have suggested that the changes in the signal
peptide aect the level of xylanase production. However, further experiments are necessary that will allow the combination of a suitable signal peptide with
the enzyme to achieve the overproduction. The reports 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 nonxylanolytic organisms has been largely restricted to
FEMSRE 646 28-6-99
428
Table 3
Homologous expression of xylanase genes
Parent strain
Bacillus pumilus IPO

Bacillus sp. (NCIM 59)
Streptomyces sp
Strain #36a
S. lividans 1326
Streptomyces EC3
Streptomyces halstedii
Host
Vector
B. subtilis MI 111
Bacillus sp. NCIM-59
S. lividans TK21
S. kasugansis G3
S. lividans
S. lividans
S. parvulus
S. parvulus
PUB110
PUC8
PIJ-702
PSK2
PIJ-702
PIJ-702
PIJ-702
PIJ-702
Xylanase activity (U ml31 )

P
0.6
66
38.9
^
5.8
600
^
4.12
1.8
128
2839
1841
380
^
^
^
Reference
[62]
[153]
[156]
[155]
[312]
[313]
P and R correspond to parent and recombinant protein.
microorganisms such as Saccharomyces, Kluyveromyces, Lactobacillus and Bacillus subtilis. These organisms have been well characterized as far as their industrial applicability is concerned. Lactobacillus
plantarum can actively produce and secrete heterologous enzymes from a variety of Gram-positive bacteria 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 ^ hemicellulose, a cheap, renewable, abundant, fermentable,
raw resource material. Saccharomyces has been considered 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 established. Saccharomyces has been used to express the
xylanases from Aspergillus and Cryptococcus. Recently cloning and expression of xylanase genes
from Meripilus giganteus, Myceloipthora thermophilum and Thielavia terrestris in Aspergillus oryzae
have been reported [160^162], illustrating the examples 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 Dictyoglomus thermophilum Rt 46 B.1 in the yeast Kluyveromyces 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 predominantly as an unglycosylated protein comprising
of 90% of the total extracellular proteins. Also xylanase from thermophilic bacterium Caldicellulosiruptor saccharolyticus was secreted to a level of 10Wg
ml31 in the same yeast. The reports on the expression 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
A. pullulans
Clostridium acetobutylicum
C. thermocellum
Cryptococcus albidus
S. cerevisiae
S. cerevisiae
L. plantarum
L. plantarum
S. cerevisiae
Pichia stipitis
S. cerevisiae
pVT 100
pYES 2
pWP 37
pWP 37
pVT 100
pJHS
pFLAGU 2
[133]
[314]
[158]
[158]
[315]
C. saccharolyticum
FEMSRE 646 28-6-99
[316]
9. Protein engineering
Biotechnological applications of the xylanases require thermostable enzyme preparation with a wide
pH and temperature range. Since the availability of
the ideal enzyme preparation is limited, the application 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 modication, X-ray crystallographic data and site-directed 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 manipulation of the gene for desired enzymatic properties.
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 groupspecic reagents, may suggest the type of residue
involved in catalysis or in substrate binding, however, 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 uorometric analysis of the xylanases from Chainia [166]
and alkaliphilic thermophilic Bacillus [167] revealed
that the tryptophan microenvironment was electronegative. Chemical modication of xylanases from
the fungus Schizophyllum commune [168] and an alkaliphilic 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 bacterial xylanases has been reported [164,165]. However,
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 residue may result in loss in activity. It is also possible
that they may participate in the formation of covalent 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 covalent 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 possible identication of specic residues involved in
substrate binding and catalysis.
Crystallization and diraction analysis of xyla . In B. puminases have been carried out at 1.5^3.0 A
lus IPO [177], the enzyme molecule is of ellipsoidal
) with a well-dened cleft down
shape (40U35U35 A
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. harzianum to be orthorhombic [179]. The crystallography studies of xylanase I from A. niger indicated a
characteristic fold which is unique for family 11 xylanases. It consists of a single domain composed predominantly 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 accommodate at least four xylose residues. Two conserved
glutamate residues Glu79 and Glu170 reach into the
cleft from opposite sides [180]. The enzymes of family 11 are single domain proteins composed of three
FEMSRE 646 28-6-99
430
antiparallel L-sheets and one K-helix, with the active

site lying between the second and third sheets. However, 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 beginning 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 conserved 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 conserved. 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 tyrosine 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 speculated 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 domains 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
serve to orient important active site residues

[185,186]. Xylopentose binds across the carboxy-terminal end of the K/L-barrel in an active site cleft
containing the two catalytic glutamates. Recent crystal 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 protected the enzyme from thermal inactivation, thermal
unfolding and proteolysis. Mutational analysis indicated 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 proteins. The essential reactive groups were identied by
selective chemical modication. Rapid developments
in molecular biology techniques have made it possible 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 substrate specicity, pH optima and to increase the thermal 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 xylanase from Bacillus pumilus with other xylanases of
known origin and the knowledge of its three-dimensional structure, the authors have proposed the residues Glu93 and Glu182 to be the most suitable candidates for the essential catalytic activity of xylanase
FEMSRE 646 28-6-99
[187]. Mutation of these residues resulted in a decrease 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 important residue in maintaining the ionization states of
two catalytic residues and in the stabilization of catalytic intermediates [189]. The three-dimensional
structures of two bacterial xylanases from B. pumilus
and B. circulans and a fungal xylanase from T. harzianum have also revealed the presence of two completely conserved glutamic acid residues corresponding to Glu87 and Glu184 of S. commune xylanase A.
The mutational and crystallographic analysis of the
active site residues of the B. circulans xylanase indicate the presence of six tyrosine and three tryptophan 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 glutamic acid residues (Glu78 and Glu172 ) are involved
in catalysis in the case of the xylanase from B. circulans. Arg112 has been implicated to play a signicant role in catalysis, and Tyr69 and Tyr80 in substrate 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 irreversible thermal denaturation studies [191].
Moreau et al. [192] have identied two acidic residues, 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. Conversely, none of the aspartic acid residues of xyla-
431
nases of family 11 were completely conserved; it appears that they are not essential for activity [188].
Roberge et al. [193] showed that two histidine residues (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 glycanases. 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 essential for protein stability and were probably involved 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 technology 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 longer than that of the wild-type enzyme, suggesting
that although the engineered xylanase had a higher
optimal temperature, the stability was not signicantly aected. Previous reports suggested that the
same substitution occurs naturally in the xylanases
produced by Bacillus sp. C-125 and C. saccharolyticum [196]. Both xylanases have an optimum temperature 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 residues that form disulde bonds spontaneously in sol-
FEMSRE 646 28-6-99
432
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 activity 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 enthalpy.
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 xylanases. The future scope includes alteration of the
residues to improve the pH stability and shift in
pH optimum of the enzyme for its commercial application at alkaline pH. SDM has also been applied
to alter the substrate specicity of proteases [200]
and glucose-xylose isomerases [201]. Designing a xylanase with multiple substrate specicities will also
lead to ecient utilization of hemicellulosic substrates.
11. Mechanism of action of the xylanases

It has frequently been suggested that the catalytic
mechanism of glycosidases resembles that of lysozyme. 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-
tion with retention of conguration involves a twostep 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]. Reactions 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 exhibit 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. circulans. The critical distance between two catalytic car in B. circulans xylanase)
boxylic acids (approx. 5.5 A
is less in retaining enzymes as compared to that in
). It was also
inverting glycosidases (approx. 10^11A
observed that the precise placement of the acid/base
catalyst is not critical, since both shortening and
lengthening this carboxyl side chain resulted in approximately the same modest decrease in kcat /Km values [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 conformational 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 consequently 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 formation of the glycosyl enzyme intermediate through
improved acid catalysis and greater nucleophilic preassociation, without aecting its rate of decomposition [84]. Hence, the residues acting as nucleophiles
have been identied on the basis of structural analysis and mutagenesis data. Tull et al. [206] have
FEMSRE 646 28-6-99
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 oxocarbonium 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
mapped the nucleophile (Glu274 ) of exoglucanase/

xylanase Cex from C. mi and Glu127 has been conrmed as the acid-base catalyst based on kinetic
studies [207].
Thus, from various reports it appears that xylanases belonging to family 11 operate via a double
displacement mechanism in which the anomeric conguration is retained. However, endoxylanase from
A. niger [84] was found to be an inverting enzyme
following the single displacement reaction mechanism (Fig. 3). Extensive NMR assignments of xylanase from B. circulans revealed the presence of potential amide-aromatic hydrogen bond between HN
of Ile118 and the indole ring of Trp71 . This interaction, which is conserved in all low molecular mass
xylanases of known structure, may play an important role in establishing the active site conformation
of these enzymes [182]. The X-ray crystallography
data on xylanases from P. uorescens [184] and kinetic 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 enzyme. However, detailed structural data available on
the xylanases is limited. A considerable sequence variability 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 applications.
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 information. HCA detects homologies between similar
three-dimensional structures of proteins having low
sequence identity. These homologies imply a struc-
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 involved 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 Bairoch have classied all the available sequences of glycosyl 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 cellobiohydrolase [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 protein folding.
12.2. Domain structure
At the molecular level the xylanase protein comprises 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 motifs 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 cellulases 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 domains [210]. Hence, it was assumed that the catalytic
domains and the substrate binding domains of the
xylanases, too, should be spatially separable or dis-
FEMSRE 646 28-6-99
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 contribute to the catalytic activity. Truncated derivatives of
xylanase A, lacking either the serine-rich linker sequence or cellulose binding domain, were found to
be less active against xylan contained in a cellulosehemicellulose 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 suggested 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 assembly [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 indicated that the full length protein contained two catalytic domains displaying similar substrate specicity
[218]. In the case of Ruminococcus avefaciens two
catalytic domains have been reported. Catalytic activity measurements and dierential scanning calorimetry of exoglucanase/xylanase Cex from C. mi
suggested that binding and catalytic domains of the
protein fold independently [92]. The amino acid sequence of the endoxylanase from Cryptococcus albidus has some homology with the catalytic region of
the egg white lysozyme [219]. Based on sequence homology it has been interpreted that the glycosidic
435
bond cleavage in xylan is similar to that of lysozyme.

However, West et al. [84] have shown that the catalytic 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 cellobiohydrolase domain of the Caldocellum bifunctional exocellulase-endocellulase (celB) catalytic domain of cellobiohydrolase from C. mi [220], xylanases from B.
subtilis C 125, C. thermocellum [221], and Cryptococcus 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 absence 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 catalytic domain. It may be that the tertiary structure of
the enzyme is such that the catalytic sites are partially 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 ancestry 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 residues 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 located within the K/L-barrel structure and three are
exposed on the surface. NMR analysis and chemical
modication studies conrmed that the exposed ar-
FEMSRE 646 28-6-99
436
omatic residues play a direct role in binding to cellulose; 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 thermocellum [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 enzymic 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 polysaccharide 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 cellulose binding domain abolished the cellulose binding capacity of the enzyme without aecting the xylan 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 directly 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 unambiguously [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 expressed 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 intrinsic 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 xylanase 1 from Rhodothermus marinus are shown to be
repeated in tandem at the N-terminus exhibiting similarity with CBD family IV [229]. It appears to be the
rst example of xylanase gene encoding a CBD family IV in combination with the catalytic domain of
glycosyl hydrolase family 10. The molecular architecture 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 modular 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 homologous 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 Thermoanaerobacterium thermosulfurigenes EM 1 has revealed 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 domain 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 domains have been closely associated with the xylanase
catalytic domain. The 180-residue domain of xylanase Y from C. thermocellum has been shown to
have 28% sequence identity with the thermostabilizing domain of xylanase A (residues 200^353) from T.
saccharolyticum. Recently sequence analysis of the
FEMSRE 646 28-6-99
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. stearothermophilus, thermophilic bacterium RT8.B4, T. maritima,
Anaerocellum thermophilum and Caldicellulosiruptor
saccharolyticus have a region similar to the thermostabilizing domain of Xyn A from T. saccharolyticum
(Fig. 4). Multiple sequence alignment of these xyla-
437
nases with Xyn A from T. saccharolyticum indicated

conserved Gly and Tyr residues in the region corresponding to the thermostabilizing domain of Xyn A
from T. saccharolyticum. The identity of xylanases
from other thermophilic organisms with the thermostabilizing domain ranged from 5 to 32%. Xylanase
C from C. thermocellum showed a maximum similarity of 32%, while xylanase from B. stearothermophilus showed very little identity. It may be possible that
these domains confer thermostability to the corresponding xylanases (Fig. 4). However, more experimental evidence is necessary to validate the concept
Fig. 4. Homology of the thermostabilizing domain of xylanase Y from Clostridium thermocellum with other thermophilic xylanases. Accession 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) Anaerocellum 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
that thermostability is conferred by a specic domain. Examination of the conserved residues in the
thermostabilizing domain reveals the absence of cysteine. A predominance of bulkier aliphatic residues is
observed, which may increase thermostability by increasing the compactness of the folded molecule
[236,237]. The occurrence of a large number of glycine residues supports the proposition that they may
be located in the loop regions of thermophilic proteins [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 domains could suggest the close evolutionary relationship between the thermophilic bacteria that might
have arisen through the lateral transfer of a single
ancestral gene between them. The thermophilic xylanase A of Thermomonospora fusca is the one
reported to date belonging to family 11 [239]. However, non-catalytic domains conferring thermostability have not yet been detected in family 11 xylanases. 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 domains are located only in xylanases and have not
been observed in a number of thermophilic endoglucanases 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 approaches for obtaining useful information about
functional and evolutionary relationships. Residues
occupying equivalent positions are believed to share
common ancestors and/or to have equivalent biological roles. HCA analysis of amino acid sequences of
xylanases and cellulases has revealed that isoenzyme
forms of these enzymes, observed in several organisms, 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 different families are often present in a single organism
suggesting that microorganisms have acquired multiple plant cell wall hydrolase genes not exclusively
through gene duplication but via extensive horizontal 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 xylanase (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 similarity with 38 other fungal and bacterial xylanases belonging 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]. Xylanase A from B. stearothermophilus 21 was 45^50%
identical to xylanases from other thermophilic organisms such as C. saccharolyticum and C. thermocellum [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 signicant 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 extensive homology with family 11 xylanases. The two
glutamic acid residues were found to be essential for
FEMSRE 646 28-6-99
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. saccharolyticum [116], xylanase B from C. stercorarium [243] and
xylanase T-6 from B. stearothermophilus T-6 [67]
showed high homology to xylanase A from alkaliphilic Bacillus C-125. B. steararothermophilus xylanase 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 originate from a common ancestral gene, i.e. these genes
were acquired by each bacterium through interspecies 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. stercorarium showed the highest homology to xylanase A
irrespective of the dierences in their thermal stabilities [244]. The amino-terminal domain of xylanase
D (Mr 90 kDa) from R. avefaciens showed signicant 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 rumen species F. succinogenes revealed much lower
identity [132]. Xylanase C from the ruminant organism F. succinogenes S85 belonged to family 10. However, domains A and B of xylanase C shared homology 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 suggested that these organisms were found in environmental niches totally dierent from the ruminal organisms, suggesting that their catalytic domains
might have had a common origin but they had diverged a long time ago. On the other hand, the enzymes from the three ruminal organisms (R. avefaciens, N. patriciarum and F. succinogenes) shared a
439
similar multiple catalytic domain structure, which

might stem from environmental selection [245]. Xylanases from two dierent strains of the rumen anaerobic bacterium, Prevotella ruminicola, showed similarities 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 unrelated 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 Bacteroides 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. thermocellum, in common with xylanases from several thermophilic bacteria, contained a family 10 catalytic domain. This observation could reect the close
evolutionary relationship between thermophilic bacteria. 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, suggesting that the cellulase system of this bacterium
arose through extensive lateral gene transfer [235].
FEMSRE 646 28-6-99
440
13.2. Evolutionary relationship of xylanases

Many xylanases and cellulases are known to possess a modular structure comprising a catalytic domain linked to a non-catalytic cellulose binding domain (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-arabinofuranosidase 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, hemicellulose 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 integration 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 catalytic properties on the enzyme. Phylogenetic analysis
has revealed a relationship between xylanases containing loop 7 and a common ancestral sequence
containing DNA insertion in the region encoding
loop 7. Since similar sequences occur in taxonomically 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 International Resource, Release 46.0) (Fig. 5). The sequences were aligned using Clustal V multiple sequence
alignment [250]. A minimum mutation distance matrix was then constructed from the pairwise comparisons of 54 sequences. The distance matrix was then
used to obtain an evolutionary tree using TAXAN
(Release 2.0, Information Resources Group, University of Maryland, USA). The evolutionary tree implies 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 observed 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 evolutionary 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 family 11 enzymes with substantial variations occurring
only in external loops, e.g. xylanases from T. reesei
and T. harzianum. These enzymes are classical illustrations of diverging evolution from a common ancestor. Recently xylanase from Streptomyces viridosporus 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].
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
FEMSRE 646 28-6-99
441
442
14. Biotechnological potentials of xylan and xylanases

During the last decade, the potential biotechnological applications of xylan and xylanases have been of
particular interest to researchers. At present, the major end products of xylan, which are of considerable
importance, are furfural and xylitol. Furfural production is derived mainly from agricultural residues
whereas xylitol is obtained from wood residues. The
hydrolysis products of xylan (xylose and oligosaccharides) have possible applications in the food industry as thickeners or as fat substitutes and as an
antifreeze food additive. In the pharmaceutical industry xylan is found suitable as an agent for `direct
tableting' and, in combination with other components, it can be used for delayed release tablet construction. The xylan hydrolysis products can be subsequently converted to liquid fuel, single cell
proteins, solvents and articial low calorie sweeteners [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]. Xylanases play an important role in debarking, deinking of recycled bers, and in the purication of cellulose 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 carbohydrate 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 investigators [255,257,258] have subsequently conrmed these
studies. Xylan and glucomannan form the basic polymers of the wood hemicellulose backbone. Xylanases and mannanases are the two main glycanases
that depolymerize the hemicellulose backbone. The
eects of puried or partially puried endo-acting Lmannanases from B. subtilis, A. niger and T. reesei
on pulp delignication have been studied. Treatment
of pine pulp with xylanase enriched with endo-L-
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, reprecipitated 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 decrease their mechanical strength. Alkaline-stable lignin-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 degradation products adversely contribute to the euent. In contrast to this, hemicellulose-degrading enzymes selectively hydrolyze polysaccharide chains
attached to lignin, thereby decreasing the amounts
of chemicals required for pulp bleaching. The xylanases assume special importance in the paper and
pulp industry as they replace toxic chemicals such
as elemental chlorine and chlorine dioxide for developing eco-friendly processes. At the same time the
high molecular mass lignin molecules, which are currently gaining more importance as a valuable byproduct of the paper and pulp industry, can be recovered 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,
FEMSRE 646 28-6-99
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 commercial 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 reducing the chlorine required to achieve a certain degree
of brightness. In the process of pulp bleaching enzymes 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 thermophilic anaerobic bacterium Dictyoglomus sp. [271]
has been evaluated for its suitability in pulp bleaching. Xylanase pretreatment at 80C and pH 6^8 resulted in an increase in brightness by 2 ISO units in
one-stage peroxide delignication. Thermostable xylanase 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 longibrachiatum 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 facilities. Thus, enzymatic pretreatment has been shown
to be fully compatible with existing industrial equip-
443
ment, which is an added advantage. Addition of an

enzymatic step to any conventional bleaching sequence results in a higher nal brightness value of
the pulp. As world pioneers, the Finnish forest companies started mill-scale trials in 1988. Since 1991,
this method has been continuously used on an industrial scale in Finland together with other low-chlorine or chlorine-free bleaching methods. The chlorine
requirement in prebleaching has been shown to be
reduced by 20^30%. As a result, the AOX (adsorbable organic halogen) load of the prebleaching euent has been reduced by 15^20%. The greatest number 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 methods 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 advantageous.
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 improve 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 preparation 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 preparation Ùltrao L' produced by a selective strain of
Humicola insolens. It is a heat-stable multi-active L-
FEMSRE 646 28-6-99
444
glucanase used in the mashing process in beer brewing 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, suggesting 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 elongation, seem to be involved in fruit softening, and are
believed to have yet undiscovered important physiological functions.
The xylanases nd application in the bakery and
the fodder industries due to the presence of substantial 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 redistribution of the water content of the bread [278], thereby
signicantly improving the desirable texture, loaf
volume and shelf life of the bread. A xylanase (Novozyme 867) has shown excellent performance in the
wheat separation process [279], since it has high activity towards soluble arabinoxylan and eects a rapid decrease in the viscosity of wheat our slurries.
The dietary hemicelluloses have little nutritional signicance 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 enzymes, absorption of the digested food and may support pathogenic conditions, especially in broiler
chicks. The use of xylanases together with other
hemicellulases corrects the problems and also increases the nutritive value of the feed. These biotechnological 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 developed for the food and paper industries which are
based on the partial hydrolysis of xylan. The longterm application of xylanases such as conversion of
renewable biomass into liquid fuels, where xylanases
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 incentive 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 obtaining 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 successfully employed for the enhanced production of cellulases by Trichoderma on both insoluble and soluble
substrates. It can be assumed that similar investigations 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 congurations of the bioreactor oer opportunities for
improvement in the fermentation process. More research 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 recovery 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
FEMSRE 646 28-6-99
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 certain 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 soluble and insoluble physical states which makes the
actual catalysis an extremely complex phenomenon.
This catalytic complexity may have resulted in multiplicity 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 developed 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. pulpzyme 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 crosslinks may prove useful for increasing the thermostability of xylanases so as to make them suitable
in biotechnological processes, its use is restricted
where disulde bonds are stable. Interestingly, xylanases 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
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 activity, much remains to be learned from natural thermophilic enzymes before precise molecular predictions can be guaranteed. Attempts have been made
to recover xylanase DNA from complex soil samples
by PCR amplication using degenerate primers. Recovered DNA, which is dierent from the known
xylanases, can be used in several ways to facilitate
the production of novel xylanases for industrial applications [282].
The transfer of xylanase genes to selected fermentative 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 understanding the basic technology involved in the biological process of hydrogen production from xylan in
plant wastes. Research eorts should be focused on
the improvement of such strains for the ecient utilization of biomass. Xylan being an abundant agricultural residue, the chemical energy of the molecule
can be meaningfully utilized if the nitrogen xation
activity can be linked to xylan degradation by coupling 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
FEMSRE 646 28-6-99
446
the nal text. A Senior Research Fellowship to N.

Kulkarni from the Council of Scientic and Industrial Research is gratefully acknowledged.
[20]
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FEMS Microbiology Reviews 23 (1999) 411^456

Molecular and biotechnological aspects of xylanases

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

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2. Scope of the present review

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sential for the complete hydrolysis of acetylxylans.

cellulosic components and hence greatly inuence the

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tion and show that the chains are organized in a

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systems resemble the natural habitats of microbes

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production of these enzymes on the medium under

5. Regulation of xylanase synthesis

hemicellulose were used together as the carbon

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uation of inducers under experimental conditions

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controlled at two levels, directly by repression of

rapid turnover as that of intracellular proteins [70].

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Clostridium stercorarium [22], Streptomyces sp. [75]

nase activity. However, enzymes having more anity

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7. Xylanases of extremophilic origin

proposed that the model could be applied to a large

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tolerant xylanase from Aspergillus scheri [100] was

high technology. Multinational companies like

8. Cloning and expression of xylanase gene(s)

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Bacillus sp. NG-27

B. pumilus [61] and C. saccharolyticum [116]. In spite

mophilum Rt46B.1 in E. coli has been reported [122];

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Aeromonas sp. 212 (ATCC 31085)

B. polymyxa NRC 2822/NRRL 8505

Ruminococcus albus #SY3

Expression of cloned xylanase

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expression of xylanases from the fungi Aspergillus

xylanase produced in E. coli had a specic activity

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of expression observed has been attributed by the