Indian Journal of Experimental Biology

Vol. 51, November 2013, pp. 875-884

Review Article

Production, purification, characterization and over-expression of xylanases from

actinomycetes
Leya Thomas*, Abhilash Joseph1, Muthu Arumugam & Ashok Pandey
Biotechnology Division, CSIR-National Institute for Interdisciplinary Science and Technology
(NIIST), Trivandrum 695 019, India
1
Department of Biotechnology & Microbiology, School of Life Science, Kannur University, Kannur 670 661, India

Xylanases are a group of depolymerizing enzymes often used for the hydrolysis of xylan (present in hemicellulose) to
monomeric sugars and comprise endo-xylanases (EC 3.2.1.8) and β-xylosidases (EC 3.2.1.37). They often act in synergy
with other enzymes for complete hydrolysis of hemicellulose. Xylanases find several industrial applications, for example in
food and feed industries, paper and pulp industries and more recently have acquired a great role in biomass to biofuels
program. Bacteria and fungi can best produce xylanases. Recent developments in rDNA technology have resulted in
molecular cloning and expression of xylanases in heterologous and homologous hosts. In view of significance of the
actinomycetes for the production of biotechnological products, attempts have been made in recent years to explore them for
the production of industrial enzymes, including xylanses, aiming to find the enzyme with novel features. This review
provides the state-of-art information and developments on the xylanases from actinomycetes, presenting the production,
purification, characterization and over-expression from various actinomycetes cultures.

Keywords: Actinomycetes, Biofuel, Biomass, Xylanase

Introduction low PI values, whereas family 11 xylanases have low

Xylan is the major constituent of hemicellulose and is
the second most abundant polysaccharide in the cell
walls of land plants, representing up to 30–35% of the
total dry weight.1,2 It is a heteropolymer composed of β-
1,4-linked xylopyranose units with branches containing
L-arabinofuranosyl and glucopyranosyl residues. The
xylanolytic enzyme system carrying out the xylan
hydrolysis is usually composed of a repertoire of
hydrolytic enzymes: β-1,4-endoxylanase, β-xylosidase,
α-L-arabinofuranosidase, α-glucuronidase, acetyl xylan
esterase, and phenolic acid (ferulic and p-coumaric acid)
esterase. All these enzymes act cooperatively to convert
xylan into its constituent sugars. The presence of such a
multifunctional xylanolytic enzyme system is quite
widespread among the fungi3,4, actinomycetes5, and
bacteria.6 Xylanases majorly fall into two glycosyl
hydrolase families, family 10 (formerly family F) and
family 11 (formerly family G) based on their amino acid
sequences and structural homologies. Family 10
xylanases have high molecular weight (>30kDa) with
_____________
*Correspondent author
Telephone: 0471-2515425
Fax: 0471-2491712;
E-mail: layam31@gmail.com
molecular weight (<30kDa) with high PI values7,8. As a
result of differential mRNA processing, partial
proteolysis or differences in the degree of amidation and
glycosylation multiple xylanases are found among many
species.
Biotechnological uses and potential applications of
xylanases include bioconversion of lignocellulose
material to fermentative products in the Kraft process
for the removal of the lignin-carbohydrate
complexes9-11, in the extraction and preparation of
beverages12; clarification of juices, detergents13,
generation of protoplast in plant cells14, production of
pharmacologically active polysaccharides for use as
antimicrobial agents or antioxidants15, production of
surfactants16, bioconversion of lignocellulosic
materials to fuels17, etc. Cellulase-free xylanases
active at high temperature and pH are important in
pulp and paper industry as alternatives to the use of
hazardous chlorinated compounds18,19. The treatment
with xylanases can improve the chemical extraction of
lignin from pulp20. This results in significant saving of
the chemicals required for bleaching, thereby
reducing the release of toxic chlorine compounds into
the environment. However, the main problem faced
by the pulp and paper industry is the lack of a suitable
876 INDIAN J EXP BIOL, NOVEMBER 2013
enzyme which is thermostable as well as highly
alkaline so as to act efficiently under the processing
conditions.
Microbes as the source of xylanases
Microorganisms are primarily responsible for xylan
degradation in the nature. Most of the xylanases
known to-date are from bacterial and fungal sources21.
Although fungi are considered as the most potent
producers of (extracellular) xylanase22, they have
often pH optima of about 5.0 for the hydrolysis of
xylan with a stability range of 2.0-9.0 and temperature
optima below 50 °C. Usually xylanases obtained from
fungal sources show high enzyme titres, a drawback,
however, often is the co-production of cellulases
along with them, which makes them un-suitable for
several applications such as paper and pulp industry.
Bacterial xylanases usually show higher pH optima
than that of fungal xylanases23, 24. Most of the
bacterial xylanases are active at alkaline pH and high
temperature. They could be cellulase-free, or
cellulase-poor as well as active at alkaline pH and
thermostable, which make them ideal candidates for
various industrial applications such as paper & pulp,
food and feed industries.
Actinomycetes  a versatile group of microbes
Actinomycetes are the organisms with
characteristics common to both bacteria and fungi but
yet possessing distinctive features to delimit them into
a distinct category. In the strict taxonomic sense,
actinomycetes are clubbed with the bacteria in the
same class of Schizomycetes and confined to the
order Actinomycetales. They are unicellular like
bacteria, but produce a mycelium which is non-
septate (coenocytic) and more slender. Like true
bacteria, they do not possess distinct cell-wall cell
wall but if the cell-wall is present, it is without chitin
and cellulose, which is common in fungi. On culture
media unlike slimy distinct colonies of true bacteria
which grow quickly, actinomycetes colonies grow
slowly, show powdery consistency and stick firmly to
agar surface. They produce hyphae and
conidia/sporangia like fungi. Certain actinomycetes
whose hyphae undergo segmentation resemble
bacteria, both morphologically and physiologically.
Actinomycetes are numerous and widely
distributed in soil25 and are next to bacteria in
abundance. They are widely distributed in the soil,
compost, etc. Plate count estimates give values
ranging from 104 108/soil. They are sensitive to
acidity/low pH (optimum pH range 6.5 to 8.0) and
waterlogged soil conditions. The population of
actinomycetes increases with the depth of soil even up
to horizon ‘C’ of a soil profiler. They are
heterotrophic, aerobic and mesophilic (25-30 °C)
organisms. Some species are commonly present in the
compost and manures growing at 55-65 °C (e.g.,
Thermoactinomycetes, Streptomyces).
Actinomycetes as source of industrial enzymes
Actinomycetes have been extensively exploited
commercially for antibacterial, antifungal,
antiparasitic and other bioactive compounds such as
immunosuppressants26 and more recently as a source
of industrial enzymes such as glucose isomerase.
Actinomycetes form an important part of the
microbial community responsible for nutrient
recycling in natural substrates. Their specific
contribution to lignocellulose degradation in the
environment has received only limited attention but
their importance can be inferred from the ubiquity and
diversity of species in which activity against
lignocellulose has been demonstrated27. Actinomycete
xylanases similarly conform to the basic patterns of
production and activity established in other bacteria
and fungi, but they have been relatively little studied.
Production of xylanases by actinomycetes
Industrial enzymes, in particular hydrolytic
enzymes have been usually produced in submerged
fermentation (SmF) as well as solid-state fermentation
(SSF) 28-31. Table 1 shows some examples of different
actinomycetes, which have been employed for the
production of xylanases in SSF or SmF using
agricultural wastes as substrate32-54. However,
relatively fewer studies have been conducted on
xylanase production by actinomycetes, particularly in
SSF using crude agricultural substrates. SSF studies
have commonly employed agro-industrial residues
such as wheat bran, rice straw, sugar cane bagasse,
Napier grass, soybean meal, etc. as substrate. Also,
different kinds of xylans such as birchwood xylan,
larchwood xylan, beechwood xylan and oat spelt
xylan have been used (cf. Table 1). In one such study
using wheat bran and eucalyptus kraft pulp as the
primary solid substrates, Streptomyces sp. QG-11-3
produced higher enzyme titres when grown in SSF
mode in comparison to SmF.39 Bajaj and Singh32
studied xylanase production by Streptomyces sp.
using wheat bran as substrate in SSF and obtained
enzyme with specific activity of 2.18. Alberton et al.39
 THOMAS et al.: XYLANASES FROM ACTINOMYCETES 877

compared the production of xylanase by a strain of in the medium containing larchwood xylan (1%, w/v),
S. viridosporus T7A in SmF and SSF using various xylanolytic activity was also observed when wheat
agro-industrial residues as substrates. They reported bran (28.4 U/mL), wheat germ (20.4 U/mL), brewer’s
highest xylanase activity (423.9 specific activity) with spent grain (16 U/mL), corn cobs (9.1 U/mL) and
a mixed substrate comprising sugar cane bagasse, paper recycling mill sludge (7.9 U/mL) were used as
Napier grass and soybean meal. A recombinant host substrates42. Saratale et al.47 reported the production
strain prepared by the cloning a DNA fragment from of biomass hydrolysing enzymes system comprising
the lignocellulolytic actinomycete S. avermitilis xylanase and others by a strain of Streptomyces sp.
CECT 3339 using a DNA probe from the xylanase MDS. The strain was able to utilize a broad range of
gene xysA of S. halstedii produced up to 400 U/mL cellulosic substrates including carboxymethyl
xylanase by (SSF) using cereal bran as substrate38. cellulose, avicel, xylan, cellobiose, filter paper, wood
The high production yields of this enzyme and its straw and rice straw. The supplementation of CaCl2
biochemical features made it a good candidate for use (5 mM) as a metal additive significantly induced the
in industrial applications. whole enzyme system. Kumar et al.48 reported the
Numerous studies have been carried out on the production of xylanase by a newly isolated
production of xylanases in SmF, which have utilised Streptomyces sp. RCK-2010 was optimized for
xylan from different sources, and/or agro-industrial varying culture conditions following one factor at a
sources such as wheat bran, rice straw, etc as the time (OFAT) and response surface methodology
source of carbon and energy. Authors studied the (RSM) approaches. Under the optimized
production of xylanases using different substrates. conditions, xylanases production was 2310 IU/mL,
Although the best production (70 U/mL) was obtained which the authors claimed as the highest reported so
Table 1Actinomycetes strains reported for the production of xylanases
Microbe Substrate Activity/Specific activity Method of Reference
(crude supernatant) production
Streptomyce sp. 7b Wheat bran 2.18 SSF 32
Streptomyces sp. PC22 Oat spelt xylan 2.39 SmF 33
Streptomyces sp. CD3 Wheat bran 14.28 SmF 34
S. cyaneus SN32 Wheat bran 396.31 SmF 35
Streptomyces sp. 234P-16I Birchwood xylan 2.03 SmF 36
Streptomyces sp. SWU10 Rice straw 0.2 SmF 37
S. avermitilis CECT3339 Cereal bran 400 U/mL SSF 38
Streptomyces sp. QG-11-3 Wheat bran and eucalyptus kraft pulp - - 39
S. viridosporus T7A Sugar cane bagasse, Napier grass and soybean 423.9 U/mL SSF 40
meal.
S. cuspidosporus Wheat bran 146 U/mL SSF 41
Streptomyces sp. AMT-3 Larchwood xylan 70 U/mL SmF 42
S. lividans Birchwood xylan - SmF 43
S. olivaceoviridis E-86 Corncob xylan 1653 U/mL SmF 44
S. chart Corncob xylan 731 U/mL SmF 45
Streptomyces sp. C1-3 Birchwood xylan 2.55 U/mL SmF 46
Streptomyces sp MDS carboxymethyl cellulose, avicel, xylan, - SmF 47
cellobiose, filter paper, wood straw and rice
straw
Streptomyces sp. RCK-2010 Wheat bran 2310 IU/mL SmF 48
Jonesia denitrificans BN-13 Xylan 10.81 U/mL SmF 49
Talaromyces thermophilus Wheat bran 10.0 U/mL SmF 50
S. lividans NRC Wheat bran 33.3 U/mL SmF 51
S.cyaneus, S. tendae, and Wheat bran >125 U/mL SmF 52
S. caelestis
S.albus Oat spelt xylan 11.97 U/mL SmF 53
S. galbus xylan of sugar cane bagasse or galactomannan - SmF 54
of palm-seeds
878 INDIAN J EXP BIOL, NOVEMBER 2013
far. A strain of Jonesia denitrificans BN-13 produced
extracellular xylanases; the best xylanolytic activity
(10.81 U/mL) was obtained in a medium containing
xylan at the end of the growth phase49. A newly
isolated thermophilic fungal strain of Talaromyces
thermophilus produced extracellular hemicellulases
when grown on various lignocellulosic substrates50.
Abd El-Nasser et al.51 described a xylanse from
Streptomyces lividans NRC which was best produced
in SmF using wheat bran as substrate. After the
supplementation of minerals and nutrients, the
enzyme production reached to 33.3. U/mL in 4-5
days. Ninawe et al.52 isolated three strains of
Streptomyces (S. cyaneus, S. tendae, and S. caelestis).
All the three strains produced large quantitites of
enzyme (>125 U/mL). Kansoh and Nagieb54 reported
a xylanase from Streptomyces. galbus using xylan of
sugar cane bagasse or galactomannan of palm-seeds
as sole carbon source. Maximum enzyme was
produced in five days when the culture was
supplemented with nutrients under optimized
conditions. A strain of Streptomyces albus ATCC
3005 produced extracellulase xylanse when grown on
oat spelt xylan and the maximum production was
achieved in 120 h (11.97 U/mL) 53.
Induction of xylanases
Xylanase activity is more widespread amongst the
actinomycetes than cellulase activity and although it is
an inherent property of cellulolytic microbes, xylanases
are clearly a distinct group of enzymes. Xylanolytic
enzymes appear to be inducible under natural
conditions and are subject to catabolic repression by
the carbon sources such as glucose or xylose. Being a
high molecular mass polymer, xylan cannot enter the
microbial cell. The induction of the enzymes is
stimulated by the low molecular fragments of xylan,
namely xyloboiose, xylotriose, xylooligosaccharides,
heterodisaccharides of xylose and glucose, and their
positional isomers, which are produced in the medium
by small amount of constitutively produced enzyme55.
Xylan is considered as the best inducer of xylanase
production in many actinomycetes56. This includes
pure xylans and other hemicellulose-rich materials and
low molecular weight carbohydrates such as xylose and
pentosan57. Induction can also be achieved by various
synthetic alkyl, aryl f3-D- xylosides and methyl f3-D-
xyloside58. These compounds enable the production of
xylanolytic enzymes in the absence of xylan and
xylooligosaccharides. Cheaper feedstocks rich in
hemicelluloses such as corncob, wheat bran, rice bran,
rice straw, corn stalk, sorghum straw, wheat straw and
sugarcane bagasse have been used as inducer for
xylanase production by actinomycetes. Wheat bran was
the best substrate for xylanase production by alkalophilic
Streptomyces T-759. In Saccharomonospora viridis, a
thermophilic actinomycete which degraded xylan but
not cellulose, both extracellular xylanase and cell-
bound β-xylosidase were induced by the growth on
xylan but not glucose or xylose. The addition of
glucose and/or xylose stimulated the growth but
depressed xylanase production by approximately 50%60.
Purification and properties of xylanases produced by
actinomycetes
Extracellularly produced xylanses have been purified
and characterized for various application studies.
Conventionally, partial purification has been done by
salt or solvent precipitation methods, followed by
chromatogarphic techniques such as gel filtration for
complete purification. Table 2 shows the properties of
the xylanases produced by different
actinomycetes33,34,48,50,65-83. Evidently, most of the
xylanses show pH optima near to neutrality or towards
slightly acidic side, although some possessed best
activity under mild alkaline conditions (from pH 5.0-6.0,
5.0-7.0 or 5.5.-8.0). The pH optimum of the enzyme
determines its application. Acidic xylanases find suitable
application in food and feed industries, while alkaline in
paper and pulp industries. Similarly, most the xylanases
show best activity at 50-65 oC. However, higher
temperature optima and stability is a key requirement for
most of the industrial applications of xylanases.
Zhu et al.53 were the first to report about the
purification and characterization of a xylanase from S.
chartreusis. The enzyme was more stable under
alkaline conditions and retained more than 80%
activity after 30 min incubation at pH 6.0-10.0. It also
showed specific activity towards different xylans. The
hydrolysis of oat-spelt and corn-cob xylans by this
xylanase yielded xylobiose and xylotriose as principle
products without the formation of xylose. These
properties indicated that the purified xylanase could
potentially be useful in biotechnological applications,
such as xylooligosaccharide preparation. In a study
carried out to characterize the xylanase from
Streptomyces sp. strain C1-3, Meryandini46 found that
the enzyme was highly thermostable and had a low
acidic pH optima (90 °C and pH 3.0, respectively).
The enzyme had preferential activity towards
birchwood and arabino xylans when used with five
kinds of xylan, i.e., birchwood, beechwood,
 THOMAS et al.: XYLANASES FROM ACTINOMYCETES 879

Table 2Properties of xylanases produced by actinomycetes

Microbe pH Temperature (°C) MW PI Km Reference

Optimum Stability Optimum Stability (kDa) mg/mL
range range
S. matensis DW 67 7.0 4.5-8.0 65 - 21.2 - 3.9 65
S. chartreusis L1105 6.7-7.7 5.0-10.0 40 40-50 - - - 66
Streptomyces sp. SU9 9.0 6.0-9.0 80 60-100 - - - 67
Streptomyces sp. CD3 8.0 8.0-10.0 50 50-70 - - - 34
Streptomyces sp.PC22 - 5.5 60 - 5 - 2.76 33
5.5-6.0 60 30 0.63
S. viridosporous 7.0-8.0 3.0-10.0 65-70 25-80 59 - 10.2-10.5 68
S. lividans 5.0-7.0 - 55-66 - - - - 69
S. flauogriseus 6.5 - 50 - - - - 70
Streptomyces sp. 5.5 - 55 - - - - 71
Thermomonospora sp. 5.5-7.7 - 65-80 - - - - 72
Thermo. curtvata 5.0-8.0 - 60-75 - - - - 73
Thermo. Fusca 5.0-8.0 - 60-75 - - - - 74
Thermo. chromogena 5.0-8.0 - 60-75 - - - 74
Saccharomonospora viridis 5.0-8.0 - 60-70 - - - - 60
Streptomyces. sp. 6.5 - 60°C - 25-50 - - 65
Streptomyces sp. AMT-3 6..0 - 55-65 - - - - 61
Nonomuraeaflexuosa (Nf Xyn11A) Nf- 6.0 5.0-7.0 50 - 23.4 - 6.0 62
Thermoascusaurantiacus (Ta Xyn10A) TaX-4.0-5.0 3.0-8.0 80 - 32.8 - 1.0

Chainia sp 5.0 - 65 - - - 8 75
Saccharomonospora viridis 5.0-8.0 - 60 - - - - 76
S. xylophagus 6.2 - 55-60 - - - - 77
S. flavogriseus (CD45-2) 6.5 - 50 - - - - 78
S. exfoliates 5.5 - 50 - - - - 79
7.0 55
5.5 55
Streptomyces sp.KT23 5.5 - 55 - 43 6.9 - 61
Streptomyces sp.3137 5.5-6.5 - 60-65 - 50 7.1 - 61
5.0-6.0 60-65 25 10.3
5.0-6.0 60-65 25 10.3
Thermomonospora sp. 5.5-7.7 - 65-80 - - - - 80
S. chartreusis 9.0 8.0-12.0 50 50-70 - - - 64
Streptomyces sp. SWU10 6.0 3.0-9.0 60 80 31 63
Thermoactinomyces thalophilus 8.5 8.5-9.0 50 50-65 - - - 81
Streptomyces sp. Ab106 7.0 - 50 - - - - 82
Streptomyces sp. CS802 12.0 7.5-13 60 - - - - 83
Talaromyces thermophilus 8.0 75 50-100 50
Streptomyces sp. RCK-2010 6.0 60 48

arabinoxylan, oat spelt and CMC. A xylanse produced
by a strain of Streptomyces sp. AMT-3 showed the
best activity at the temperature range from 55-65 °C
and pH 6.0. The enzyme retained 50% of its activity
after 20 h at 55 °C. Hence, this xylanase could be
considered as a thermotolerant enzyme suitable for
the biotechnological applications61.
Zhang et al.62 purified the xylanases from
Nonomuraea flexuosa (Nf Xyn11A) and Thermoascus
aurantiacus (Ta Xyn10A) by heat treatment and gel
permeation chromatography. The latter showed higher
hydrolytic efficiency with birchwood glucuronoxylan,
insoluble oat spelt arabinoxylan and hydrothermally
pretreated wheat straw, resulting more reducing sugars.
The mode of action of both the xylanases on
glucuronoxylan and arabinoxylan showed
typical production patterns of endo-xylanases belonging
to GH11 and GH10, respectively. A xylanase produced
880 INDIAN J EXP BIOL, NOVEMBER 2013
by Streptomyces sp. SWU10 showed very broad pH
optima, ranging from low acidic pH (3.0) to alkaline pH
(8.0). The enzyme was optimally active at 60 °C but
showed partial activity retention at 80 °C as well63.
Thomas et al.64 have recently isolated a strain of S.
chartreusis which produced a novel xylanase showing
pH and temperature optima as 9.0 and 70 °C.
The enzyme was substantially active at pH 12.0
and 70 °C, with a good stability between pH 8.0-12.0.
Simkhada et al.83 also reported an extremely alkaline
novel xylanase from a newly isolated Streptomyces
strain cultivated in corncob medium, which showed
optimal activity at pH 12.0 and temperature of 60 °C.
Biotechnological approach for the over-expression of
recombinant xylanases
The lack of hyper-producing potent cultures has
resulted in the attempts to over-express the xylanase
genes from the available cultures. Gene manipulation
has the advantage of producing microbial strains with
selected enzyme machinery. Current developments in
the recombinant DNA techniques offer new tools of
research for the construction of genetically modified
microbial strains with selected characteristics for
enzyme production. In this respect, the isolation and
cloning of the xylanase gene represent an essential
step in the engineering of the most efficient
microorganism16. The biotechnological approach for
over-expression of recombinant xylanases involves
the up-gradation of fermentation process of
industrially important xylose fermenting microbes by
introducing the genes for xylanase and xylosidase,
aiming at direct fermentation of xylan, as well as the
construction of xy1anolytic enzyme producers devoid
of cellulase activity4. Cloning helps in the introduction
of novel gene as well as amplification of existing
expression.
The selection of host expression system is
predominantly chosen by the compatibility of gene
expression machinery, fast multiplication of host in
relatively in expansive medium, simple transformation
technique, easy isolation and purification. Bacterial,
yeast, fungal and plant expression systems have been
used for heterologus expression of xylanase gene for
the production of recombinant xylanase. However,
bacterial expression system has been mainly used for
the general expression studies. Although, bacterial
expression system offers many attractive features, its
application has remained limited due to lack of the
specific post-translational modification machinery for
the expression of heterologus xylanase. The yeast
constitutes an ideal expression system because of the
presence of post-translational modifications and the
secretary system84. Plant can also be used as
heterogonous expression system for the over-
expression of many xylanase. Some example include
endo-xylanases of N. patriciarum expressed in
Brassica napus85, Bacillusendo-xylanase A expressed
in chloroplast of tobacco plant86, etc. Hernández et al.87
cloned S. avermitilis CECT 3339 using a DNA probe
from the xylanase gene xysA of S. halstedii. The
nucleotide sequence analysis revealed two potential
ORFs, xyl30 and hd30, encoding a deduced multi-
modular F/10 xylanase with a binding domain and a
secreted glycoxyl hydrolase, respectively. They further
reported that in S. lividans, carrying the subcloned
DNA fragment, two xylanase activity bands with
estimated molecular masses of 42.8 and 35 kDa were
detected by zymograms and SDS-PAGE. The two
xylanases had identical N-terminal sequences,
suggesting that Xyl30 "1" was derived from Xyl30 "h"
by C-terminal processing in the culture supernatant38. A
thermostable xylanase produced by a Streptomyces sp.
(claimed to produce 1155 IU/mL/day as highest
volumetric activities of xylanases reported so far
among Streptomyces) was most active at 60 °C and pH
6.0 and almost 40% stable after 4 h at the optimum
temperature48.
Strain improvement
Several studies have been undertaken on the
improvement of microbial strains using the classical
approaches for mutagensis or rDNA technique. Strain
improvement is a process which aims to increase the
production of xylanase or alter the physio-chemical
properties of the natural wild type strains.
Mutatagenesis is one of the preferred methods for
strain improvement because of its high efficiency.
Different mutagenic agents such as ultraviolet (UV)
rays, X-rays, gamma radiation, ethyl methane sulfonate
(EMS), N-methyl-N-nitro-N-nitrosoguanidine (NTG)
are commonly used to get mutant organism with
improved enzyme production. Abdel-Aziz et al.88
achieved 161 % over-production of xylanse by a UV
mutant of S. pseudogriseolus as compared to wild type
strain.
Strain improvement using recombinant DNA
techniques can be readily applied to actinomycetes,
particularly Streptomyces sp. Protoplast
transformation and fusion techniques and the
construction of plasmid and phage cloning vectors
strategies have also been used for improving the
 THOMAS et al.: XYLANASES FROM ACTINOMYCETES 881

Table 3Cloning of xylanase genes from Streptomyces sp. into suitable hosts
Microbe Xylanase gene Expression system Host Reference
Streptomyces sp. EC3 xln pIJ702 S. parvulus 90
Streptomyces sp.TN119 xynA119 - Escherichia coli BL21 (DE3) 95
S. halstedii JM8 xln pJM9 S. parvulus 96
S. flavogriseus xln pUC8 E. coli lysogenic for λcI857 70
S. lividans 66 xln pIJ702 xylanase negative double mutant of S. lividans 101
Streptomyces sp. S27 xynBS27 - Pichia pastoris 97
Streptomyces sp. S9 xynAS9 - E. coli BL21 (DE3) 98
S. olivaceoviridis A1 XYNB - E. coli and P. pastoris 99
S. thermoviolaceus OPC-520 STX-I STX-II pUC18, pUC19, M13mp18, E. coli JM109 and S. griseus PSR2 100
M13mp19, pIJ702 pIJ702
Thermomonospora alba UL JB1 xylA - S. lividans 80
S. viridosporous T7A svxA pBluescript SK DH5aMCR 68

strains for xylanase production. Improving enzyme
production by gene amplification and derepression,
and the construction of recombinant strains, which
can selectively delignify or completely solubilise the
lignocellulose have been amongst the most important
objectives for the strain improvement studies reported
potential of rDNA technology to overcome the
problems of insufficient xylanase yield by
Streptomycetes89. In the Streptomycetes, hybrid
antibiotic synthesis and the cloning of antibiotic
biosynthesis genes have already been achieved,
indicating that our understanding of actinomycete
molecular genetics has progressed to the point where
real biotechnological applications are tenable89.
Over-expression of actinomyces xylanase
Over-expression followed by efficient secretion of
xylanase has been considered as the viable alternative
to produce the xylanase in economical terms and to
meet the demand of industrial application. In this
regard many Streptomyces sp. xylanase has been
cloned and over- expressed in homologous and
heterologus expression system. There are several
reports regarding genetic manipulation of xylanase
producing microorganisms90. The early studies on the
cloning of xylanase gene included the works on
Bacillus sp.4. There are reports regarding the cloning of
xylanases from various organisms such as
Streptomyces sp. strain EC390, S. thermoviolaceus
OPC-52091, Actinomadura sp. strain FC792 and S.
lividans93. Table 3 shows the cloning of xylanase genes
from Streptomyces sp. into suitable hosts68,70,80,90,95-101.
The thermostabi1ity of S. lividans xylanase B (SIxB-
cat) was significantly increased by the replacement of
its N-terminal region with the corresponding region
from Thermomonosporafusca xylanase A (TfxA-cat)
without observing a decrease in enzyme activity by
DNA shuffling technique96. The homologous cloning
of the xylanase gene from S. lividans by functional
complementation resulted the over-production of the
extracellular enzyme (380 IU/mL) 96.
Summary and future perspectives
From the above, it is evident that actinomycetes
xylanases offer potential industrial applications, in
particular for paper and pulp industry, and food and
feed industry. Evidently Actinomycetes could be
unique source of novel xylanases. Since the wild type
strains do not show all the desired properties making
them ideal for industrial applications (e.g. high pH and
temperature optima together with high enzyme
activities for paper and pulp industry), it would be
necessary to adopt the biotechnological tools for strain
improvement. Streptomyces strains are less amenable
to transformation but the use of the techniques such as
electrotransformation has paved the way for genetic
engineering of the organism. Recombinant DNA
techniques can be readily applied to actinomycetes,
particularly for the construction of recombinant
Streptomyces strain, which produce the enzyme
capable of complete solubilisation of the
lignocellulose. The understanding of actinomycetes
molecular genetics can help further to do alterations in
such a way that it becomes a suitable candidate for
industrial application. Thus, the improvement of the
strains by recombinant DNA technology and site-
directed mutagenesis appear to be the most promising
options and should be explored in future works.
Acknowledgement
One of the authors (LT) is grateful to the
University Grants Commission, New Delhi for the
award of Junior Research fellowship.
882 INDIAN J EXP BIOL, NOVEMBER 2013
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