Production and biochemical characterization of xylanase from an alkalitolerant novel sp Aspergillus niveus RS2

Abstract

The novel fungus Aspergillus niveus RS2 isolated from rice straw showed relatively high xylanase production after 5days of fermentation. Of the different xylan-containing
agricultural by-products tested, rice husk was the best substrate; however, maximum xylanase production occurred when the
organism was cultured on purified xylan. Yeast extract was found to be the best nitrogen source for xylanase production, followed
by ammonium sulfate and peptone. The optimum pH for maximum enzyme production was 8 (18.2U/ml); however, an appreciable level
of activity was obtained at pH 7 (10.9U/ml). Temperature and pH optima for xylanase were 50°C and 7.0, respectively; however
the enzyme retained considerably high activity under high temperature (12.1U/ml at 60°C) and high alkaline conditions (17.2U/ml
at pH 8 and 13.9U/ml at pH 9). The enzyme was strongly inhibited by Hg2+, while Mn2+ was slight activator. The half-life of the enzyme was 48min at 50°C. The enzyme was purified by 5.08-fold using carboxymethyl-sephadex
chromatography. Zymogram analysis suggested the presence of a single candidate xylanase in the purified preparation. SDS-PAGE
revealed a molecular weight of approximately 22.5kDa. The enzyme had K
m and V
max values of 2.5 and 26μmol/mg per minute, respectively.

Full text

Abstract The novel fungus Aspergillus niveus RS2
isolated from rice straw showed relatively high xylan-
ase production after 5 days of fermentation. Of the
different xylan-containing agricultural by-products
tested, rice husk was the best substrate; however,
maximum xylanase production occurred when the
organism was cultured on purified xylan. Yeast extract
was found to be the best nitrogen source for xylanase
production, followed by ammonium sulfate and pep-
tone. The optimum pH for maximum enzyme pro-
duction was 8 (18.2 U/ml); however, an appreciable
level of activity was obtained at pH 7 (10.9 U/ml).
Temperature and pH optima for xylanase were 50C
and 7.0, respectively; however the enzyme retained
considerably high activity under high temperature
(12.1 U/ml at 60C) and high alkaline conditions
(17.2 U/ml at pH 8 and 13.9 U/ml at pH 9). The en-
zyme was strongly inhibited by Hg
2+
, while Mn
2+
was
slight activator. The half-life of the enzyme was 48 min
at 50C. The enzyme was purified by 5.08-fold using
carboxymethyl-sephadex chromatography. Zymogram
analysis suggested the presence of a single candidate
xylanase in the purified preparation. SDS-PAGE re-
vealed a molecular weight of approximately 22.5 kDa.
The enzyme had K
m
and V
max
values of 2.5 and
26 lmol/mg per minute, respectively.
Keywords Alkalitolerant Æ Aspergillus niveus Æ
Production Æ Thermostable Æ Xylanase Æ Zymogram
Introduction
Xylanases produced by microorganisms have attracted
a great deal of attention during the past few decades
because of their potential biotechnological applications
in various industries, including the food, feed, fuel,
textile and paper and pulp industries, and in waste
treatment (Beg et al. 2001). In the paper and pulp
industries xylanases can be used for bleaching kraft
pulp. Treatment with xylanases facilitates the chemical
extraction of lignin from pulp and leads to a significant
reduction in the use of hazardous chemicals required
for bleaching while simultaneously preserving the
paper strength properties, brightness, fibrillation,
drainage, among others. For biobleaching applications,
the candidate xylanase should be thermostable, alka-
litolerant and stable on kraft pulp, and its various
properties, such as effective molecular weight, net ionic
properties and specific action pattern, must suit the
process requirements. Moreover, to avoid damage to
cellulose pulp, enzyme preparations should be free
from cellulase activity.
Lignocellulose, the most abundant and renewable
biomass available on earth, comprises three major
groups of polymers – cellulose, hemicellulose and lig-
nin. The primary component of the hemicellulose is
xylan, which is the most abundant noncellulosic poly-
saccharide in hardwoods and annual plants, where it
accounts for 20–35% of the total dry weight. Xylan is a
heterogeneous polysaccharide consisting of a homo-
polymeric backbone of 1,4 linked b-
D-xylopyranose
units and short chain branches, including acetyl 4-O-
methyl-
D-glucuronosyl, L-arabinofuranosyl, D-glucuro-
nyl, O-acetyl, p-coumaric side chains and ferulic acid
cross linkages. Due to the structural heterogeneity of
R. Sudan Æ B. K. Bajaj (&)
Department of Biotechnology, University of Jammu,
Jammu 180006, India
e-mail: bkbajaj1@rediffmail.com
World J Microbiol Biotechnol (2007) 23:491–500
DOI 10.1007/s11274-006-9251-0
123
ORIGINAL PAPER
Production and biochemical characterization of xylanase
from an alkalitolerant novel species Aspergillus niveus RS2
Raki Sudan Æ Bijender Kumar Bajaj
Received: 4 January 2006 / Accepted: 8 March 2006 / Published online: 4 November 2006
 Springer Science+Business Media B.V. 2006

the xylans, the xylan-degrading enzyme system consists
of two main families of hydrolytic enzymes, the b-1,4
endoxylanases (E.C 3.2.1.8), which attack the main
chain, and the b-xylosidases (E.C 3.2.1.37), which
hydrolyze xylooligosaccharides to xylose, as well as
several accessory enzymes necessary for debranching
the substituted xylans (Kuhad and Singh 1993). Xy-
lanases are produced by diverse genera and species of
bacteria and fungi (Subramaniyan and Prema 2000).
Filamentous fungi are particularly interesting producer
of xylanases from an industrial point of view due to the
fact that they excrete xylan-degrading enzymes into the
medium, thus eliminating the need for cell disruption.
In addition, fungi are easy to cultivate and can be
cultured on cheap raw materials. Furthermore, fungal
cultures typically produce larger amounts of xylanase
than yeast and bacterial cultures. In addition to the
xylanases, fungi normally produce several auxiliary
enzymes that are necessary for debranching the
substituted xylans (Haltrich et al. 1996).
The present study was aimed at optimization of the
fermentation process for novel xylanase-producing
fungus Aspergillus niveus RS2, which has been isolated
from decaying rice straw, and subsequent character-
ization and purification of xylanase.
Materials and methods
Culture media, microorganism and xylanase
production
Samples of alkaline soil, sugar cane bagasse, rice straw,
cow dung manure, poultry waste, sawdust, among
others, were collected from alkaline, hot and humid
locations in the vicinity of decaying organic matter and
used for the isolation of xylanolytic fungi. Xylanolytic
activity was tested by Congo red staining, as described
previously (Sharma and Bajaj 2005), on xylan agar
(0.3% ammonium sulfate, 0.3% potassium dihydrogen
phosphate, 0.6% ammonium acetate, 0.5% oat spelt
xylan and 2.0% agar, pH 8; all w/v). The same medium
without agar was used as fermentation medium for
xylanase production in a submerged fermentation
protocol. The composition of the production medium
was varied by using alternative carbon sources or
supplementing it with different nitrogen sources, when
required. The selected organisms were grown on po-
tato dextrose agar plates for 3 days, and four to six
discs (3–4 mm each) were cut and inoculated into the
enzyme production medium (100 ml) in 250-ml
Erlenmeyer flasks. The flasks were then incubated on a
shaker-incubator at 45C and 180 rpm. After varying
lengths of culture period, a suitable volume of the
fermentation broth was withdrawn under aseptic con-
ditions and centrifuged at 10,000 g for 5 min at 5C
(rotor number 12154-H; Sigma, St. Louis, Mo.). The
supernatant was considered to be equivalent to the
crude enzyme and used for xylanase assay.
Xylanase assay and protein estimation
Xylanase activity was assayed using a 0.5% oat spelt
xylan solution prepared in Tris buffer (50 mM, pH 8),
at 45C. The reducing sugars that were released were
assayed by the dinitrosalicylic acid method using xylose
as the standard (Miller 1959). One unit of enzyme
activity was defined as the enzyme necessary to release
1 lmol of reducing sugar or xylose equivalent per
minute under assay conditions. Specific activity was
expressed as enzyme units per milligram of protein.
The reaction conditions for assaying cellulase
activity were same as those of the xylanase assay ex-
cept that the substrates used contained either 0.5%
carboxymethyl cellulose (CMC), cellulose powder or
filter paper powder (HiMedia Laboratories, Mumbai,
India). The reducing sugars that were released were
assayed using the dinitrosalicylic acid method using
glucose as the standard. One unit of cellulase activity
was defined as the enzyme amount necessary to release
1 lmol of glucose equivalent per minute at 45C. The
method of Lowry et al. (1951) was used to estimate the
protein level using bovine serum albumin (BSA) frac-
tion V as the standard. Protein content was also
determined using the Christian-Warburg method
(Sharma and Bajaj 2005).
Effect of carbon and nitrogen sources on xylanase
production
The effect of different carbon sources on xylanase
production was tested by replacing xylan in the pro-
duction medium with wheat bran, wood powder, rice
husk, sugarcane bagasse, starch, rice straw or wheat
straw at the rate of 1% (w/v), and with glycerol or
mannitol at the rate of 1% (v/v). Crude substrates were
first steam hydrolyzed for 20 min in an autoclave, then
dried and used. In order to study the effect of various
nitrogen sources on xylanase production, we supple-
mented the basic production medium containing 1%
xylan as the carbon source with alternative nitrogen
sources such as albumin, gelatin, peptone, soy meal,
tryptone, urea and yeast extract at the rate of 0.5%
(w/v) and with ammonium sulfate at the rate of 0.3%
(w/v).
492 World J Microbiol Biotechnol (2007) 23:491–500
123

Effect of initial medium pH
The production medium with 1% xylan as the carbon
source and 0.5% yeast extract as nitrogen source was
used to study the effect of the initial pH of the medium
on enzyme production. Fermentation was carried out
in production media adjusted with different pH (5, 6, 7,
8 and 9), and the resulting xylanase production was
observed after 5 days.
Characterization of the xylanase
A crude enzyme preparation was obtained by culti-
vating the organism in production medium (pH 8)
containing 1% xylan as the carbon source and 0.5%
yeast extract as the nitrogen source for 5 days at 45C
(180 rpm); this preparation was used to study the effect
of various parameters on enzyme activity.
The effect of temperature on enzyme activity was
assayed by incubating the enzyme assay reaction mix-
ture at different temperatures – 30,40,50,60 and
70C – and then determining the xylanase activity. The
effect of pH on enzyme activity was measured by using
buffers of different pH, ranging from 3 to 10 (citrate
buffer, pH 3, 4, 5 and 6; Tris buffer, pH 7, 8 and 9;
glycine-NaOH buffer, pH 10). The thermostability of
the enzyme was tested by incubating the crude enzyme
preparation at temperatures ranging from 50 to 70C
for different intervals of time before using it for assay.
Effect of various metal ions and other additives on
xylanase activity.
Various additives – Fe
2+
,NH
4
+
,Co
2+
,Ca
2+
,Zn
2+
,
Hg
2+
,
Mg
2+
,Mn
2+
and phenylmethylsulphonylfluoride
(PMSF) at a final concentration of 10 mM – were used
in the enzyme assay reaction mixture to study their
effect on xylanase activity.
Xylanase purification
The fungal culture was grown in the production med-
ium, and the biomass was separated by centrifugation
at 10,000 g for 10 min. The supernatant was subjected
to ammonium sulfate precipitation at different satura-
tion levels (25–75%). The protein precipitate obtained
was dissolved in Tris buffer (50 mM, pH 8.0) and dia-
lyzed against the same buffer. The dialyzed enzyme
preparation was applied to an ion exchange column
(20·1 cm) packed with carboxymethyl-sephadex (CM-
Sephadex), regenerated as per the standard method
and pre-equilibrated using 10 volumes of Tris buffer
(50 mM, pH 8). The column was washed with the same
buffer to remove the unbound protein components.
Protein was then eluted out using solutions of varying
salt concentrations (range: 0.1–0.5 M NaCl) and the
different fractions were collected. Any protein sus-
pected to be still bound was eluted using 25 ml of 1 M
NaCl. The collected fractions were tested for xylanase
activity and protein content.
Native and sodium dodecyl sulphate-
polyacrylamide gel electrophoresis (SDS-PAGE)
Electrophoresis (SDS and PAGE) was carried out as
described by Sambrook et al. (1989). The resolving gel
consisted of 12.5% polyacrylamide in Tris-HCl (1.5 M,
pH 8.8), while the stacking gel consisted of 4.5%
polyacrylamide in Tris-HCl (1.0 M, pH 6.8). Zymo-
gram studies were carried out using native-PAGE.
After casting and solidification of the polyacrylamide
gel, samples of the ammonium sulfate-precipitated
fraction and partially purified enzyme obtained by ion
exchange chromatography were loaded in appropriate
wells. The electrophoresis was performed at 200 V for
1 h in Tris-glycine buffer (pH 8.3), following which the
polyacrylamide gel was over-layered onto an agar gel
(1.5%, w/v) pre-seeded with 0.5% xylan and incubated
for 1 h at 45C. The agar gel was then stained with
0.1% Congo red to determine the location of xylanase
protein.
The polyacrylamide gel was dipped into fixative
agent (ethanol:acetic acid:water, 30:10:60) overnight,
then washed twice with 30% ethanol and deionized
water and stained with a 1% silver nitrate solution for
30 min. The staining solution was removed and the gel
dipped into 2.5% sodium carbonate and 0.02% form-
aldehyde solution for a few minutes until a dark band
appeared. The reaction was then stopped by dipping
the gel into a 1% acetic acid solution. The gel was then
observed on a white illuminator. SDS-PAGE was
performed to determine the molecular weight of
xylanase using different molecular weight markers
(Fermentas, Vilnius, Lithuania).
Kinetic studies
The partially purified enzyme preparation obtained by
ion exchange chromatography was used for studying
kinetic parameters (K
m
and V
max
). For determining the
reaction rate, different substrate (oat spelt xylan)
concentrations were used, ranging from 1.25 to
11.25 mg/ml. The reaction rate versus substrate con-
centration was plotted to determine whether the en-
zyme obeys Michaelis-Menten kinetics, and K
m
and
V
max
were determined from the Lineweaver-Burk plot.
World J Microbiol Biotechnol (2007) 23:491–500 493
123

Results and discussion
Isolation and identification of high xylanase-
producing fungal isolates
Selected fungal isolates, following preliminary screen-
ing for xylanolytic activity by Congo red staining, were
subjected to submerged fermentation in xylanase pro-
duction medium. The isolate RS2 showed the highest
xylanase production and was subsequently ear-marked
and identified as Aspergillus niveus. It was later des-
ignated as Aspergillus niveus RS2. Its identification was
based on its macro (colony, growth, exudates, reverse
color, etc.) and micro (heads, condiphores, phialides,
vesicles, conidia etc.) morphological characteristics and
comparing these with the taxonomic keys of Raper and
Fennell (1965).
Large number of Aspergillus species have been
reported to be producers of xylanase (Subramaniyan
and Prema 2000; Fialho and Carmona 2004; Shah and
Madamwar 2005) but to our knowledge this is the first
report of xylanase production by Aspergillus niveus.
Habitats with degrading lignocellulosic materials are
considered to be the best niches for isolating xylanolytic
microorganisms, as the xylanases are inducible en-
zymes. Many researchers have isolated xylanase-pro-
ducing fungi from natural habitats rich in degrading
organic matter. Shah and Madamwar (2005) isolated
xylanase-producing Aspergillus foetidus from decaying
agricultural waste. Kango et al. (2003) isolated a
themotolerant, xylanase-producing fungus, Emericella
nidulans, from bird nesting material. Fialho and
Carmona (2004) isolated Aspergillus giganteus from
Brazilian soil and purified and characterized the xy-
lanases from this strain. Ruckmanl and Rajendran
(2001) isolated a xylanase-producing alkalitolerant
strain of Aspergillus flavus from decomposed leaf litter.
Effect of cultivation period on xylanase production
Xylanase production by A. niveus RS2 was studied in a
submerged fermentation culture system for over a
week. The production of the enzyme started from on
the second day of cultivation and reached a maximum
on the fifth day (17.6 U/ml). There was a slight de-
crease in activity on the sixth day of cultivation
(15.25 U/ml) (Fig. 1), which may have been due to the
activation of proteases.
Ruckmanl and Rajendran (2001) and Carmona
et al. (2005) obtained maximal xylanase production on
the fifth day of cultivation using A. flavus and
A. versicolor, respectively. Shah and Madamwar (2005)
reported maximum xylanase production from
A. foetidus after 4 days of cultivation. It would appear
that the maximum enzyme production stage of any one
organism is largely dependent upon the type of
microbial strains and their genetic make-up as well as
on cultural and environmental conditions during the
growth of the organism.
Effect of carbon source on xylanase production
Since the cost of the substrate plays a crucial role in the
economics of the xylanase production process, it is
desirable to use low-value crude agriculture-based raw
materials for xylanase production. We tested various
carbon sources, such as wheat bran, rice husk, wood
powder, sugarcane bagasse, rice straw, wheat straw and
starch, as the sole carbon source for xylanase produc-
tion. Aspergillus niveus
RS2 showed the maximum
xylanase production when xylan was used as the sole
carbon source. Rice husk and wheat bran supported
moderate xylanase production, while the other sub-
strates supported very small or negligible xylanase
production (Fig. 2). Even xylose induced considerable
0
2
4
6
8
10
12
14
16
18
20
135
Time (da
y
s)
Enzyme activity (U/ml)
246
Fig. 1 Time course of xylanase production in shake flask culture
at 45C. Each data point is the mean value of three replicates
0
2
4
6
8
10
12
14
16
18
Wheat bran
Rice husk
Saw dust
Bagasse
Starch
Xylan
Rice straw
Wheat straw
Xylose
Glycerol
Mannitol
Carbon sources
Enzyme acitivity (U/ml)
Fig. 2 Effect of carbon sources on xylanase production as
determined after 3 days (
) and 5 days ( ) of fermentation
in shake flask culture at 45C. Data are the means of three
replicates
494 World J Microbiol Biotechnol (2007) 23:491–500
123

xylanase production, which is contrary to published
reports (Haltrich et al. 1996).
Investigations reported in the literature (Gilbert and
Hazlewood 1993; Haltirch et al. 1996) indicate that
purified xylans can be excellent carbon sources,
resulting not only in an increased yield of xylanases but
often in the selective induction of xylanase with no or
only low concomitantly formed cellulases. Bakir et al.
(2001) and Shah and Madamwar (2005) reported that
xylan was the most effective carbon source for xylanase
production by Rhizopus oryzae and A. foetidus,
respectively. Similarly, Ruckmanl and Rajendran
(2001) reported that A. flavus produced higher levels of
xylanase when cultured on xylan than on wheat bran.
Conversely, Kango et al. (2003) found higher xylanase
activity by Emericella nidulans on wheat bran than on
xylan.
Xylanase excretion is potentiated in the presence of
oligosaccharides with a higher degree of polymeriza-
tion and substitution, such as xylans. The backbone
structure of xylan is preferentially attacked, inducing
then excretion of xylanases. Substrates like wheat bran,
rice husk, wood powder, sugarcane bagasse, rice straw
and wheat straw are heterogeneous and complex and
their use as the sole carbon source results in a lower
yield activity, possibly because the extracellular de-
graditve enzyme system is not sufficient to degrade the
xylans in the complex substrates (Haltrich et al. 1996).
Effect of nitrogen source on xylanase production
The mechanisms which govern the formation of
extracellular enzymes are influenced by the availability
of precursors for protein synthesis. Furthermore, the
nitrogen source can significantly affect the pH of the
medium during the course of fermentation and, in turn,
may substantially influence the enzyme activity.
Among the different nitrogen sources tested, the
addition of yeast extract to the production medium
resulted in maximum xylanase production, followed by
ammonium sulfate, peptone and soymeal. No signifi-
cant effect on xylanase production was reported when
the medium was supplemented with other nitrogen
sources (Fig. 3). Similarly, Ruckmanl and Rajendran
(2001) and Bakri et al. (2003) reported yeast extract to
be the best nitrogen source for maximum xylanase
production. Further, Bakri et al. (2003) found that a
combination of yeast extract and peptone gave still
better results. In contrast, Bakir et al. (2001) found
soybean meal to be the best nitrogen source for xy-
lanase production from fungus Rhizopus oryzae. Shah
and Madamwar (2005) obtained maximum xylanase
activity from A. foetidus when proteose peptone was
used as the nitrogen source. Badhan et al. (2004)
employed ammonium sulfate and ammonium acetate
in the production medium for obtaining maximum
xylanase activity from Myceliophthora sp.
Cellulase activity
It is worth mentioning here that when the intended
application of the xylanase preparations is in the pulp
and paper industries, they should be free of cellulolytic
activities, otherwise the cellulase may damage the
cellulose fibers. An interesting result from our inves-
tigation was that the xylanase preparation from
Aspergillus niveus RS2 had negligible cellulase activity
[0.077 U/ml filter paperase (Fpase) and 0.1 U/ml
CMCase]. Such enzyme preparations may have po-
tential to be used in pulp and paper industries. Many
researchers have reported moderate or negligible or
even a total absence of cellulase activity in xylanase
preparations (Bakir et al. 2001). However, other
researchers have reported considerably high contami-
nation of xylanase preparations with cellulases (Singh
et al. 2003; Xiong et al. 2004), which may be desirable
if enzyme preparations are intended to be used for
alternative applications, i.e. for the food and feed
industry, lignocellulose biotransformation industries
and silage preparation.
Effect of initial medium pH
The organism showed a greater xylanase production at
a neutral and slightly alkaline pH (pH 8) than at an
acidic pH and high alkaline pH. Maximum xylanase
activity was reported when the fermentation was
0
2
4
6
8
10
12
14
16
18
20
Albumin
Gelatin
Peptone
Soyameal
Tryptone
Urea
Yeast Extract
Ammonium sulp
hate
Nitro
g
en sources
Enzyme activity (U/ml)
Fig. 3 Effect of various nitrogen sources on xylanase production
as determined after 3 days (
) and 5 days ( ) of fermentation
in shake flask culture at 45C. Data are the means of three
replicates
World J Microbiol Biotechnol (2007) 23:491–500 495
123

carried out in a medium with pH 8.0, and xylanase
activity decreased on both sides of this pH (Fig. 4).
Although the organism is alkalitolerant, the production
of the enzyme was maximal only under the neutral or
slightly alkaline conditions (pH 8.0). In the case of
fungi, the majority of researchers have reported an
acidic pH to be the most appropriate for maximum
enzyme production (Subramaniyan and Prema 2000;
Fialho and Carmona 2004; Xiong et al. 2004; Carmona
et al. 2005). However, Ruckmanl and Rajendran
(2001) obtained maximum xylanase production by
A. flavus in pH 9 medium, while Purkarthofer et al.
(1993) found that Thermomyces lanuginosus showed a
better xylanase production at pH 7.5 than at pH 6.5.
The relatively poor production of xylanases at the
higher pH may be due to the proteolytic inactivation of
xylanase.
Effect of temperature on enzyme activity
Temperature has a profound influence on enzyme
activity and, in general, it is highly desirable that
enzymatic preparations intended for use in industrial
applications must not only be thermostable but must
have a high activity at elevated temperatures as most of
the industrial enzymatic operations are carried out at
high temperatures. Aspergillus niveus RS2 xylanase
showed maximum activity at 50C, while the relative
activity was 83.9% at 40C and 67% at 60C; however,
xylanase activity was reduced drastically at 70C
(Fig. 5).
Microbial xylanases typically have temperature
optima of about 50C (Srinivasan and Rele 1995).
Ruckmanl and Rajendran (2001), Fialho and Carmona
(2004) and Shah and Madamwar (2005) observed
temperature optima of 50C for xylanases obtained
from A. flavus and A. giganteus, and A. foetidus,
respectively. The thermostability of xylanases could be
enhanced by protein engineering (Xiong et al. 2004).
Effect of pH on enzyme activity
The majority of xylanases reported to date are opti-
mally active in the acidic or neutral pH range (Haltirch
et al. 1996; Subramaniyan and Prema 2000). From the
application point of view, xylanases active and stable in
the alkaline pH range are very important. In the
present study, maximum xylanase activity was found at
pH 7, decreasing slightly (by 2.5%) at pH 6. A con-
siderable amount of enzyme activity was observed
even under high alkalinity, i.e. 84.8% and 68.7% at
pH 8 and 9, respectively (Fig. 6). However, at pH 10
the enzyme activity decreased significantly to 35.2% of
the maximum activity. Thus, this enzyme has sub-
stantial level of alkali tolerance, which is a desirable
features for applications in the biopulping industry.
The optimum pH for xylan hydrolysis by most of the
fungal xylanases is in the acidic range (Bakir et al.
0
2
4
6
8
10
12
14
16
18
20
0 1020304050607080
Temperature (°C)
Enzyme acitvity (U/ml)
Fig. 5 Effect of temperature on xylanase activity. Data are the
means of three replicates
0
5
10
15
20
25
01012
pH
Enzyme activity (U/ml)
2468
Fig. 6 Effect of pH on xylanase activity. The buffers used were
citrate buffer (pH 3, 4, 5, 6); Tris buffer (pH 7, 8, 9); glycine-
NaOH (pH 10). Data are the means of three replicates
0
2
4
6
8
10
12
14
16
18
20
9876543210 10
Initial medium
p
H
Enzyme activity (U/ml)
Fig. 4 Effect of initial medium pH on xylanase production after
5 days of fermentation in shake flask culture at 45C. Data are
the means of three replicates
496 World J Microbiol Biotechnol (2007) 23:491–500
123

2001; Fialho and Carmona 2004). Belancic et al. (1995)
and Cesar and Mrsa (1996) reported pH optima of 7
for xylanase from Penicillium purpurogenum and
Thermomyces lanuginosus, respectively.
Thermostability of xylanase
The enzyme preparation was incubated at tempera-
tures ranging from 50 to 70 C for different intervals of
time and the residual activity assayed. At 50C the
enzyme retained 97, 88.9 and 70.9% of the initial
activity after 20, 30 and 40 min of incubation, respec-
tively, following which the activity decreased steeply
and was totally lost at 120 min. At 60C the residual
activity after 20 min was 52.9%, and after 60 min a
complete loss of activity was observed. The loss in
enzyme activity was more drastic at 70C. The half-life
of the enzyme was found to be 48 min at 50C (Fig. 7).
Shah and Madamwar (2005) reported residual
activity of 71 and 20% after 30 min of exposure at 50
and 60C, respectively, while at 70 C the enzyme was
completely inactivated within 30 min. Fialho and
Carmona (2004) studied the thermal stability of two
xylanases from A. giganteus and reported that xylanase
I was more stable with a half-life of 22.5 min as com-
pared to xylanase II, which had half-life of 17.5 min at
50C. However, both xylanases showed the same half-
life of 1.5 min at 60C. Similarly, Carmona et al. (2005)
reported half-lives of 17 min and 1.7 min for
A. versicolor xylanase I and xylanase II, respectively, at
60C.
Effect of various additives on enzyme activity
The excretion of xylanase and its extracellular perfor-
mance also depend directly on the type of ions present
in solution, since they affect the overall kinetics of the
enzyme. A crude enzyme preparation was used to
study the effect of various ions and other additives on
xylanase activity. The presence of Mn
2+
ions enhanced
the activity by 16.3%, while Hg
2+
ions strongly inhib-
ited the enzyme and resulted in a total loss of enzyme
activity. Enzyme activity was reduced to 66–67% of its
baseline level in the presence of Co
2+
and Ca
2+
, and to
62% in the presence of NH
4
+
, while the presence of
Fe
2+
and Mg
2+
caused only a slight decrease in activity
(17–18%). Phenylmethylsulphonylfluoride (PMSF)
caused a 32.8% reduction in activity; this may have
been caused due to the involvement of serine and/or
cysteine residues in the catalytic process (Fig. 8).
Carmona et al. (2005) studied the effect of various
additives on the activity of A. versicolor xylanase and
reported that Hg
2+
and Cu
2+
ions are strong inhibitors
of the activity of this xylanase at a 10 mM concentra-
tion. They also reported that PMSF, Ca
2+
and Co
2+
were moderate inhibitors of enzyme activity and that
the Mn
2+
ion was an activator. In contrast, Sharma and
Bajaj (2005) reported Ca
2+
and Fe
3+
to be slight stim-
ulators of enzyme activity.
Xylanase purification
The crude enzyme preparation obtained after growing
the organism in production medium with 1% xylan as
the carbon source and 0.5% yeast extract as the
nitrogen source was subjected to ammonium sulfate
precipitation (25–50% saturation). The specific activity
of xylanase in the ammonium sulfate-precipitated
fraction was 20.8 U/mg protein compared to that of
9.50 U/mg protein in the crude preparation. Thus, en-
zyme purification increased the activity by 2.18-fold
(Table 1).
The ammonium sulfate-precipitated fraction was
dissolved in a small quantity of Tris buffer (50 mM,
0
20
40
60
80
100
120
140
Additives
Relative activity (%)
Control PMSF Hg
2+
Mg
2+
Mn
2+
Co
2+
NH
4
+
Ca
2+
Zn
2+
Fe
2+
Fig. 8 Effect of various additives on xylanase activity at final
concentration of 10 mM in the enzyme assay mixture. PMSF
Phenylmethylsulphonylfluoride. Data are the means of three
replicates
0
20
40
60
80
100
120
0 20 40 60 80 100 120 140
Time (min)
Relative activity (%)
Fig. 7 Thermostability profile of xylanase at 50C(d), 60C(n)
and 70C(m). Data are the means of three replicates
World J Microbiol Biotechnol (2007) 23:491–500 497
123

pH 8) and dialyzed for 24 h. The dialyzed enzyme
preparation was then loaded onto a pre-equilibrated
column of CM-Sephadex, the column was washed with
Tris buffer (50 mM, pH 8) and the run-through was
collected. Elution was carried out at different salt con-
centrations (0.1–0.5 M NaCl, and the final elution was
effected with 1 M NaCl). The enzyme and the protein
contents of the different eluted fractions were then
determined. The maximum enzyme activity was ob-
tained in the fraction eluted at 0.3 M NaCl (specific
activity: 48.33 U/mg protein), indicating that the en-
zyme was purified by 5.08-fold. Bakir et al. (2001) re-
ported a 2.48-fold purification of R. oryzae xylanase,
while Shah and Madamwar (2005) achieved a 4.98-fold
purification of xylanase from A. foetidus using ammo-
nium sulfate fractionation. Carmona et al. (2005) used
ion exchange and gel filtration chromatography to
achieve a 28-fold purification of xylanase II from
A. versicolor.
Zymogram analysis and molecular weight
determination
Native PAGE was performed for zymogram analysis.
When the native PAGE gel was over-layered onto the
agar gel seeded with substrate xylan, a clear zone on
the agar gel indicated the presence of xylanase and
helped localize the xylanase (Fig. 9a, b). Zymogram
analysis revealed the presence of a single xylanase in
the enzyme preparation obtained after CM-Sephadex
chromatography.
Jiang et al. (2004) also used this technique for zymo-
gram analysis in addition to studying the zymogram in
SDS-PAGE. These researchers copolymerized the xy-
lan in the acrylamide gel and, following electrophoresis,
the SDS was removed by soaking the gel in isopropanol,
which renatured the xylanase protein. However, in the
present study we could not perform zymogram analysis
in SDS-PAGE. Badhan et al. (2004) also could not study
the zymogram using SDS-PAGE and used instead
nondenaturing PAGE.
Table 1 Purification of xylanase from Aspergillus niveus
Purification
step
Total
protein
(mg)
Total
activity
(U)
Specific
activity
(U/mg
protein)
Purifica-
tion
fold
Recovery
(%)
Crude
enzyme
210.5 2000 9.50 1 100
Ammonium
sulfate
precipita-
tion
(25–50%)
12.5 260 20.8 2.18 13
CM-Sephadex
chromato-
graphy
3.0 145 48.33 5.08 7.25
Fig. 9 Zymogram (a), native PAGE (b) and SDS-PAGE (c)
showing the location of xylanase. A Ammonium sulfate-
precipitated fraction, P carboxymethyl-sephadex-purified prepa-
ration, M molecular weight marker
498 World J Microbiol Biotechnol (2007) 23:491–500
123

SDS-PAGE was performed to determine the
molecular weight of the enzyme. Purified enzyme
preparation by CM-Sephadex had only one band that
corresponded to a molecular weight of approximately
22.5 kDa (Fig. 9c).
The molecular mass of xylanase from different
fungal species varied. Carmona et al. (2005) docu-
mented a molecular mass of 32 kDa for Aspergillus
versicolor xylanase. Lin et al. (1999) reported a
molecular weight of 23 kDa for xylanase from Ther-
momyces lanuginosus, while Bakir et al. (2001) re-
ported a molecular weight of 22 kDa for xylanase from
Rhizopus oryzae.
Kinetics study
The purified xylanase preparation obtained by ammo-
nium sulfate precipitation followed by ion exchange
chromatography was used for determining K
m
. Sub-
strate (oat spelt xylan) concentration was varied from
1.25–11.25 mg/ml in the reaction mixture. Initial reac-
tion rates versus substrate concentration showed that
the enzyme obeys Michealis-Menten kinetics and has a
low K
m
of 2.5 mg/ml. The V
max
was found to be
26 lmol/mg per minute. Bakir et al. (2001) reported
K
m
and V
max
values of 18.5 mg xylan/ml and 90 IU/mg
protein, respectively, for xylanase from Rhizopus ory-
zae, while Carmona et al. (2005) reported a K
m
and
V
max
of 6.5 and 1.440 lmol/mg per minute for xylanase
I and 2.3 and 5.6 lmol ml per minute for xylanase II,
respectively, from A. versicolor.
Conclusion
From the above results it is clear that the novel
xylanase-producing fungus Aspergillus niveus RS2 has
the ability to grow and produce xylanase under con-
ditions of high temperature and alkaline pH. This
xylanase was able to withstand moderately high tem-
peratures and pH. Further studies on understanding
the molecular basis of thermostability and alkali
stability of fungal xylanses should be initiated.
Acknowledgements The authors wish to thank the Head,
Department of Biotechnology, University of Jammu, Jammu, for
the laboratory facilities, and Dr. Yash Pal Reader, Department
of Botany, University of Jammu, Jammu, for kindly helping in
the identification of the fungus.
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