Studies on Xylan degrading fungal strains

A project report
Submitted in partial fulfillment of the requirement for the award of the degree of

Bachelor of Technology
in
Biotechnology
Submitted by

Shinjini Barik
Under the Guidance of

Prof. R.P. Singh

Department of Biotechnology

Indian Institute of Technology Roorkee

Roorkee-247667

Uttarakhand, India

May, 2009

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 Certificate
This is to certify that report entitled “Studies on Xylan degrading fungal strains” submitted by
Ms. Shinjini Barik for the partial fulfillment of requirement for the award of degree of Bachelor
of Technology in Biotechnology of Indian Institute of Technology, Roorkee, is a record of the
candidate’s own work carried out by her under my supervision and guidance. The matter
embodied in this project report has not been for the award of any degree or diploma. It is further
certified that she has worked from Jan 2009 to May 2009 for preparing this report at the institute.

Dr. R.P. Singh

Professor, Dept. of Biotechnology

Indian Institute of Technology, Roorkee

Roorkee, Uttarakhand-247667

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 Acknowledgement
I express my esteemed regards to my supervisor, Dr. R.P. Singh for his supervision, invaluable
guidance and encouragement during the entire course of this investigation. It was an honorable
and great pleasure for me to work with him. I am very thankful to him for providing the
equipments necessary for the completion of my work.

I am extremely grateful to Prof. Ritu Barthwal, Head of the Department of Biotechnology, and
all the faculty members for providing me with all the facilities required for the completion of this
work.

I would also like to thank Research scholar Ms. Nidhi Pareek, Ms. Samta Saroj, and my
classmate Latika Gupata for their support throughout the work.

Date: 19th May 2009

Place: Roorkee (Shinjini Barik)

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 Abstract
Xylanases is a group of enzymes that act by hydrolyzing xylan which is one of the major
components of plant cell wall. Because of their ability to degrade xylan, xylanases are important
from industrial point of view. Out of large array of enzymes two enzymes selected for study
were endoxlanase and acetyl esterase. Soil sample collected from forest soil was screened for
strains producing xylanase. Out of the three strains that were selected, the strain with highest
levels of both enzymes was selected for further study.

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 Table of contents

1. Introduction

2. Review of litereature

a. Historical background

b. Structure and distribution of xylan

c. Xylanases and their application

3. Material and methods

a. Microorganisms

b. Chemicals

c. Effect of temperature and pH

d. Estimation of xylanase activity

e. Product analysis by gas chromatography

f. Scanning electron microscopy

4. Results and Discussion

5. Conclusion and future prospects

6. References

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 Introduction
Due to the enormous advancement in the field of chemical technology where the manufacture of
a variety of products on a large scale has resulted in serious effluent and hazardous waste
disposal problem, thus necessitating a need for safer and environment friendly technologies. This
has resulted in scientists across the world attempting to learn from natural processes and
therefore a new aspect of microbiology and biotechnology has rapidly begun to gain ground.
Microorganisms perform their myriads of biochemical reactions under ambient conditions with
little or no toxic and hazardous by-products therefore, enzymes secreted by these
microorganisms are now being employed as an alternative to the polluting chemical technologies
in various areas, one of which is pulp and paper industry where the quantities of raw materials
processed are huge, as well as the use of naturally hazardous chemicals are also very large.

Wood is one of the most abundant natural resources in the bio-based industry and yet it is one of
the most complex materials, composed of lignin and carbohydrates that are physically and
chemically bound together. Major components of wood are:

1. Cellulose: Comprising nearly half of the woody plant composition by weight, cellulose is
a carbohydrate polymer composed of glucose chains.

2. Hemicellulose: Hemicellulose is composed of carbohydrates based on pentose sugars,

mainly xylose, as well as hexose sugars, such as glucose and mannose. Hemicelluloses
comprise 25-30% of the dry weight of wood.

3. Lignin: Lignin is a phenylpropane polymer that holds together cellulose and

hemicelluloses components of wood. It constitutes 15-25% of the dry weight of wood.

Pulping of wood primarily aimed at removal of lignin, converts wood to a reduced fibrous mass.
Pulping can be achieved either by chemical, thermal, mechanical or by combination of these
treatments. Pulping using organic solvents in aqueous solution known as organosolv is widely
used process. Chlorine utilized in the traditional bleaching sequences reacts with lignin and
forms soluble chlorlignins in the basic medium. The low molecular weight-compounds (AOX)
formed in high amounts own toxic mutagenic or carcinogenic character. Environmental laws

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have put restrictions in use of chlorine in bleaching process and hence hydrogen peroxide is used
as oxygen-based bleaching chemicals in the paper and pulp industry.

Enzymes have shown to be a biotechnological alternative in bleaching of pulps, used together

with conventional chemical agents. A number of microorganisms that live on decaying plant
matter produce enzymes those break down hemicelluloses and in some cases lignin. It should be
possible to use enzymes from such organisms to develop appropriate biocatalysts for use in the
major phases (pulping and bleaching) of paper manufacture, in other words to substitute enzymes
for chemicals in what can be described as biopulping and biobleaching processes.

Biopulping - Paper is made from cellulose fibres, which must be separated from a tough wood
fibre called lignin. The step by step process used to separate cellulose from lignin and other
wood components is known as pulping. It is a time and energy consuming process, involving the
mechanical processing of wood or the treatment of wood with harsh chemicals. In biopulping,
cellulase and xylanase enzymes made by lignin-degrading fungi are used to pre-treat wood and
break down the lignin fibres. Removing lignin prior to further wood pulping saves time and
energy, and decreases the quantities of chemicals used. So, far two enzyme based approches
have been investigated. One is the use of hemicellulases and another is the use of lignolytic
enzymes.

Hemicellulase
Hemicellulase, especially xylanases are responsible for removal of hemicellulosic layer of plant
cell wall. Thus they cause loosening of cell wall hence facilitate the removal of lignin. The
second one is somewhat direct approach and cause degradation of lignin layer of plant cell wall.
Enzymatic hydrolysis of xylan renders the structure of the fibre more permeable, allowing lignin
and lignin carbohydrates to diffuse more easily into the bleaching liquor. An alternative
explanation is that xylanases attack the bonds that exists between xylan and lignin release lignin,
which can then diffuse more easily into the bleaching powder. Xylanase aided bleaching
decreases the consumption of bleaching chemicals and the release of adsorbale organic halides
(AOX) and total organic halides (TOX) in bleach plant effluent. Therefore, it is an “Ecofriendly
process”. Microbial xylanases are preferred catalysts for xylan hydrolysis due to their high
specificity, mild reaction conditions, and negligible substrate loss and side product generation.

Lignolytic enzymes
These enzymes, unlike xylanases attack lignin directly. White-rot fungi are the main producers
of lignolytic enzymes. These fungi secrete a number of oxidative enzymes and unknown
mediators into their environmental together effecting a slow but continuous degradation. The
enzymatic treatments based on xylanase cause modification in the carbohydrate structures
leading to enhanced delignification. Thus the removal of lignin from the pulp, without affecting
much of the cell wall cellulose, not only improves the discoloration characteristics of the

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resultant pulp but also yields better quality paper with increased brightness by decreasing the
overall viscosity of the pulp and kappa number (measure of the residual lignin content).

A pre-requisite, the paper and pulp industry, is the use of xylanases that are active and stable at
high temperature and alkaline pH due to prevailing conditions in pulp and paper industry.
Furthermore, the enzymatic processes also require cellulose free xylanases in order to generate
superior quality dissolving pulps by causing little or no damage to fibres.

With this view in mind, this project was designed with the following objectives:

1. Isolation of xylanase producing strains from forest soil

2. Selection of strain with maximum enzyme activity

3. Time course study for enzyme production

4. Evaluation of best fermentation conditions for enzyme production

a. Temperature

b. pH

5. Product analysis by gas chromatography

6. Morphological studies by scanning electron microscope

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 Literature Review
Historical background
Man’s use of enzymes dates back to the earliest times of civilization. Important human activities
in the primitive communities such as production of certain types of food, beverages, tanning of
hides and skins to produce leather, involved application enzymes activities. However, not until
the 19th century with the development of biochemistry and the pioneering work of a number of
scientists did the nature of enzymes and how they work begin to be clarified. During 20 th century
the recognition that enzymes are proteins along with the design of techniques for their
purification and analysis, principally the work of James B. Sumner and Kaj Linderstrom-Lang,
paved the way for development of procedures for their industrial production and use.

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Enzymes are found in naturally occurring microorganisms, such as fungi, bacteria, and yeast all
of which may or may not be genetically modified. Large quantities of enzymes are often needed
for industrial use, so microorganisms are multiplied through a process called fermentation. With
help of various techniques of biotechnology these naturally occurring enzymes can be modified
for desired results. Today wide array of enzymes are being used for various purposes in different
industries. Carbohydrate active enzymes are used not in food industry but also in paper and pulp,
textile, detergents, etc.

Historically enzymes have found some uses in paper industry, but these have been mainly
confined to areas such as modifications of raw starch. However, a wide range of applications in
the paper and pulp industry has grown rapidly since 1980s. While many applications of enzymes
in the paper and pulp industry are still in the research and development stage, several
applications have found their way into the mills in an unprecedented short period of time.
Interest in hemicellulolytic enzymes has increased remarkably during recent years. This is
mainly due to the new areas of application of these enzymes within the pulp and paper industry.
Among these, the most promising seems to be utilization of hemicellulases, especially xylanases,
to increase the bleachability of kraft pulps. This is partly due to the great potential of an
environmentally safe method. The main enzymes needed in the enzyme-aided bleaching have
been shown to belong to the group of endo-β-xylanases. Xylanases act mainly on the relocated,
reprecipitated xylan on the surface of the pulp fibers. Enzymatic hydrolysis of this specific type
of xylan renders the structure of the fibers more permeable, allowing enhanced extraction of
residual lignin from the fibers. The hydrolysis of hemicelluloses in the inner fiber layers may
also enhance the bleachability. The main goals in the enzyme-aided bleaching of kraft pulps have
been the reduction of consumption of chlorine chemicals in the bleaching process, and
consequent lowering of the AOX of the effluents. In the production of totally chlorine-free pulps,
enzymes have also been successfully used for increasing the brightness of pulp. Other suggested
enzymatic modifications of fibers are aimed at improved drainage (water removal) in the paper
machine, improvement of fiber properties or production of dissolving pulps.

Xylanases are produced by a plethora of microorganisms like bacteria, fungi, algae, protozoa,
arthropods. Most of the bacteria and fungi secrete extracellular xylanases which act on the
hemicellulosic material to liberate xylose as a directly assimiable end product allowing the
organisms to grow on xyaln. However unlike xylanase, β -xylosidase may be extracellular or
cell bound depending on the organisms and the cultivation conditions. In almost all bacteria and
yeats, β -xylosidase is cell associated (Godden et al) whereas some fungi secrete enzyme in the
extracellular medium (Wong et al).

Chemical structure and Distribution of Xylan in plant biomass

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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 hemicelluloses representing 20-35% of the total lignocellulosic
biomass (de Vries and Visser, 2001). The major hemicellulose in cereals is and hardwood is
xylan, while the main hemicellulose in softwood 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).

Xylan is a major structural component of plant cell wall 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 softwood from gymnosperms, accounting
for approximately 15-30% and 7-12% of their 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. Xylan interacts with
lignin and cellulose via covalent and non-covalent linkages, these interactions being of
importance for both protecting cellulose microfibrils against biodegradation and maintaining
structural integrity of cell walls.

Xylan is a complex polysaccharide composed of backbone of β -1,4-linked xylopyranosyl

residues that depending on plant source can be variably substituted by side chains of arabinosyl,
glucuronosyl, methyglucuronosyl, acetyl, feruloyl and p-coumaroyl residues. Although
homoxylan have been found in some plants and seaweeds, in the latter case also containing
xylose β -1,3- linkages, xylans consisting exclusively xylose residues are not widespread in
nature (Beg et al, 2001). Xylan from most plant sources occurs as hetereopolysaccharide and the
terms glucuronoxylan and glucuronoarabinoxylan are commonly used to describe xylan from
hardwood and softwood respectively. These two types of xylan have 4-O-methyl α -D-
glucuronic acid residues attached to C-2 of xylose backbone. 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 α -L-arabinofuranose
on C-3 of approximately 13% of the xylose backbone residues (Coughlan and Hazelwood,
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 acetylated, this substitution occurring on around 70%
of the xylose units at C-2, C-3 or both (Coughlan and Hazelwood, 1993). The presence of acetyl
groups maikes xylan significantly more soluble in water (Biely, 1985). Xylan from grasses is
usually referred 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 acetyleated and also has glucuronic acid
present, albeit at a lower content compared to hardwoods. Feruloyl and coumaroyl residues are
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

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molecules. As a consequence of thes 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).

Enzymatic degradation of xylan

Due to its heterogeneity and complex nature, the complete breakdown of xylan requires the
action of a large variety of hydrolytic enzymes (Biley, 1985; Coughlan and Hazelwood, 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 (β -1,4 Endoxylanase) and β -xylosidases that release xylose
units from xylobiose and xylooligomers. Removal of xylan side chains is catalysed by α -L-
arabinofuranosidases, α -D-glucuronidases, acetyl xylan esterases, ferulic acid esterases and p-
coumaric acid esterases (Fig1). Xylan degradation is quite widespread among saprpphytic
microorganism, 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). Synergims between xylan degrading enzymes has been extensively studied and found to
frequently occur between and also in between different xylanases (Coughlan et al, 1993; deVries
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.

Main chain cleaving enzymes

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1. Endoxylanases (endo-β -1,4-xylanase EC3.2.1.8): cleave the xylan backbone into
smaller oligosaccharides. They are the key enzymes for xylan degradation and differ in
their specificites toward the xylan polymer. Many cleave only at unsubstituted regions
whereas others have a requirement for side chains in the vicinity of the cleaved bonds
(Coughlan and Hazelwood, 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 hydrolyze internal linkages in
xylan and act by a random attack mechanism yielding a mixture of xylooligosaccharises
from the polymer. As xylan is a large polymer that cannot penetrate into cells, xylanases
have to be secreted into the extracellular environment to reach and hydrolyze it.
Generally, xylanases are induced in most microorganisms during their growth on
substrates containing xylan. Many xylan degrading microorganism produce a multiplicity
of xylanases with different but overlapping specificities (Wong and Saddler, 1988).
Multiplicity of xylanases can arise from different post-translational processing of same
gene product (Ruiz-Arribas et al, 1995). These may have diverse physiochemical
properties, structures, specific activities and yields, as well as overlapping but dissimilar
specificities, thereby increasing the efficiency and extent of hydrolysis and also the
diversity and complexity of the enzymes. Typical examples of microorganism which
produce xylanase isoenzymes include Aspergillus.niger, which produces fifteen extra
cellular xylanases, and Trichoderma viride which secretes thirteen.

2. β -xylosidase(EC3.2.1.37): As mentioned above, efficient degradation of xylan requires

not only the action of xylanases but also the cooperation of other enzymes such as β -
xylosidase and chain degrading enzymes. β -xylosidase (EC3.2.1.37) hydrolyze
xylobiose and short chan xylooligosaccharides generated by the action of xylanases,
releasing xylose from the non-reducing end. The affinity of β -xylosidases for
oligosaccharides decreases with the increasing degree of polymerization of the latter.
These enzymes do not usually hydrolyze xylan but they can hydrolyze artificial substrates
such as p-nitrophenyl- β -D-xylopyroanoside which is frequently used as substrate for
β -xylosidase activity assay.

3. α -L-arabinofuranosidases: are exo-acting enzymes that catalyze the cleavage of

terminal arabinose residues from the side chains of xylan and other arabinose-containing
polysaccharides (Saha, 2000). The apparent release of arabinose by some xylanses gave
rise to a classification of xylanases into debranching and non-debranching enzymes
depending on whether or not they produced free arabinose in addition to cleaving the
xylan backbone (Matte and Forsberg, 1992).

4. α -D-glucuronidases: these enzymes (EC 3.2.1.131) hydrolyze the linkages between 4-

O-methylglucuronic acid and xylose residues in glucuronoxylan, and are found
exclusively in glycoside hydrolase family 67.
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5. Acetyl xylan esterases: Acetyl xylan esterase (EC 3.1.1.72) removes the acetyl groups
from acetylated xylan. These enzymes are a late discovery due to the lack of appropriate
substrates. Although xylans can be highly acetylated, most of the substrates used to study
enzymatic degradation were obtained by alkali extraction, a method that tends to strip the
acetyl groups from xylan (Sunna and Antranikain, 1997). Acetyl xylan esterases play an
important role in the hydrolysis of xylan since acetyl groups can hinder the approach of
enzymes that cleave the xylan backbone; hence removal of these substituents facilitates
the action of xylanases (Coughlan et al, 1993).

6. Hydroxycinnamic acid esterases: Ferulic acid and p-coumaric acid esterases cleave the
ester bonds between arabinose side chains and feruloyl or p-coumaroyl residues
respectively (Williamson et al, 1998). These residues can cross-link xylan molecules to
eachother or to lignin.

Xylanase classification

First of all Wong et al (1998), classified xylanases on the basis of their physiochemical
properties and proposed two groups: one with a low molecular weight (<30kDa) and
basic pI, second with a high molecular weight (>30kDa) and acidic pI. Later on a more
complete classification system was introduced which allowed the classification of not
only xylanases, but also glycosidases in general. This system is based on primary
structure comparison and sequence similarities of catalytic domains only (Henrissat and
Coutinho, 2001).

Within this classification system initially xylanses were confined to families 10 (formerly
F) and 11 (formerly G). Table1 gives the major differences between xylanases from two
families.

Table1: Differences between xylanases of family 10 and 11

Family 10 Family 11

1. This family consists of endo- This family consists of soley

1,4-β -xylanases and of endo-1,4-β -xylanases
cellobiohydrolases

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 2. Members of this family are Member of this family are
not entirely specific for monospecific, they are true
xylan xylanases and exclusively
active on D-xylose
containing substrastes

3. This family contains high This family contains low

molecular weight xylanases molecular weight xylanases
(>30kDa) (<30kDa)

4. Xylanases of this family are Xylanases of this family are

with low isoelectric point with high isoelectric point

Mode of action

1. Endoxylanases: The mode of action of endoxylanases has been extensively studied

(Coughlan and Hazelwood, 1993; Biely et al, 1997). In general, these enzymes cleave
the internal β -1,4 linkages of the xylan backbone and release mainly xylobiose,
xylotrisoe and unsubstituted oligomers having two or four residues. It has been
reported that most endoxylanases cleave the xylan backbone leaving the substituent at
the non-reducing end of the xylosyl chain or the oligosaccharide (Dekker, 1985).
Xylanase has two proximal carboxylate, GLU172 and GLU78, which act as an acid
catalyst and nuleophile respectively. The rate of hydrolysis of xylooligomers by
xylanase enzyme with chain length (Ananad and Vithayathil, 1996). There is
involvement of double displacement mechanism for the anomeric retention of the
product. This mechanism includes the following features:

a. An acid catalyst protonates the substrate

b. A carboxyl group of the enzyme positioned on a covalent glycosyl enzyme

intermediate with this carboxylate in which the anomeric configuration of the
sugar is opposite to that of the substrate.

c. This covalent intermediate is reached from both directions through transition

states involving oxo-carbonium ions.

d. Various non-covalent interactions providing most of the rate enhancements.

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 2. Acetyl xylan esterases: Acetyl xylan esterases deacetylate partially acetylaed- 4-O-
methyl-D-glucuronxylan, or the fragments generated upon action of β -1,4-
endoxylanases.

Microbial sources of xylanases

A wide variety of microogranims including fungi, bacteria, actinomycetes, algae, protozoa and
yeast are known for their xylanase producing ability. Although xylanase producing ability is
reported higher in bacteria and yeast but fungi are better producers because their enzyme level is
higher as compared to yeast and bacteria. They secrete these enzymes into the medium and
fungal xylanases are multifunctional as compared to bacteria that produce monofunctional
xylanases. However, fungal xylanases are associated with cellulose that is one drawback with
fungal xylanases. Selective production of xylanase is possible using only xylan as carbon source.
Some of the sources are: Aspergillus fischeri, Aspergillus niger, Fusarium oxysporium,
Trichoderma reesi, Trichosporon cutaneum SL409, Bacillus amyloliquefaciens,
Thermoanaerobacterium sp, Streptomyces avermitilis UAH30.

Regulation of xylanase synthesis

Xylanase production by various bacteria and fungi has been shown to be inducible. But rare
examples of constitutive xylanase expression have also been reported. In general the xylanase
induction is a complex phenomenon and the level of response to an individual inducer varies

16
with the organism. An inducer producing maximum xylanase activity in one species may be the
inhibitor of activity in another species. The subsrate derivaties and the enzymatic end products
may often play a key positive role in the induction of xylanases; they can also act as the end
product inhibitors, possibly at much higher concentrations. Xylan, being a high molecular weight
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 (Biely et al, 1998). The repression of xylanase production due to increasing
soluble products in the media has been reported in a number of organisms. The monosaccharides
originating by xylan decomposition inhibit the production of xylanase. It has been seen that at
high substrate concentration, initial rapid increase in activity is followed by a steady state decline
due to the repressing effect of accumulated low molecular weight oligosaccharides (Coughlans
and Hazelwood, 1993). Many environmental factors in addition to the culture medium affect the
growth and production of xylanase by fungi. In addition to the substrate, medium composition
also affect the xylanase production, which include mineral salts e.g. KH2PO4 ,MgSO4.

Effect of Temperature

The optimum temperature for endoxylanases from bacterial and fungal sources varies between
40°C to 60°C. Fungal xylanases are thermostable than bacterial xylanases. By applying the
temperature shift during a laboratory cultivation xylanase activity could almost be doubled
whereas the xylanase/cellulose ration was threefold higher in comparison to cultivation at a
constant temperature of 28°C. Some xylanases have been reported to exhibit higher thermal
stability and optimal ability ranging from 80°C to 100°C.

Effect of pH

An important environmental factor which significantly affects the production of xylanases is the
pH-value during cultivation. It has been shown for several fungi that cultivating the organism at
unfavorable pH, a condition which may favor limited growth rate, results in increased yields of
xylanase (Denison S). The importance of the pH-value for the production of xylanase by
A.awamori has also been pointed out in another publication (Smith and Wood, 1991). Acid pH-
values favored xylanase production with this organism, while pH 5.0 and higher caused a
dramatic reduction in enzyme production. The reason for poor production at higher pH was
probably proteolytic inactivation of the xylanase. Recently thermostable and alkaline tolerant
cellulose free xylanases have been produced from agricultural wastes which have the desired
properties for use in pulp bleaching (Charin Techapun, 2003).

Applications of xylanolytic enzymes

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Microbial hemicellulases, especially xylanases, have important applications in industry due to
their enormous potential to modify and transform the lignocellulos and cell wall materials
abundant in vegetal biomass which is used in a wide variety of industrial processes. The
biotechnological applications of xylanases began in the 1980s in the preparation of animal feed,
and later expanded to the food, textile and paper industries. At present, xylanases together with
cellulases and pectinases account for 20% of the global industrial enzyme market (Polizeli et al,
2005).

1. Bioconversions

Xylanases are gaining importance fot the bioconversion of lignocellulosic biomass to

xylose and other fermetable sugars for the production of biological fuels (ethanol) (Lee,
1997). Bioconversion of xylan to the low calorie sweetner xylitol is a promising field
where xylanases can play a key role (Polizeli et al, 2005).

2. Textile

Another potential application of cellulose free xylnolytic systems is in processing of plant

fiber sources such as flax and hemp. Xylanases are used in retting of jute, flax and hemp
the process, which involves removal of binding, material present in plant tissues.

3. Feed

Xylanases are used as additives in animal feeds for monogastric animals, together with
cellulases, pectinases and many other depolymerizing enzymes. Enzyme degradation of
arabinoxylans, commonly used ingredients of feeds, reduces the viscosity of the raw
materials this facilitating better mobility and absorption of other components of the feed
and improving nutritional value (Polizeli et al, 2005). The incorporation of xylanae into
the rye-or-wheat-based diets of broiler chickens resulted in an improvement in weight
gain of chicks and their feed conversion efficiency (Bedford and Classen, 1992).

4. Food and juice industry

The application of xylanases along with pectinases in the juice and wine industries
facilitates the extraction and clarification of the final products (Bhat, 2000). These
enzymes can also increase the stability of fruit pulp and release aroma precursors. As
regards the latter, a recombinant yeast strain expressing fungal xylanase produced a wine
with increased fruity aroma (Ganga et al, 1999). As baking additives, xylanases degrade
four hemicelluloses resulting in a redistribution of water from pentosans to gluten, thus
giving rise to an increase in bread volume and crumb quality, and an antistailing effect
(Linko et al, 1997).

5. Paper and pulp industry

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The major current industrial application of xylanases is in the pulp and paper industry
where xylanase pretreatment facilitates chemical bleaching of pulps, resulting in
important economic and environmental advantages over the non-enzymatic process
(Viikari et al, 1994; Bajpai, 2004). Degradation of xylan in xylan-lignin complexes or
reprecipitated on the surface of fibers after Kraft cooking, allows a more efficient
extraction of lignin by the bleaching chemicals. Xylanase-boosted bleaching results in
upt0 20-25% savings on chlorine-based chemicals and a reduction of 15-20% in the
generation of pollutant organic chlorine compounds from lignin degradation (absorbable
organic halogens, AOX) (Viikari et al, 1994; Bajpai, 2004).

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