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CHAPTER 5
XYLANASES: MOLECULAR PROPERTIES
AND APPLICATIONS
F. I. JAVIER PASTOR
1∗
, ÓSCAR GALLARDO
1
, JULIA SANZ-APARICIO
2
AND PILAR DÍAZ
1
1
Department of Microbiology, Faculty of Biology, University of Barcelona, Spain and
2
Instituto de Química-Física Rocasolano, CSIC, Madrid, Spain
∗
fpastor@ub.edu
1. INTRODUCTION
The plant cell wall is a highly organized network of lignocellulose, made up of
cellulose and cross-linked glycans embedded in a gel matrix of pectic substances
and reinforced with structural proteins and aromatic compounds. Cellulose and
hemicelluloses are the major components of cell wall polysaccharides, with hemicel-
luloses representing up to 20–35% of the total lignocellulosic biomass (de Vries and
Visser, 2001). The major hemicellulose in cereals and hardwoods is xylan, while
the main hemicellulose in softwoods is galactoglucomannan. Other less abundant
hemicelluloses include glucomannan, xyloglucan, arabinogalactan and arabinan,
the latter polymers often being found as side chains of pectins (de Vries and
Visser, 2001). The degradation of hemicelluloses is mostly carried out by micro-
organisms that can be found either free in nature or as a part of the digestive tract
of higher animals. The hydrolytic enzymes produced by these micro-organisms are
the key components for the degradation of plant biomass and carbon flow in nature
(Shallom and Shoham, 2003).
Xylan is a major structural component of plant cell walls and, after cellulose, is
the second most abundant renewable polysaccharide in nature (Collins et al., 2005).
It is the main hemicellulose in hardwoods from angiosperms and is less abundant in
softwoods from gymnosperms, accounting for approximately 15–30% and 7–12%
of their total dry weights, respectively (Wong et al., 1988). In woody tissues
xylan is located mainly in the secondary cell wall where, together with lignin,
it forms an amorphous matrix that includes and embeds cellulose microfibrils.
65
J. Polaina and A.P. MacCabe (eds.), Industrial Enzymes, 65–82.
© 2007 Springer.
66 PASTOR ET AL.
Xylan interacts with lignin and cellulose via covalent and non-covalent linkages,
these interactions being of importance for both protecting the cellulose microfibrils
against biodegradation and maintaining the structural integrity of cell walls.
Xylan is a complex polysaccharide composed of a backbone of -1,4-linked
xylopyranosyl residues that, depending on the plant source, can be variably
substituted by side chains of arabinosyl, glucuronosyl, methylglucuronosyl, acetyl,
feruloyl and p-coumaroyl residues. Although homoxylans have been found in
some plants and seaweeds, in the latter case also containing xylose -1,3 linkages,
xylans containing exclusively xylose residues are not widespread in nature (Beg
et al., 2001). Xylan from most plant sources occurs as a heteropolysaccharide
and the terms glucuronoxylan and glucuronoarabinoxylan are commonly used to
describe xylan from hardwoods and softwoods, respectively. These two types of
xylan have 4-O-methyl -D-glucuronic acid residues attached to C-2 of the xylose
backbone units. Hardwoods have this substitution on approximately 10% of the
xylose residues, while in softwoods around 20% of xylose residues are branched
with glucuronic acid. Softwood xylan is also substituted with -l-arabinofuranose
on C-3 of approximately 13% of the xylose backbone residues (Coughlan and
Hazlewood, 1993). The degree of polymerization is variable among xylans, being
greater in hardwoods (150–200) than in softwoods (70–130) (Kulkarni et al., 1999).
While xylan from softwoods is not acetylated, xylan from hardwoods is highly acety-
lated, this substitution occurring on around 70% of the xylose units at C-2, C-3 or
both (Coughlan and Hazlewood, 1993). The presence of acetyl groups makes xylan
significantly more soluble in water (Biely, 1985). Xylan from grasses is usually
referred to as arabinoxylan because of its large content in arabinosyl residues, which
are linked to xylose at C-2 or C-3 or both. This xylan is acetylated and also has
glucuronic acid present, albeit at a lower content compared to hardwoods. Feruloyl
and coumaroyl residues ester-linked to C-5 of arabinose side chains are found in
xylans from different sources, and may be involved in the covalent cross-linking
of xylan molecules with lignin or with other xylan molecules. As a consequence of
all these features, xylans constitute a very heterogeneous group of polysaccharides
showing microheterogeneity with respect to the degree and nature of branching in
each category. Xylans containing rhamnose and galactose residues have also been
described from different plant sources (Wong et al., 1988).
2. ENZYMATIC DEGRADATION OF XYLAN
Due its heterogeneity and complex nature, the complete breakdown of xylan requires
the action of a large variety of hydrolytic enzymes (Biely, 1985; Coughlan and
Hazlewood, 1993). These enzymes can be classified into two main groups: those
acting on the xylose backbone, and those cleaving the side chains. Degradation of
the xylose backbone depends on xylanases, that cleave bonds within the polymer,
and -xylosidases that release xylose units from xylobiose and xylooligomers.
Removal of xylan side chains is catalysed by -l-arabinofuranosidases, -d-
glucuronidases, acetyl xylan esterases, ferulic acid esterases and p-coumaric acid
esterases (Fig. 1). Xylan degradation is quite widespread among saprophytic
XYLANASES: MOLECULAR PROPERTIES AND APPLICATIONS 67
Figure 1. Structure of xylan and the sites of attack by xylanolytic enzymes. The backbone of xylan
chains is composed of -1,4-linked xylopyranose residues. This backbone can be variously substituted by
side chains of arabinosyl, glucuronosyl, methylglucuronosyl, acetyl, feruloyl and p-coumaroyl residues.
Hydrolysis of the xylan backbone is carried out by xylanases that hydrolyse internal linkages in xylan,
and -xylosidases that release xylose units from xylobiose and xylooligomers, while removal of xylan
side chains is catalysed by -l-arabinofuranosidases, -d-glucuronidases, acetyl xylan esterases, ferulic
acid esterases and p-coumaric acid esterases
micro-organisms, including bacteria and fungi, as well as in the rumen micro-
biota, that possess complete xylanolytic enzyme systems (Biely, 1985; Sunna
and Antranikian, 1997; Krause et al., 2003). Synergism between xylan degrading
enzymes has been extensively studied and found to frequently occur between
xylanases and side chain cleaving enzymes, between xylanases and -xylosidases,
and also between different xylanases (Coughlan et al., 1993; de Vries et al., 2000).
In this way, xylan degradation can proceed despite that the access of xylanases to
their targets in the xylan backbone may be obstructed by side chain substituents
and that these substituents may be more readily released from xylan fragments than
from the polymeric substrate.
3. XYLANASES
3.1. Function, Expression and Multiplicity
Xylanases (endo--1,4-xylanases, EC 3.2.1.8) cleave the xylan backbone into
smaller oligosaccharides. They are the key enzymes for xylan degradation and
differ in their specificities toward the xylan polymer. Many cleave only at unsub-
stituted regions whereas others have a requirement for side chains in the vicinity
of the cleaved bonds (Coughlan and Hazlewood, 1993). Most xylanases are also
active on xylooligomers of degree of polymerization greater than 2, showing
increasing affinity for xylooligomers of increasing length. Xylanases are endo
type enzymes that hydrolyse internal linkages in xylan and act by a random
attack mechanism yielding a mixture of xylooligosaccharides from the polymer.
68 PASTOR ET AL.
Nevertheless, characterization of Aeromonas xylanases which produce only one
oligosaccharide type as a reaction product from xylan suggested an alternative
mode of hydrolysis: an exo type mechanism from one end of the polymer
(Kubata et al., 1995) similar to the processive mode of action of exocellulases in
cellulose degradation (Lynd et al., 2002). However, as this type of mechanism
would first require depletion of the side chains from xylan, the occurrence of true
exoxylanases for xylan degradation seems unlikely.
As xylan is a large polymer that cannot penetrate into cells, xylanases have to
be secreted to the extracellular environment to reach and hydrolyse it. Generally,
xylanases are induced in most micro-organisms during their growth on substrates
containing xylan. Small soluble oligosaccharides released from xylan by the action
of low levels of constitutively produced enzymes are transported inside cells where
they induce xylanase expression (Kulkarni et al., 1999). In the fungus Cryptococcus
albidus, xylobiose is considered to be the natural inducer or the direct precursor of
compounds that induce xylanase expression (Biely, 1985). Regulation of xylanase
synthesis is often coordinated with the expression of cellulases, as in the case
of Aspergillus in which several regulatory proteins including the transcriptional
activator XlnR and the carbon catabolite repressor CreA have been identified and
characterized (de Vries and Visser, 2001). Although most xylanases are extra-
cellular enzymes, usually secreted by the Sec-dependent pathway, periplasmic
xylanases have been described in some rumen bacteria and in Cellvibrio mixtus
(Fontes et al., 2000). These periplasmic xylanases are probably involved in the
breakdown of large xylooligosaccharides and protected in this location from extra-
cellular proteases. Recently, a cytoplasmic xylanase that may represent a new type of
enzymes involved in xylan degradation has been characterized from Paenibacillus
barcinonensis (Gallardo et al., 2003). In common with three other xylanases charac-
terized from Bacillus and Aeromonas, the P. barcinonensis enzyme lacks a signal
peptide for export outside the cytoplasm. These four enzymes constitute a group
of highly homologous xylanases whose proposed role is the hydrolysis of the short
oligosaccharides resulting from extracellular xylan hydrolysis once they have been
transported inside cells.
Many xylan degrading micro-organisms produce a multiplicity of xylanases with
different but overlapping specificities (Wong and Saddler, 1988). This has been
evidenced for important xylanase producers like Aspergillus, Trichoderma, Strepto-
myces and Bacillus amongst others (Beg et al., 2001; Sunna and Antranikian, 1997).
Multiplicity of xylanases can arise from different post-translational processing of
the same gene product (Ruiz-Arribas et al., 1995), though very often several
xylanase-encoding genes have been isolated from a defined microbial strain. In this
regard, at least 4 xylanase genes have been isolated from Fibrobacter succinogenes
(Jun et al., 2003) and 6 have been characterized in Cellvibrio japonicus (formerly
Pseudomonas cellulosa) (Emami et al., 2002). The production of a multienzyme
system of xylanases, in which each enzyme has a specific function, represents a
strategy to achieve efficient hydrolysis of xylan.
XYLANASES: MOLECULAR PROPERTIES AND APPLICATIONS 69
Cellulosomes are secreted multienzyme complexes found in anaerobic bacteria
that mediate the attachment between cells and cellulose particles to enhance the
efficiency of cellulose and hemicellulose degradation (Bayer et al., 2004). They
contain numerous enzymes, most of them cellulases, kept together through interac-
tions between pairs of dockerin and cohesin domains located on the enzymes and on
a non-catalytic scaffolding protein. Xylanases are also found among the component
enzymes of most known cellulosomes. Xylan degradation can be essential in making
cellulose available for enzymatic hydrolysis, since cellulose is present in close
proximity with xylan in the plant cell wall matrix. By analogy, the term xylanosome
has been proposed for extracellular protein aggregates predominantly composed of
xylanases reported in several bacteria, though these have not been as well charac-
terized as cellulosomes (Beg et al., 2001; Jiang et al., 2005).
3.2. Molecular Architecture and Classification
According to their molecular architecture, xylanases, like cellulases and other
carbohydratases, can be classified into two types: single domain and multidomain
enzymes. Xylanases of the first type contain a single catalytic domain, whereas
multidomaim xylanases have a modular structure that comprises, in addition
to a catalytic domain, several ancillary domains joined by linker sequences
(Gilkes et al., 1991; Gilbert and Hazlewood, 1993). These domains may fold and
function independently and can mediate binding to cellulose (cellulose binding
domains,CBD), xylan (xylanbinding domains),or to cellulosomescaffolding proteins
(dockerin domains); or they may have functions that are not yet fully identified such
as Fn3 domains (Kataeva et al., 2002) or SLH domains (Ali et al., 2001).
Cellulose binding domains, the most abundant non-catalytic domains in
xylanases, have been characterized and grouped into several families (Tomme
et al., 1998). They promote binding to different forms of crystalline or amorphous
cellulose and may disrupt cellulose microfibrils to facilitate degradation by cellu-
lases (Linder and Teeri, 1997). Modules that mediate binding to xylans have
also been characterized (Black et al., 1995). They include previously designated
thermostabilizing domains, found in some thermophilic (Lee et al., 1993) and also
in mesophilic xylanases (Blanco et al., 1999), that have subsequently been shown
to promote binding to xylan (Sunna et al., 2000). Identification of domains that
promote binding to other carbohydrates such as chitin and starch has prompted the
term carbohydrate binding module to group all domains that mediate binding to
carbohydrates (Boraston et al., 2004).
The heterogeneity and complex nature of xylan has resulted in a diversity of
xylanases with varying specificities, sequences and folds. Wong et al. (1988)
classified xylanases into two types according to their physicochemical properties: a
group of low molecular weight (<30 kDa) and basic pI, and a second group of high
molecular weight (>30 kDa) and acidic pI. Although many xylanases fit in these
groups, the number of xylanases described since then has increased enormously,
and at present many xylanases of intermediate properties have been identified that
70 PASTOR ET AL.
do not fit either of these categories. A more complete classification system was
introduced in 1991 for the glycoside hydrolases (EC 3.2.1.x), a group of enzymes
in which xylanases are included, which is based on primary structure comparison
of catalytic domains and the grouping of enzymes in families of related sequences
(Henrissat, 1991). From the 35 families identified in 1991, the classification has
been updated regularly with newly sequenced enzymes and comprises over 100
families (GH1 to GH106) at present (Coutinho and Henrissat, 1999; Carbohydrate
Active enZYmes CAZY server at http://afmb.cnrs-mrs.fr/CAZY/). Most families
comprise enzymes with the same substrate specificity, though several families are
polyspecific and include enzymes active on different carbohydrates. The finding of
related structures in different families has resulted in the introduction of a higher
hierarchical level of classification known as the superfamily or clan. A clan is a
group of families that are believed to share a common ancestor and show related
tertiary structures together with conservation of the catalytic residues and catalytic
mechanism (Henrissat and Bairoch, 1996).
Xylanases are usually classified into glycoside hydrolase families 10 (formerly F)
and 11 (formerly G). These two families include, respectively, xylanases of the
high MW/low pI and low MW/high pI groups previously mentioned, but each
of the families includes many other xylanases with physicochemical properties
widely different from these two groups (Sunna and Antranikian, 1997; Beg
et al., 2001). Family 10 includes cellobiohydrolases (exocellulases) and endo--1,3-
xylanases besides xylanases (endo--1,4-xylanases), while family 11 is monospe-
cific, comprising solely xylanases. A small number of recently characterized
xylanases do not show sequence similarity to families 10 or 11. Instead, these
xylanases exhibit homology to enzymes belonging to glycoside hydrolase families
5, 7, 8 and 43. Accordingly, the group of families containing xylanases should be
expanded to include these new enzymes (Collins et al., 2005).
3.3. Structure
The three dimensional structures of many bacterial and fungal xylanases from
families 10 and 11 have been reported. In addition, crystal structures from new
xylanases belonging to families 5 and 8 have been solved within the last few years.
Glycoside hydrolase families 5 and 10 are members of clan GH-A which includes
17 glycoside hydrolase families. Despite large differences in size and sequence,
members of this clan possess a catalytic domain of 250–450 amino acids which
shares a common (/
8
TIM-barrel fold and a remarkable conservation of the
3D structure of the active site (Fig. 2a). Many family 10 enzymes are modular,
containing a carbohydrate binding module connected to the catalytic domain by a
flexible linker. To date, the only crystal structures of full length xylanases known
for this family are those of Xyn10A from Streptomyces olivaceoviridis (Fujimoto
et al., 2000) which displays a small substrate binding domain linked by a Gly/Pro-
rich region, and Xyn10C from Cellvibrio japonicus which bears a family 15 carbo-
hydrate binding module (Pell et al., 2004a). In both cases the linker is not visible