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Xylanase is an industrial enzyme having applications in a wide range of processes. It is a glycoside hydrolase that catalyzes the
hydrolysis of xylan producing mainly xylobiose, xylotriose, and a small fraction of xylooligosacharides with higher degree of
polymerization. The present investigation aims at isolation of a potent xylanolytic bacterial strain and study of the production,
purification, characterization, immobilization and application of the xylanase secreted by this strain. Several reviews have been
published on xylanases (Gilbert and Hazlewood, 1993; Bajpai et al., 1994; Wong et al., 1988; Srinivasan and Rele, 1999; Bhat, 2000;
Bajpai, 2004; Beg et al., 2001; Subramaniyan and Prema, 2002; Sá-Pereira et al., 2003; Collins et al., 2005; Pollizeli et al., 2005;
Dhiman et al., 2008b; Juturu and Wu, 2011; Kuhad and Singh, 1993). This chapter presents a brief review of the literature pertaining
mainly to isolation, production, purification and characterization, immobilization, and application of bacterial xylanases.
2.1 Chemical structure and distribution of xylan (the substrate for xylanase)
Xylan is the substrate for xylanase. It a major component of hemicelluloses present in plant cell walls and is the second most
abundant polysaccharide next to cellulose in nature, accounting for approximately one-third of all renewable organic carbon on earth
(Biely, 1985; Prade, 1995). The term hemicellulose refers to a group of non-cellulosic polysaccharides which include xylan,
xyloglucan (a heteropolymer of D-xylose and D-glucose), glucomannan (a heteropolymer of D-mannose and D-glucose),
galactoglucomannan (a heteropolymer of D-galactose, D-glucose and D-mannose), arabinogalactans (a heteropolymer of D-galactose
and arabinose) (Shallom and Shoham, 2003). In plant cell wall, hemicelluloses occur in association with cellulose and lignin
constituting lignocellulose which on an average contains 35-45% cellulose, 20-30% hemicellulose and 8-15% lignin (a polymer of
phenylpropanoid residues). β-1,4-Xylans are mainly found in secondary walls, the major component of mature cell walls in woody
tissue. Although they also represent the major hemicellulose in the primary walls of monocots, xylans generally constitute a minor
component of the primary walls in dicots. Xylan functions as an adhesive by forming covalent and non-covalent bonds with lignin,
cellulose and other polymers essential to the integrity of the cell wall. Xylan is considered to form an interface between lignin and
cellulose (Kulkarni et al., 1999; Beg et al., 2001). The phenolic residues of lignin are linked to xylan by an ester linkage to 4-O-
methyl-D-glucuronic acid residues. Lignocellulosic materials in the form of agro-residues can serve as excellent substrate for inducing
xylanase production. Xylan is found in large quantities in hardwoods from angiosperms, softwoods from gymnosperms and annual
plants constituting 15–30%, 7–10% and <30% of the total dry weight, respectively (Whistler and Richards, 1970; Singh et al., 2003).
Xylan is a highly branched heteropolysaccharide having β-1,4-linked xylopyranosyl residues forming a backbone which is
substituted to varying degrees with α-L-arabinofuranosyl residues, D-glucuropyranosyl or its methyl derivative 4-O-methyl-D-
glucuropyranosyl residues (at C-2 of a xylosyl residue in the main chain), acetyl groups (at either C-2 or C-3 but often at C-3),
feruloyl and/or p-coumaroyl. The hardwood and softwood xylans differ in their structure, degree of polymerization and degree of
branching. The hardwood xylans contain 150-200 xylopyranosyl residues, 70% of which are acetylated (for instance, birchwood xylan
contains more than one mol of acetic acid per two mol of xylose) and are more branched. In contrast, softwood xylans contain 70-130
xylopyranosyl residues and are not acetylated and less branched. The softwood xylans have, instead of acetyl groups, α-L-
arabinofuranosyl residues (either as single residues or as short side chains) attached to C-3 of a xylopyranosyl residue in the xylan
backbone via α-1,3-glycosidic bond (Puls and Schuseil, 1993). The arabinosyl substituents occur on almost 12% of the xylosyl
residues (Wong et al., 1988). Wood xylan exists as O-acetyl-4-O-methylglucuronoxylan in hardwoods and as arabino-4-O-
methylglucuronoxylan in softwoods, while xylans in grasses and annual plants are typically arabinoxylans (Kulkarni et al., 1999). A
schematic representation of the structure of xylan from hardwood and softwood is shown in Fig. 2.1.
(A)
-------------------β(1 4)D-Xyl-β(1 4)D-Xyl-β(1 4)D-Xyl-β(1 4)D-Xyl---------------------
2 3(2)
1 1
4-O-Me-α-D-GlcA- OAc
(B)
--------------------β(1 4)D-Xyl3-β(1 4)D-Xyl-β(1 4)D-Xyl7-β(1 4)D-Xyl-----
2 3
1 1
(4-O-Me-α-D-GlcA)3 α-L-Arabinose
Fig. 2.1 Schematic representation of xylan structure: A) Softwood xylan and (B) Hardwood xylan (Woodward, 1984)
Xyl= xylopyranose residue; Me= Methyl group; GluA=Glucuronic acid residue; Arab= L-Arabinofuranosyl residue,
and Ac=Acetyl group
B. licheniformis Wheat bran Corn steep liquor 50 7.0 756 Archana and
Satyanarayana, 1998
B. mojavensis A-21 Barley bran NH4Cl 34.08 30 8.0 7.5 Haddar et al., 2012
B. mojavensis Oat bran Peptone, Yeast 48 37 8.0 302.46 Sepahy et al., 2011
AG137 extract
B. pumilus Oat spelt xylan Peptone, Yeast 96 35 9.0 580 Poorna and Prema,
Wheat bran extract 72 430 2006
B. pumilus ASH Wheat bran Peptone, Yeast 26 37 8.0 5407 Battan et al., 2007
extract, KNO3
B. pumilus B20 Wheat bran, Peptone, Yeast 36 37 7.5 313 Geetha and
extract Gunasekaran, 2010
B. pumilus MK001 Wheat bran Peptone, Yeast 48 37 9.0 1220 Kapoor et al., 2008
extract
B. pumilus SV-85S Wheat bran Peptone, Yeast 48 37 6.0 2900 Nagar et al., 2010b
extract, KNO3
B. pumilus SV 34S Wheat bran Beef extract, 48 37 7.0 3454 Mittal et al., 2013
(NH4)2H2PO4
Bacillus sp. Wheat bran Peptone 48-60 50-55 8.0 4.0 Sharma et al., 2011
Bacillus sp. AG20 Wheat bran - 42-48 35 8.5 2.4-3.7 Azeri et al., 2010
Bacillus sp. SSP-34 Oat spelt xylan Yeast extract and 96 35 8.5 380 Subramaniyan et al.,
peptone 2001
Bacillus sp. V1-4 Birchwood Corn steep liquid 48 37 10 49 Yang et al., 1995
xylan
B. subtilis Oat spelt xylan 18 50 6.0 12 Sa-Pereira et al.,
2002
B. subtilis Oat spelt xylan Peptone, Yeast 36 55 9.0 128 Annamalai et al.,
extract 2009
B. subtilis ASH Wheat bran Peptone, Yeast 48 37 7.0 410 Sanghi et al., 2009
extract, KNO3
B. subtilis 276NS Xylan Yeast extract 24 37 8.0 360 Ali et al., 2013
Chromohalobacter Sugarcane Feather 140 40 9.0 250 Prakash et al., 2009
sp. TPSV 101 bagasse hydrolyzate
Flavobacterium sp. Larchwood Yeast extract, 30 6.8 6.64 Bhatt et al., 1994
xylan Peptone, Casein
hydrolyzate and
Nitrates
Geobacillus Wheat bran Tryptone 72 70 7.5 12.95 Sharma et al., 2007
thermoleovorans
Gracilibacillus sp. Birchwood Yeast extract and 60 30 7.5 18.44 Giridhar and
TSCPVG xylan Tryptone Chandra, 2010
Paenibacillus sp. Xylose (NH4)2HPO4 72 50 9.0 52.30 Pathania et al., 2012
N1
Table 2.2 Production of xylanases from bacteria in solid state fermentation (SSF)
Bacillus sp. GRE7 Wheat bran - 55 °C 7.0 3,950 Kiddinamoorthy et al., 2008
Bacillus sp. JB-99 Rice bran Yeast extract, 72 50°C 10.0 3,644 Virupakshi et al., 2005
beef extract
B. stearothermophilus Wheat bran - 8.0 3,446 Dhiman et al., 2008a
SDX
B. subtilis ASH Wheat bran Yeast extract 72 37°C 7.0 8,964 Sanghi et al., 2008
Gessesse (1998) purified two xylanases (Xyl-A and Xyl-B) from the alkaliphilic Bacillus sp. strain AR-009 using the DEAE-
Sepharose column chromatography. Their molecular mass by SDS-PAGE was found to be 23 and 48 kDa, respectively.
Ratanakhanokchai et al. (1999) purified an extracellular xylanases to homogeneity by affinity adsorption/desorption on insoluble
xylan from Bacillus sp. strain K-1. The molecular mass of purified xylanase was approximately 23 kDa. Metal ions such as Fe+2, Ca+2,
and Mg+2 increased the xylanase activity, whereas Mn+2 strongly inhibited it. Archana and Satyanarayana (2003) purified a xylanase
from B. licheniforms A99 by 14.4- fold with 38% yield using ammonium sulfate precipitation and ion-exchange chromatography
through DEAE-Sephadex A-50.
Wang et al. (2009) purified a xylanase by fast protein liquid chromatography (FPLC) and had a molecular mass of 24 kDa. The
enzyme was active over a concentration range of 0–20% sodium chloride in culture broth, although its activity was optimal in 5%
sodium chloride. Xylanase activity was maximal at pH 8.0 and 40°C. The xylanase had Km 3.45 mg/ml and Vmax 387 μmol/min/mg.
The deduced internal amino acid sequence of Bacillus sp. NTU-06 xylanase resembled the sequence of β-1,4-endoxylanase, which is a
member of glycoside hydrolase family 11. Monisa et al. (2009) partially purified a xylanase from B. pumilus by 3.79-fold with 66%
recovery using ammonium sulfate precipitation. Partially purified enzyme preparation exhibited a specific activity of 0.69
μM/min/mg, Km 4.0 mg/ml, Vmax 0.068 × 10-4 mM/min/mg and molecular mass 19 kDa.
Sanghi et al. (2010) purified an extracellular cellulase-free xylanase to homogeneity from B. subtilis ASH by 10.5-fold with
43% recovery in a single step using CM-Sephadex C-50 column chromatography. It showed an optimum pH at 7.0 and was stable
over the pH range 6.0-9.0. The optimum temperature of the enzyme was 55 °C. The purified enzyme revealed a single band on SDS-
PAGE gel with a molecular mass of 23 kDa. The Km and Vmax of the enzyme for birch wood xylan were found to be 3.33 mg/ml and
100 IU/ml, respectively. The purified enzyme was stable for six weeks at 4°C and lost 20% activity after 10 weeks. The enzyme
activity was strongly inhibited by Hg2+ and Cu2+ but stimulated by Co2+ and Mn2+.
Shrinivas et al. (2010) purified a highly thermostable xylanase from Bacillus sp. JB 99 to 25.7-fold and 43.5% recovery using
two step purification strategy involving chromatography through DEAE-Sepharose and Sephadex G-100. The purified enzyme
exhibited a molecular mass of 20 kDa, Km 4.8 mg/ml and Vmax 218.6 μM/min/mg for oat spelt xylan.
Menon et al. (2010) used (NH4)2SO4 fractionation, DEAE-Cellulose, and Sephadex-G-200 chromatography to purify xylanase
from B. pumilus strain, GESF1 resulting in 21.21-fold purification with a specific activity 112.42 U/mg protein, Km 5.3 mg/ml and
Vmax 0.42 μmol/min/ml. Prakash et al. (2009) reported the partial purification of a xylanase from Chromohalobacter sp. TPSV 101
using the protein concentrator. The partially purified enzyme had a molecular mass 15 kDa, optimum temperature 65°C and pH
optimum at 9.0.
Yin et al. (2010) reported the purification of xylanase from Bacillus sp. YJ6 using Sephacryl S-100 HR chromatography with
3.5% recovery and 678.1-fold purification. Xylanase had an optimal pH at 5.0 but was stable over the pH range 5.0-9.0. The enzyme
showed an optimum temperature of 50 ºC and was stable at temperatures less than 50 ºC. Xylanase activity was inhibited by Cu+2,
Fe+3, Hg+2, phenylmethyl sulfonyl fluoride (PMSF), N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), N-ethylmaleimide, and
leupeptin, but activated by K+, Na+, Co+2, Mg+2, β-mercaptoethanol, and glutathione. The purified xylanase had high specificity for
beechwood, birchwood, and oat spelt xylans. The DNA fragment encoding this xylanase, corresponding to 213 amino acids, exhibited
about 95% homology with seven strains of Bacillus in the NCBI database. The purified enzyme had a molecular mass of 19 kDa and
specific activity 1436.0 U/mg protein.
Bai et al. (2010) cloned the gene (xynA4) encoding xylanase from Alicyclobacillus sp. A4 and expressed it in E. coli. It
encoded a 338-amino acid residue polypeptide with a calculated molecular mass of 42.5 kDa. Amino acid sequence was similar to
(53% identity) an endoxylanase from Geobacillus stearothermophilus belonging to family 10 of the glycoside hydrolases.
Recombinant XynA4 exhibited maximum activity at 55°C and pH 7.0, had broad pH stability (retaining 80% activity after incubation
at pH 2.6–12.0 for 1 h at 37°C), and was highly thermostable retaining 90% activity after incubation at 60°C for 1 h.
Zhang et al. (2010a) cloned the xylanase gene (xyn10) from alkaliphilic Bacillus sp. N16-5 and expressed it in Pichia pastoris.
The deduced amino acid sequence had 85% identity with xylanase xyn10A from B. halodurans and contained two potential N-
glycosylation sites. The glycosylated Xyn10 with MW 48 kDa could hydrolyze birchwood and oatspelt xylans. The enzyme had
optimum activity at pH 7.0 and 70°C, with the specific activity of 92.5U/mg. The Xyn10 retained over 90% residual activity at 60°C
for 30 min but lost all activity at 80°C over 15 min.
Chi et al. (2012) purified an extracellular xylanase from the culture broth of Bacillus sp. MX47 using two chromatographic
steps. The xylanase had an apparent molecular mass of 26.4 kDa with an amino terminal sequence (Gln-Gly-Gly-Asn-Phe) distinct
from that of reported proteins, implying that it was a novel enzyme. The optimum pH and temperature for xylanase activity were 8.0
and 40 °C, respectively. The enzyme activity was severely inhibited by many divalent metal ions and EDTA at 5 mM. The xylanase
was highly specific for beechwood and oat spelt xylans, however, not active on carboxymethyl cellulose (CMC), avicel, pectin, and
starch. Analysis of the xylan hydrolysis products by Bacillus sp. MX47 xylanase indicated that it was an endo-β-1,4-xylanase. The Km
and Vmax values toward beechwood xylan were 3.24 mg/ml and 58.21 μmol/min/mg protein, respectively.
Nagar et al. (2012a) purified an extracellular xylanase from B. pumilus 85S to apparent homogeneity by 25.3-fold with 63.2%
recovery using (NH4)2SO4 fractionation and CM-Sephadex column chromatography. The enzyme purity was tested by polyacrylamide
gel electrophoresis and HPLC. The purified enzyme revealed a molecular mass of 23.6 kDa estimated by SDS-PAGE. The Km and
Vmax values of the purified xylanase were 1.0 mg/mL and 333.3 IU/mL, respectively.
Mittal et al. (2013) reported the purification of Bacillus sp. SV-34S xylanase by 12.94-fold with a recovery of 13.4% and a
specific activity of 3417.2 IU/mg protein employing (NH4)2SO4 fractionation followed CM-Sephadex C-50 column chromatography.
The purified enzyme showed a molecular mass of 27 kDa, optimum temperature 50°C, optimum pH at 6.5, Km 3.7 mg/mL, and Vmax
133.33 IU/mL with birchwood xylan as the substrate.
Sharma et al. (2013) purified a xylanase secreted from Paenibacillus macquariensis RC 1819 using (NH4)2SO4 fractionation,
ion exchange chromatography using DEAE-cellulose, and gel filtration chromatography over Sephadex G-200 and Sephadex G-100.
The purified enzyme had a specific activity of 25.2 units/mg protein, molecular mass 31kDa, optimum pH at 8.6, optimum
temperature 50°C, and Km 2.2 mg/ml for birchwood xylan. Metal ions such as Co+2 and Mn+2 stimulated whereas Hg+2 inhibited the
enzyme activity.
Verma et al. (2013) cloned the xylanase encoding gene (1,224 bp) from Geobacillus thermodenitrificans in pET28a (+) vector
and successfully expressed in E. coli BL21 (DE3). Xylanase was purified by Ni2+-affinity chromatography. The eluted protein
appeared as a single band on 15% SDS-PAGE. The deduced amino acid sequence analysis revealed homology with that of glycosyl
hydrolase family 10 with a high molecular mass (50 kDa). The purified recombinant xylanase was optimally active at pH 9.0 and 70
°C with half-life of 10 min at 80 °C, and retained greater than 85% activity after exposure to 70°C for 180 min. The xylanase was
quite stable in the presence of the detergents tested. The recombinant xylanase showed Km 0.625 mgml−1 and Vmax 555.5
μmol/mg/min for birchwood xylan.
Textile industry
The xylanolytic complex can be used in the textile industry to process plant fibres, such as of flax, hemp, jute, sisal and bast,
cotton and jute. For this purpose, the xylanase should be free of cellulolytic enzymes. Relatively little research has been done on the
enzymatic pretreatment of textile fibers, and yet this appears to be a promising market demanding the development of new techniques.
A bacterial xylanase was used in bioprocessing of fabrics (Dhiman et al., 2008b). Garg et al. (2012) evaluated the bioscouring of jute
fabric using the alkalo-thermostable xylanase from Bacillus pumilus ASH. An enzyme dose of 5 IU/g of oven dried jute fabric resulted
in release of more reducing sugar and weight loss as compared to control when incubated at 55º. An incubation time of 120 min was
sufficient to increase the whiteness and brightness of fabric up to 3.93 and 10.19%, respectively and to decrease the yellowness by
5.57%. Addition of chelating and wetting agent greatly enhanced the fabric properties. Bioscouring of jute fabric with xylanase
enzyme along with EDTA and Tween-20 resulted in an increase of 9.63, 4.28 and 10.71% of reducing sugars, whiteness and
brightness, respectively as compared to conventional process. Bleaching of bioscoured jute fabric, further improved the various
properties like tenacity, brightness, yellowness and whiteness.
Baking industry
Xylanases are also used in wheat flour for improving dough handling and quality of baked products (Chen and Li, 2010).
Enzymatic hydrolysis of non-starch polysaccharides leads to the improvement of Rheological properties of dough, bread specific
volume and crumb firmness. Xylanases break down the hemicellulose in wheat-flour, helping in the redistribution of water, increase
the bread volumes, delay crumb formation, improve resistance to fermentation, leaving the dough softer and easier to knead (Harbak
and Thygesen, 2002; Camacho and Aguilar, 2003). For application of xylanase in the baking industry, it must be active at
temperatures below 35 ºC (Juturu and Wu, 2011). The enzyme can even substitute the addition of different emulsifiers and other
chemical additives used in bread production (Butt et al., 2008). Bajaj and Manhas (2012) studied the application of xylanase in dough
prepared from wheat flour and observed that xylanase treatment of dough resulted in enhanced dough-raising as compared to the
control. Xylanase improves the bread quality with an increase in specific bread volume. Xylanases are used as additives in the baking
industry to increase the elasticity of the gluten network. Elasticity improves handling and stability of the dough. Thereafter, the
enzyme gets denatured and inactivated during bread baking. The addition of xylanases to wheat flour for bread making causes changes
in arabinoxylans resulting in more voluminous loaf. Several xylanases from bacterial and fungal sources are used in baking industries
(Pariza and Johnson, 2001).
Animal feed
Xylanases are used in animal feed industry along with others enzymes to break down arabinoxylans present in the feed,
reducing the viscosity of the raw material. The arabinoxylan found in the cell walls of grains have an antinutrient effect on poultry.
Xylanase is added to feed containing maize and sorghum (both of which are low viscosity foods) so as to improve the digestion of
nutrients in the initial part of the digestive tract, resulting in a better use of energy (Polizeli et al., 2005). Endoxylanase and cellulase
treatment of forages produces better quality silage that improves the subsequent rate of plant cell wall digestion by ruminants. As a
result of endo-1,4-β-xylanase treatment, there is increased nutritive sugar and that is useful for digestion in cow and other ruminants.
Thermostable xylanase would be beneficial in animal feeds if added to the feeds before the pelleting process (typically carried out at
70–95 ºC). In addition, for this latter application the enzyme must be highly active at the temperature (approximately 40 ºC) and pH
(approximately pH 4.8) of the digestive tract (Collin et al., 2005). Babalola et al. (2006) observed improved apparent nitrogen and
fiber absorption as well as feed transit time by the application of xylanase in poultry feed. Moreover, the enzyme addition in boiled
castor seed meal (up to 150g/kg) was found to be acceptable and showed no adverse effect on growth performance or blood
constituents. Nagar et al. (2012d) showed that the immobilized Bacillus pumilus SV-205 xylanase could enhance the digestibility of
poultry feed. Deswal et al. (2012) studied the application of thermostable and alkalostable xylanase from B. pumilus MK001 for
upgradation of chick feed. Xylan the main anti-nutritional factor present in the chick feed was removed to an extent of 11.6 mg/g
xylose sugars at 50°C using the thermostable xylanase. Besides, the treatment with thermostable xylanase also brought about a release
of 0.85 mg/g of soluble phosphorous.
Alltech, Inc, (USA) Fibrozyme A. niger & T. viride (SSF) Upgrading animal feed
Amano Pharmaceutical Amano 90 A. niger (SSF) Pharmaceutical, food and feed industry.
Co, Ltd (Japan)
A/S Resinase - Cellulose and paper industry
Ciba- Geiby Ltd Irgazyme 40 T. longibrachiatum (SmF) Pulp and paper industry, animal feed
(Switzerland)
Danishco Ingredients Grindazym PF A. niger Supplementation of poultry and piglet food
(Denmark) Grindazym GP
Gamma Chemie GmBH Gammafeed X T. longibrachiatum (SmF) Production of wheat starch, baking and
(Germany) Gammazym brewing industry, feed and brewing industry
X4000L T. reesei (SmF)
Gencor International Multifect XL T. longibrachiatum (SmF) Food industry
Europe Ltd (Finland)
Hankyo Bioindustry Xylanase 250 T. viride (SSF) Baking industry & for macerating vegetables
Co. Ltd (Japan) and fruits
Hemicellulose 100 A. niger Improving the filtration speed of saccharified
cereal solutions and fruit juices
Logen Corp (Canada) Xylanase GS35 T. reesei (SmF) Pulp bleaching, Pulp cleaning and animal
feed processing
Novozymes Bio-Feed-Plus Humicola insolens (SmF) Animal feed
(Denmark) Novozym 431 T. longibrachiatum (SmF) Animal feed
Pulpzyme Bacillus sp. Cellulose and paper industry
Primalco Ltd Biotec Ecopulp X-200 T. reesei (SmF) Improve the bleach ability of softwood and
(Finland) hardwood kraft pulps
Quest International Bioxylanase T. reesei (SmF) Brewing and animal feed industry
(Ireland)
Röhm GmbH Rohalasa 7118 Aspergillus sp. and Reduction of viscosity in starch processing
(Germany) Vernon 191 Trichoderma sp. (SmF) Baking industry
(Sources: Beg et al., 2001; Haltrich et al., 1996; Octavia Loera et al., 2006; Corral and Ortega, 2006 and Polizeli et al., 2005)