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Original Research Paper

Enhanced production of cellulase-free, thermo-alkali-solvent-stable


xylanase from Bacillus altitudinis DHN8, its characterization and
application in sorghum straw saccharication
Dharmesh N. Adhyaru
a
, Nikhil S. Bhatt
a,n
, H.A. Modi
b
a
P.G. Department of Microbiology, Gujarat Vidyapeeth, Sadra-382 320, Gujarat, India
b
Department of Life Sciences, School of Sciences, Gujarat University, Ahmadabad, Gujarat, India
a r t i c l e i n f o
Article history:
Received 16 September 2013
Received in revised form
7 October 2013
Accepted 11 October 2013
Available online 23 October 2013
Keywords:
Bacillus altitudinis DHN8
Cellulase-free xylanase
Submerged fermentation
Partial purication
Chemical pretreatments
Sorghum straw saccharication
a b s t r a c t
A newly isolated Bacillus altitudinis DHN8 was assessed for xylanase production by utilizing sorghum
straw. The highest xylanase production was recorded at sorghum straw 3% w/v, inoculum size 1% v/v,
inoculum age 18 h, incubation time 42 h, pH 7.0, temperature 35 1C and agitation speed 250 rpm.
Moreover, xylose 0.5%, gelatine 0.5% and KNO
3
0.3% (w/v) further enhanced the production. The detailed
optimization study resulted in a 3.74-fold increase in xylanase production as compared to that of
the initial conditions. The partially puried xylanase showed 70% pH stability after 18 h at pH range of
610. Thermostability study revealed more than 60% xylanase activity at temperature range 4565 1C
after 60 min. The presence of metal ions (10 mM CaCl
2
, MnCl
2
and FeCl
3
) and solvents (10% v/v
isopropanol, methanol, ethanol and acetone) were increased xylanase activities remarkably. During
saccharication study, 3% alkaline hydrogen peroxide treatment was found to be benecial for the
maximum enzymatic hydrolysis of sorghum straw (34.94 mg/g reducing sugar) after 36 h. As such, this
xylanase could be considered as a cellulase-free, thermo-alkali-solvent stable biocatalyst being important
tool for many biotechnological industries.
To the best of our knowledge, this is the rst report on the production of xylanase by this Bacillus
species.
& 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Xylan is the principal hemicellulose which is the major compo-
nent of plant cell wall. It is the second plentiful polysaccharide after
cellulose, in both hard woods and annual plants (Qie et al., 2010;
Polizeli et al., 2005) and accounts for 2035% of the total dry weight
in tropical plant biomass. Endo-xylanases (EC 3.2.1.8) are an impor-
tant group of industrial enzymes responsible for complete hydrolysis
of xylan in to short xylooligosaccharides and xylose in synergism
with other accessory enzymes such as -xylosidase, -L-arabinofur-
anosidase and -glucuronidase (Chavez et al., 2006; Collins et al.,
2005). Xylanases have been reported from bacteria, fungi, actino-
mycetes and yeast (Qie et al., 2010; Sunna and Antranikian, 1997)
but bacteria due to their high metabolic diversity are widely used for
xylanase production. Several Bacillus sp. are known to secrete high
levels of extracellular xylanases which are either poor or free in
cellulase activity. In recent years, xylanases have attracted a great
deal of attention because of their biotechnological potential in the
various industrial processes such as, in food industry in order to
enhance the digestibility of animal feed, in textile industry and in
the paper and pulp industry for bleaching purpose resulting in a
decrease of chlorine utilization and consequently lowering environ-
mental impact (Chivero et al., 2001). Xylanases in synergism with
several enzymes can be used for the generation of biological fuels,
such as ethanol and xylitol from lignocellulosic biomass (Cormana
et al., 2005; Beg et al., 2001).
The major obstacle for wide range application of the xylanases
in industries is high cost of the production. Pure substrates being
highly expensive and thus it cannot be afforded at the industrial level
bulk production of enzymes. Therefore, it is necessary to explore
cheap substrates for cost-effective enzyme production. Agricultural
residues represent one such cheap raw material for industrial
production of enzymes. Furthermore, each bacterium has its own
special conditions for maximum enzyme production. Thus, the hyper
production of industrial enzymes by optimizing various physiological
and nutritional parameters is of prime importance because an
improper optimization of these growth parameters leads to a lower
enzyme production. In addition of the above problems, majority
of the reported xylanases do not withstand the robust industrial
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/bab
Biocatalysis and Agricultural Biotechnology
1878-8181/$ - see front matter & 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bcab.2013.10.003
n
Corresponding author. Tel.: 91 98794 83847/079 23274274;
fax: 079 232 74 272.
E-mail addresses: bhattnikhil2114@gmail.com, bhattnikhil_brc@yahoo.in
(N.S. Bhatt).
Biocatalysis and Agricultural Biotechnology 3 (2014) 182190
process conditions of high alkaline pH and high temperatures
(Sharma and Bajaj, 2005). Therefore, search for still better enzymes
which comply well with the industrial processes is in progress
(Kumar and Satyanarayana, 2011).
In view of the demand for novel xylanases with industrially
important properties, an attempt has been made to assess the
potentiality of newly isolated Bacillus species for the xylanase
production using low cost agricultural residue under submerged
fermentation. The partially puried enzyme was characterized
for few properties and further examined for its application in
the saccharication of sorghum straw.
2. Material and methods
2.1. Isolation, screening and identication of xylanase producing
strain
2.1.1. Primary screening
Different soil samples were collected from the active compost
pit (10 cm depth) during mid of the April 2011. Each sample was
mixed thoroughly and 1 g of that was suspended in 50 ml sterile
distilled water. Mixtures were allowed to settle down and then serial
dilutions were prepared. From each dilution, 0.1 ml was spread on
oat spelt xylan (1% w/v) agar plates and incubated at 37 1C for 48 h.
Those colonies showed a clear zone of xylan hydrolysis were isolated
and retained for further secondary screening.
2.1.2. Secondary screening
The isolates from the primary screening were cultured in basal
liquid media containing (g/l): Oat spelt xylan, 10.0; beef extract, 3.0;
NaCl, 5.0; KNO
3
, 2.0; K
2
HPO
4
, 1.0 and MgSO
4
7H
2
O, 0.5 at pH 7.0 in
100 ml Erlenmeyer asks and incubated at 37 1C under shaking
(150 rpm). After 48 h of incubation the fermented broth was
centrifuged at 10,000g for 10 min and the supernatant was used
for enzyme assay.
2.1.3. Bacterial identication
A total of 15 bacterial cultures were selected for the xylanase
production based on the secondary screening. The most promising
isolate DHN8 was identied on the basis of its morphological and
biochemical characterization. Further conrmation was done by
16S rRNA gene sequencing at Gujarat State Biotechnology Mission
(GSBTM), Gandhinagar, India. The gene sequence has been sub-
mitted to Gene Bank at NCBI (GenBank accession no. KF258832).
The sequence was compared with sequences available in NCBI
database and dendrogram was generated using Mega 5.2 software.
2.2. Selection of agro-residue for enhanced cellulase free xylanase
production
For enhanced xylanase production various agro-residues such
as caster shell, sugarcane bagasse, saw dust, rice straw, barley
straw, sorghum straw, wheat straw, groundnut shell and maize
straw were evaluated. Xylanase production was carried out using
2% (w/v) agro-residue in the 100 ml basal liquid medium (pH 7.0)
containing same medium components but without oat spelt
xylan. The asks were sterilized at 121 1C for 15 min, cooled and
inoculated with 1% (v/v) of bacterial culture. The contents were
incubated at 37 1C under shaking condition (150 rpm) for 48 h.
The fermented broth was centrifuged at 10,000g for 10 min at 4 1C
and the clear supernatant was analysed for enzyme assay.
2.3. Analytical methods
All the experiments were done in triplicates and the values
presented are mean values7SD.
Chemicals such as oat spelt xylan, carboxy methyl cellulose
(CMC), bovine serum albumin (BSA) and dinitrosalicylic acid
(DNSA) were purchased from Hi Media Laboratories Ltd. All the
other chemicals, media, salts and reagents used were of analytical
grade (Sigma- Aldrich, Hi Media, Qualigens and Merck).
The xylanase activity was measured according to Bailey et al.
(1992). The reaction mixture consisting 450 ml of 1% oat spelt xylan
in 50 mM sodium phosphate buffer (pH 7.0) and 50 ml of enzyme
was incubated for 10 min at 50 1C. The amount of reducing sugar
released was quantied using 3,5-dinitrosalicylic acid (DNS) method
as described by Miller (1959). The CMCase and FPase activities were
determined according to IUPAC recommendation (Ghose, 1987). One
unit of xylanase or cellulase activity was dened as the amount of
enzyme required to liberate 1 mmol of xylose or glucose equivalent
per min under the specied conditions.
The protein content was measured according to Lowry et al.
(1951) with BSA as the standard.
2.4. Assessment of process parameters for cellulase free xylanase
production
2.4.1. Optimization of physiological parameters
2.4.1.1. Inoculum size. To study the effect of inoculum size, produc-
tion media were inoculated at a level of 0.5, 1.0, 2.0, 3.0, 4.0 and
5.0% (v/v) from the 12 h old bacterial culture broth.
2.4.1.2. Inoculum age. To study the effect of inoculum age on
xylanase production, inoculum was prepared by inoculating 50 ml
of basal liquid medium with bacterium DHN8 and incubated at
37 1C under shaking condition (150 rpm). At regular interval of 6 h
incubation, 1% (v/v) of inoculum was transferred to 100 ml fresh
fermentation medium followed by incubation at 37 1C for 48 h
under shaking at 150 rpm. The culture ltrate was centrifuged and
nally used for enzyme assay.
2.4.1.3. Incubation time. In order to achieve maximum xylanase
production, incubation time was varied from 12 to 60 h. The crude
enzyme was extracted and assayed at regular interval of 6 h.
2.4.1.4. pH. Initial pH of the fermentation mediumwas varied from
3 to 10. The pH was adjusted by using 1 MHCl or NaOH prior to
autoclaving.
2.4.1.5. Temperature. Xylanase production was studied at different
temperatures ranging from 25 to 50 1C.
2.4.1.6. Agitation speed. The rate of agitation was studied at 0 to
300 rpm with a difference of 50 rpm.
2.4.2. Optimization of nutritional parameters
2.4.2.1. Inuence of carbon sources. Inuence of various carbon sources
on the xylanase production were assessed at a concentration of
0.5% (w/v).
2.4.2.2. Inuence of organic and inorganic nitrogen sources. Various
organic and inorganic nitrogen sources were evaluated for enhan-
ced xylanase production at concentration of 0.5 and 0.3% (w/v),
respectively.
D.N. Adhyaru et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 182190 183
2.5. Partial purication and characterization of xylanase
Solid ammonium sulphate was slowly added to the culture
supernatant in an ice bath under constant stirring to achieve 70%
saturation. After centrifugation at 10,000g for 10 min at 4 1C,
the supernatant was removed and the precipitate was resus-
pended in 10 ml of 50 mM sodium phosphate buffer (pH 7.0).
The nal solution was dialysed against the same buffer overnight
at 4 1C with three intermittent changes of the buffer.
2.5.1. Effect of temperature on xylanase activity and stability
The optimum temperature was determined by incubating the
enzyme extract in 1% oat spelt xylan solution at various tempera-
tures (3575 1C). For thermostability assay, enzyme was incubated
at different temperatures in the absence of substrate for 120 min.
Aliquots were withdrawn at regular time intervals to test the
residual xylanase activity.
2.5.2. Effect of pH on xylanase activity and stability
The pH optima was determined by measuring relative activity at
various pH values using sodium citrate (pH 36), sodium phosphate
(pH 68) and glycine-NaOH (pH 810) buffers (50 mM). The stability
of xylanase was assessed by incubating enzyme samples for 24 h in
different buffers. At regular interval of 3 h samples were withdrawn
and residual enzyme activity was determined.
2.5.3. Effect of metal ions on xylanase activity
The effect of metal ions on enzyme activity was determined by
incubating the xylanase preparations in 10 mM metal solution for
30 min. Residual activity was measured by standard enzyme assay.
2.5.4. Effect of various solvents on xylanase activity
The enzyme was incubated with various alcoholic and non
alcoholic solvents at 10% (v/v) concentration for 30 min and residual
activity was measured by the standard assay procedure.
2.6. Chemical pretreatment of sorghum straw
Prior to the enzymatic hydrolysis, the sorghum straw was
given three separate chemical pretreatments. Ten grams of dried
and powdered sorghum straw was taken for each pretreatment
experiment. The biomass was mixed with 200 ml of 1 M NaOH (for
24 h), 1 M HCl (for 12 h) and 3% alkaline hydrogen peroxide, pH
11.0 (for 12 h) separately to obtain a solid:liquid ratio of 1:20. The
contents were kept under shaking (100 rpm) at 50 1C. The pre-
treated substrates were washed until neutrality and dried in oven
at 50 1C till constant weight was achieved.
2.7. Enzymatic hydrolysis
Enzymatic hydrolysis of sorghum straw was carried out in
50 ml Erlenmeyer ask with 2.5% (untreated and pre-treated)
substrate and 20 ml of appropriately diluted crude enzyme at
50 1C, 100 rpm for 48 h. Controls were kept in which active
enzyme was replaced with heat inactivated enzyme. The reaction
system was supplemented with 0.005% sodium azide to prevent
microbial growth. Aliquots were taken at regular intervals of 12 h,
centrifuged and the supernatant was assayed for total reducing
sugar by 3,5-dinitrosalysilic acid method (Miller, 1959).
2.8. Analysis of hydrolysed products by TLC
The products of enzymatic hydrolysis of sorghum straw were
examined by ascending thin-layer chromatography (TLC) on pre-
coated silica gel plates (60 F254, Merck) using acetone/ isopropyl
alcohol/water (6:3:1.5, v/v/v) as mobile phase. At dened times,
reaction mixtures were sampled, the enzyme activity was stopped
by boiling for 10 min and 3.0 ml samples were applied on TLC
plates. The separated products were detected by spraying the plate
with -naphthol (3.5% in ethanol and 10% sulphuric acid) followed
by heating at 100 1C.
3. Results and discussion
3.1. Isolation and identication of strain DHN8
The newly isolated DHN8 strain was Gram positive, strictly
aerobic, spore forming, rod shaped and peritrichous. It reacted
positively in the catalase and oxidase tests. According to these
results, it was clear that the bacterium belonged to the genus
Bacillus species. Based on the results of 16S rRNA gene sequencing
the bacterium was identied as Bacillus altitudinis (accession no.
Fig. 1. The phylogenetic dendrogram of Bacillus altitudinis DHN8 was labelled with an aster and related species by the neighbour-joining approach. Bootstrap values obtained
with 1000 resamplings are indicated as percentages at all branches. The scale bars represent 0.05 substitutions per nucleotide position. Numbers following the names of the
strains are accession numbers of published sequences.
D.N. Adhyaru et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 182190 184
KF258832) and designated as B. altitudinis DHN8. Through the
alignment of homologous sequences of known bacteria, phyloge-
netic position of the strain is shown in Fig. 1.
3.2. Effect of various agro-residues on xylanase production
Among tested agro residues, B. altitudinis DHN8 exhibited clear
preference towards sorghum straw for highest xylanase produc-
tion (65.4972.18 IU/ml) followed by wheat straw, sugarcane
bagasse, barley straw and rice straw (62.0071.91, 47.0071.68,
42.6871.16 and 34.7371.21 IU/ml, respectively). However, lower
xylanase activities were observed in presence of maize straw,
caster shell, groundnut shell and saw dust (Fig. 2). The higher
xylanase production in presence of sorghum straw might be due
to the nature of hemicellulose, the presence of some activators
in the carbon source, surface, pore size and favourable degrad-
ability of carbon source (Gomes et al., 1993). However, many
research groups used hemicellulosic substrates such as wheat
bran, rice straw, wheat straw, soybean akes, rice bran, sugarcane
bagasse and groundnut shells for xylanase production (Battan
et al., 2007; Sharma et al., 2007; Taneja et al., 2002; Gawande
and Kamat, 1999). These data suggested that the xylanase induc-
tion is a complex phenomenon and the level of response to
different inducers varies with the strains. This study highlighted
that choosing an appropriate agro-residue can improve enzyme
production markedly.
Further different concentrations of sorghum straw ranging
from 1.0 to 6.0% (w/v) were tested under submerged fermentation.
The maximum xylanase production (68.4972.37 IU/ml) was
achieved at 3% (w/v) sorghum straw (Fig. 3). The gradual decrease
in xylanase production was observed above 3% sorghum straw
concentration. The possible reason behind such xylanase produc-
tion behaviour could be formation of thick suspension in presence
of higher substrate concentration resulted in improper mixing of
the substrate under agitation condition.
3.3. Effect of inoculum size, inoculum age and incubation period
An optimum inoculum level is necessary for maintaining balance
between the proliferating biomass and available nutrients to produce
maximum enzyme level. The highest xylanase production (72.847
2.64 IU/ml) was achieved when the production medium was inocu-
lated with 1% (w/v) of 12 h old inoculum (Fig. 4). The enzyme titre
declined drastically with increase in inoculum size beyond the
optimum values could be due to faster nutrient consumption. In
earlier studies, Battan et al. (2007) and Nagar et al. (2010) also showed
the use of 1.05.0% (v/v) inoculum size for hyper xylanase production.
Higher concentration of inoculum is not preferable in industrial
fermentations (Lincon, 1960).
The effect of inoculum age was studied from 6 to 48 h old
culture of B. altitudinis DHN8. The maximum xylanase production
91.3872.68 IU/ml was obtained with 18 h old inoculum (Fig. 5)
but decreased thereafter. These results obtained might be due to
the fact that, the maximum enzyme titre is produced during early
to late exponential phase of the organism. It also suggests that the
partial association existed between growth of the organism and its
enzyme production pattern. Kumar et al. (2012) and Nagar et al.
(2010) also showed the use of 18 h old inoculum for xylanase
production.
The time of fermentation for maximum enzyme production
varies among different bacteria and is dependent upon the organism
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CS SB SD RS BS SS WS GS MS
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Agro-residues
Xylanase activity Protein
Fig. 2. Effect of various agro residues on xylanase production. CS: caster shell,
SB: sugarcane bagasse, SD: saw dust, RS: rice straw, BS: barley straw, SS: sorghum
straw, WS: wheat straw, GS: groundnut shell, MS: maize straw. The fermentation
medium containing 2% w/v agro residue, beef extract, 3.0; NaCl, 5.0; KNO
3
, 2.0;
K
2
HPO
4
, 1.0 and MgSO
4
7H
2
O, 0.5 at pH 7.0 was inoculated with 12 h old bacterial
culture at 1% v/v. The enzyme production was carried out at 37 1C under shaking
(150 rpm) for 48 h.
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Sorghum straw concentration (%)
Xylanase activity Specific activity Protein
Fig. 3. Effect of sorghum straw concentration (16% w/v) on xylanase production.
The fermentation medium (pH 7.0) with varying concentration of sorghum straw
was inoculated with 12 h old bacterial culture at 1% v/v. The enzyme production
was carried out at 37 1C under shaking (150 rpm) for 48 h.
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Inoculum size (% v/v)
Xylanase activity Specific activity Protein
Fig. 4. Effect of inoculum size on xylanase production. The 3% w/v sorghum straw
containing fermentation medium (pH 7.0) was inoculated with 12 h old bacterial
culture at 1 to 5% v/v. The enzyme production was carried out at 37 1C under
shaking (150 rpm) for 48 h.
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Inoculum age (h)
Xylanase activity Specific activity Protein
Fig. 5. Effect of inoculum age on xylanase production. The fermentation medium
(pH 7.0) was inoculated at 1% v/v level with 12 to 60 h old bacterial culture. The
enzyme production was carried out at 37 1C under shaking (150 rpm) for 48 h.
D.N. Adhyaru et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 182190 185
type, its enzyme production pattern, cultural conditions and genetic
makeup of the organism. During course of incubation period, high-
est xylanase production (81.5672.85 IU/ml) was obtained at 42 h
of incubation and thereafter it declined gradually (Fig. 6). The
reduction in xylanase yield could be due to nutrients depletion or
due to proteolysis (Flores et al., 1997). Xylanase produced by Bacillus
sp. was found to be growth-associated, reaching a maximum after
24 h and the enzyme production remained more or less constant
up to 48 h (Anuradha et al., 2007). On the other hand, Bacillus
halodurans PPKS-2 (Prakash et al., 2011) and Bacillus SSP-34
(Subramaniyan and Prema, 2000) produced maximum xylanase
after 48 and 96 h, respectively.
3.4. Effect of pH, temperature and agitation speed
The B. altitudinis DHN8 was cultivated in the production medium
adjusted at different pH (3.010.0). The bacterium did not produce
satisfactory xylanase yield at initial medium pH 3.0 to 5.0 but at pH
6.0 xylanase titre was increased and reached maximum at pH 7.0
(85.7372.23 IU/ml). Moreover, afterwards from pH 8.0 to 10.0
substantial xylanase production was observed (Fig. 7). Xylanase
production by various bacteria and fungi has been shown to be
markedly dependent on pH (Wong et al., 1982). Acidic pH (4.06.0)
generally favours fungal xylanases (Bajpai, 1997) while higher pH
favours bacterial xylanases (Bisaria and Ghose, 1991). In tuning with
this nding, Bacillus pumilus ASH (Battan et al., 2007) and Bacillus
subtilis ASH (Sanghi et al., 2008) showed elevated xylanase produc-
tion at pH 7.0. However, Bacillus mojavensis AG137 (Sepahy et al.,
2011) and Bacillus NT 9 (Han et al., 2004) showed maximum
xylanase production at medium pH 8.0 and 10, respectively.
The xylanase production was conducted at different temperatures
(2550 1C). The maximum xylanase production (91.1472.01 IU/ml)
was obtained at 35 1C (Fig. 8) and reduced sharply at higher
temperature range of 40 to 50 1C. Although the physiological changes
induced by high temperatures during enzyme production are not
completely understood, it has been suggested that at higher tem-
peratures microorganisms may synthesize only a reduced number
of proteins essential for growth and other physiological processes
(Gawande and Kamat, 1999). These results might also be corresponds
to the growth prole of the microorganism where as no other
temperature was suitable to such extant for growth and enzyme
secretion. Many researchers reported 37 1C temperature for max-
imum xylanase production from Bacillus sp. (Nagar et al., 2010;
Sanghi et al., 2008; Battan et al., 2007).
During submerged fermentation process, agitation and aeration
are two important parameters used for uniform mixing of the
nutrients and to meet the oxygen demand. The B. altitudinis DHN8
exhibited signicant amount of xylanase production in the range
of 150 to 300 rpm agitation speed with optimum xylanase yield
(103.8773.46 IU/ml) at 250 rpm. However, under static and lower
agitation (50 rpm) conditions only 10.21 and 45.31% of the maximum
xylanase activity was observed (Fig. 9). The lower xylanase produc-
tion under static to low agitation conditions may be attributed to the
dissolved oxygen (DO) limitation, improper mixing of media compo-
nents and cell clumping. Various researchers showed maximum
xylanase production at an agitation rate of 200250 rpm (Kumar
et al., 2012; Battan et al., 2007; Taneja et al., 2002; Beg et al., 2001;
Gawande and Kamat, 1999).
3.5. Inuence of carbon and nitrogen sources
Carbon source is one of the most important factors during
the growth and metabolic process of microorganisms. The presence
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v
i
t
y

(
I
U
/
m
g
)

Incubation time (h)
Xylanase activity Specific activity Protein
Fig. 6. Effect of incubation time on xylanase production. The fermentation medium
(pH 7.0) was inoculated at 1% v/v level with 18 h old bacterial culture. The enzyme
production was carried out at 37 1C under shaking (150 rpm) and samples were
analysed at 6 h intervals.
0
0.5
1
1.5
2
2.5
0
10
20
30
40
50
60
70
80
90
100
3 4 5 6 7 8 9 10
P
r
o
t
e
i
n

c
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n
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t
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t
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(
m
g
/
m
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)

X
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a
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(
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/
m
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)
;

S
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a
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(
I
U
/
m
g
)

pH
Xylanase activity Specific activity Protein
Fig. 7. Effect of pH on xylanase production. The fermentation medium containing
different pH from 3 to 10 was inoculated at 1% v/v level with 18 h old bacterial
culture. The enzyme production was carried out at 37 1C under shaking (150 rpm)
for 42 h.
0
0.5
1
1.5
2
2.5
3
0
10
20
30
40
50
60
70
80
90
100
25 30 35 40 45 50
P
r
o
t
e
i
n

c
o
n
c
e
n
t
r
a
t
i
o
n

(
m
g
/
m
l
)

X
y
l
a
n
a
s
e

a
c
t
i
v
i
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y

(
I
U
/
m
l
)
;

S
p
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i
f
i
c

a
c
t
i
v
i
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y

(
I
U
/
m
g
)

Temperature ( C)
Xylanase activity Specific activity Protein
Fig. 8. Effect of temperature on xylanase production. The fermentation medium
(pH 7.0) was inoculated at 1% v/v level with 18 h old bacterial culture. The enzyme
production was carried out at different temperature range from 25 to 50 1C under
shaking (150 rpm) for 42 h.
0
0.5
1
1.5
2
2.5
3
0
20
40
60
80
100
120
0 50 100 150 200 250 300
P
r
o
t
e
i
n

c
o
n
c
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t
r
a
t
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(
m
g
/
m
l
)

X
y
l
a
n
a
s
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a
c
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v
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(
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/
m
l
)
;

S
p
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i
f
i
c

a
c
t
i
v
i
t
y

(
I
U
/
m
g
)

Agitation speed (rpm)
Xylanase activity Specific activity Protein
Fig. 9. Effect of agitation speed on xylanase production. The fermentation medium
(pH 7.0) was inoculated at 1% v/v level with 18 h old bacterial culture. The enzyme
production was carried out at 35 1C under static and shaking (0 to 300 rpm)
conditions for 42 h.
D.N. Adhyaru et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 182190 186
of carbon sources in the fermentation medium exerted profound
effect on the enzyme production behaviour of the bacterium. Among
various monosaccharide and disaccharides tested only xylose,
sucrose and starch (145.1373.85, 115.4173.66 and 111.073.91 IU/
ml, respectively) stimulated xylanase production. However, other
carbon sources failed to support high yield of xylanase production as
compared to control (Table 1). The most plausible explanation for
decrease in xylanase yield with other carbon sources is that these
sources exerted catabolite repression. Such carbon source dependent
enzyme production regulation has been noticed in different xylanase
producing microbial strains (Lakshmi et al., 2009; Oliveira et al.,
2006; Gassesse and MaMo, 1999).
To check the effect of nitrogen sources on xylanase production
different organic and inorganic nitrogen sources were added
individually to the fermentation medium (Table 2).
Amongst various organic nitrogen sources, maximum xylanase
production was obtained with gelatine (197.9476.00 IU/ml) followed
by urea, yeast extract, tryptone and beef extract. However, casein,
meat extract, malt extract and peptone did not showed stimulatory
effect on xylanase production. The enhanced production of xylanase
in presence of gelatine as organic nitrogen sources may be attributed
to organic nitrogen source mediated regulation of microbial growth
and metabolism (Gupta et al., 2000). Moreover, it has been also
observed that nitrogen can signicantly affect the pH of the medium
during the course of fermentation (Haapala et al., 1994) thereby
inuences the microbial metabolism. Bajaj and Manhas (2012)
reported 24.0% xylanase improvement in the presence of gelatine.
The most suitable organic nitrogen source for the xylanase production
by Geobacillus thermoleovorans (Sharma et al., 2007) and Bacillus
circulans AB16 (Dhillon and Khanna, 2000) was tryptone. B. moja-
vensis AG147 yielded maximum xylanase when tryptone and yeast
extract was used in combination (Sepahy et al., 2011).
During the evaluation of inorganic nitrogen sources KNO
3
showed
maximum xylanase production (245.0473.15 IU/ml). All other tested
inorganic nitrogen sources also showed increase in xylanase produc-
tion except (NH
4
)
2
H
2
PO
4
(103.7974.76 IU/ml) as compared to the
control set devoid of any nitrogen source. In accordance with these
results, Nagar et al. (2012) observed stimulatory effect of KNO
3
in
combination with peptone and yeast extract on xylanase production.
3.6. Partial characterization of xylanase produced by B. altitudinis
DHN8
3.6.1. Effect of temperature on activity and stability
The partially puried xylanase was active in the broad range
of temperatures 35 to 75 1C, with temperature optima at 50 1C.
The enzyme exhibited more than 60% of its activity in the range
from 45 to 65 1C. The xylanase activity was dropped at temperature
values above 70 1C and only 20% of relative xylanase activity was
detected at 75 1C (Fig. 10).
The enzyme thermostability depends upon molecular interac-
tions such as hydrogen bonds, electrostatic and hydrophobic inter-
actions, disulde bonds, and metal binding which can promote a
superior conformational structure for the enzyme with a higher
packing efciency, reduced entropy of unfolding, conformational
strain release and stability of -helices (Li et al., 2005). Thermo-
stability studies of B. altitudinis DHN8 xylanase showed residual
activity between 70 and 90% at 45 to 60 1C temperature after 60 min
of incubation. Even at 70 1C after 30 min of incubation 47% of the
residual activity was noticed. The obtained results clearly indicate
the nature of xylanase as thermostable. The suitable temperature
range for industrial application of present xylanase lies in 35 to 65 1C
(Fig. 11). Most of the bacterial xylanases showoptimumactivity at 50
to 60 1C (Bajaj and Singh, 2010). Identical temperature optima
at 50 1C were reported for the xylanases from Bacillus sp. (Nagar
et al., 2010; Polizeli et al., 2005). However, xylanase produced by
B. halodurans exhibited activity over 30100 1C, with an optimum at
Table 1
Inuence of carbon sources on cellulase-free xylanase production by Bacillus
altitudinis DHN8.
Carbon
sources
Xylanase activity
(IU/ml)
Protein
(mg/ml)
Specic activity
(IU/mg)
Control
a
103.1872.80 2.5470.09 40.5371.11
Glucose 62.0773.04 2.1070.06 29.6072.29
Maltose 83.9372.29 2.2870.07 36.7170.99
Starch 111.0073.91 2.2870.07 36.7172.30
Cellulose 68.3672.86 2.4470.09 27.9370.16
Sucrose 115.4173.66 2.9370.15 39.4170.80
Fructose 88.2972.66 2.2670.11 39.1171.69
Xylose 145.1373.85 3.1370.13 46.3172.02
Galactose 68.8773.70 2.3370.16 29.6672.72
Lactose 62.1271.71 2.3370.14 26.7272.36
Mannitol 85.3374.01 2.9370.06 29.1371.38
a
The control medium used was devoid of carbon source.
Table 2
Inuence of nitrogen sources on cellulase-free xylanase production by B.
altitudinis DHN8.
Nitrogen
source
Xylanase activity
(IU/ml)
Protein
(mg/ml)
Specic activity
(IU/mg)
Organic
Peptone 133.9673.13 3.0470.13 44.1071.40
Beef extract 144.2974.61 3.3370.07 43.3572.04
Yeast extract 173.6476.38 3.5470.08 48.9970.75
Casein 83.0372.66 2.5070.11 33.1470.54
Malt extract 101.0672.52 2.9070.10 34.8572.00
Trypton 154.7672.40 3.4970.14 44.3471.67
Gelatine 197.9476.00 3.8770.10 51.1772.12
Skim milk powder 144.8273.47 3.4270.09 42.2771.13
Meat extract 94.7873.94 2.3870.11 39.7971.21
Urea 183.8477.93 3.1870.08 57.6971.87
Inorganic
(NH
4
)
2
HPO
4
195.6578.69 3.8670.11 50.6770.79
(NH
4
)
2
H
2
PO
4
103.7974.76 3.2670.14 31.8470.93
KNO
3
245.0473.15 4.0070.17 61.3171.95
(NH
4
)
2
S
2
O
8
163.7372.49 3.6470.14 45.0071.10
NH
4
NO
3
213.8874.90 3.9170.16 54.6471.25
NH
4
Cl 177.3176.09 3.1070.09 57.1172.93
NaNO
3
198.1372.18 3.6670.15 54.0871.70
(NH
4
)
2
SO
4
207.0673.27 4.0870.19 50.8372.64
(NH
4
)
2
HPO
4
195.6578.69 3.8670.11 50.6770.79
Control
a
135.3973.87 3.1370.14 43.2371.08
a
The control medium used was devoid of nitrogen source.
0
20
40
60
80
100
120
35 40 45 50 55 60 65 70 75
R
e
l
a
t
i
v
e

a
c
t
i
v
i
t
y

(
%
)

Temperature ( C)
Fig. 10. Effect of temperature on the activity of partially puried xylanase. The
enzyme extract was incubated in 1% oat spelt xylan solution at different tempera-
tures (35 to 75 1C). The relative activity was measured using standard assay after
10 min.
D.N. Adhyaru et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 182190 187
80 1C (Kumar and Satyanarayana, 2011). B. halodurans PPKS-2
xylanase showed optimum temperature of 70 1C (Prakash et al.,
2011). Thermostability of different Bacillus sp. xylanases varies
between 55 and 80 1C (Kumar and Satyanarayana, 2011).
3.6.2. Effect of pH on activity and stability
Enzyme activity is markedly affected by pH because substrate
binding and catalysis are often dependent on charge distribution
of both substrate and particularly enzyme molecules. The xylanase
of B. altitudinis DHN8 exhibited the highest xylanase activity at pH
7.0 and retained more than 60% relative activity over broad range
of pH 5 to 10 (Fig. 12).
pH stability study revealed that the present xylanase was stable
in pH range of 6 to 10 and showed 70% residual activity after
18 h of incubation (Fig. 13). The results indicated that the partially
puried xylanase was alkali stable in nature. In similar study,
xylanase from Bacillus sp. GRE7 showed pH stability over the pH
range of 511 for 30 min (Kiddinamoorthy et al., 2008). Bacillus
stearothermophilus T-6 xylanase had stability at pH 6.510.0 for
5 min (Khasin et al., 1993). The differences in pH and temperature
stability for extracellular xylanases might be due to the post
transcriptional modications in xylanase excretion process, such
as glycosylation, that improved stability in more extreme pH and
temperature conditions (Savitha et al., 2007).
Thus, B. altitudinis DHN8 xylanase possessed cellulase-free
nature, broad pH stability spectrum and stability at elevated
temperature, could be important tool for application in many
industrial processes.
3.6.3. Effect of metal ions and additives on xylanase activity
Metal ions can be involved in enzyme catalysis in a variety
of ways: they may accept or donate electrons; they may them-
selves act as electrophiles; they may mask nucleophiles to prevent
unwanted side reactions; they may bring together enzyme and
substrate by coordinate bonds; they may hold the reacting groups
in the required 3D orientation and they may simply stabilize
a catalytically active conrmation of the enzyme (Palmer, 2001).
As shown in Table 3, xylanase activity was assayed in the presence
of several metal solutions (10 mM). The xylanase activity was
markedly stimulated in the presence of metal ions such as CaCl
2
(168.00%), MnCl
2
(126.95%) and FeCl
3
(106.01%). However, signi-
cant inhibition in xylanase activity was found in the presence
of other metal compounds. HgCl
2
was acted as strong inhibitory
compound (64% inhibition), suggesting that there is an impor-
tant cystein residue in or close to the active site of the enzyme
(Khandeparkar and Bhosle, 2006). Kumar and Satyanarayana
(2011) reported that the activity of B. halodurans xylanase was
strongly inhibited by Sn
2
, Hg
2
, Cu
2
and Cd
2
. More than
a quarter of all known enzymes require the presence of metal
atoms for fully catalytic activity (Palmer, 2001). These effects
reveal which kind of ions should be precluded or included in
industrial processes.
The reports on the solvent stable xylanases are very rare.
The inuence of various alcoholic and non alcoholic solvent additives
on the activity of xylanase is shown in Table 3. The xylanase activity
was stimulated in the presence of isopropanol (126.07%), methanol
(117.81%), ethanol (108.69%) and acetone (102.96%). However, pre-
sence of 1-butanol, 2-butanol, benzene and toluene resulted in
reduced xylanase activity. Similar observations for xylanase stimula-
tion and suppression in presence of straight chain alcohols were
reported by Li et al. (2010). Biocatalysis in organic media offers several
0
20
40
60
80
100
120
0 30 60 90 120
R
e
s
i
d
u
a
l

a
c
t
i
v
i
t
y

(
%
)

Time (min)
35 C
40 C
45 C
50 C
55 C
60 C
65 C
70 C
75 C
Fig. 11. Effect of temperature on the stability of partially puried xylanase.
The enzyme extract was incubated at different temperatures (35 to 75 1C) for
120 min. The residual activity was measured after each 30 min intervals.
0
20
40
60
80
100
120
3 4 5 6 7 8 9 10
R
e
l
a
t
i
v
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a
c
t
i
v
i
t
y

(
%
)

pH
pH 3-6 pH 6-8 pH 8-10
Fig. 12. Effect of pH on the activity of partially puried xylanase. The enzyme
extract was incubated in different buffers: sodium citrate (pH 36), sodium
phosphate (pH 68) and glycine-NaOH (pH 810) buffers (50 mM).
0
20
40
60
80
100
120
0 3 6 9 12 15 18 21 24
R
e
s
i
d
u
a
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a
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t
i
v
i
t
y

(
%
)

Time (h)
pH 3
pH 4
pH 5
pH 6
pH 7
pH 8
pH 9
pH 10
Fig. 13. Effect of pH on the stability of partially puried xylanase. The enzyme
extract was incubated for 24 h in different buffers (pH 3 to 10) for 24 h. At regular
interval of 3 h samples were withdrawn and residual enzyme activity was
determined.
Table 3
Effect of metal compounds and solvents on cellulase-free xylanase activity.
Metal compound
(10 mM)
Relative xylanase
activity (%)
Solvents
(10% v/v)
Relative xylanase
activity (%)
Control
a
100.0071.42 Control 100.0071.42
CoCl
2
72.7072.45 Methanol 117.817 5.04
FeCl
3
106.017 4.98 Ethanol 108.6972.56
MnCl
2
126.9573.21 Isopropanol 126.0774.90
CaCl
2
168.7278.66 1-Butanol 89.3472.47
HgCl
2
46.8772.03 2- Butanol 92.5673.83
ZnCl
2
62.3673.00 Acetone 102.9672.81
NiCl
2
32.3571.21 Toluene 67.2172.99
MgCl
2
62.9571.27 Benzene 76.1071.60
a
The control medium used was devoid of any modulator or solvent.
D.N. Adhyaru et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 182190 188
advantages viz. higher solubility of hydrophobic substrate enabaling
their effective reactions, reduced microbial contamination and reusa-
bility (Sardessai and Bhosle, 2004). These results provide essential
data for the application of xylanase in an organic phase industrial
reactions.
3.7. Enzymatic hydrolysis of sorghum straw
The utilization of enzymatic hydrolysis to obtain sugars from
agricultural residues is of great interest in modern biotechnology
particularly for bio-solvent production. Fig. 13 depicts the effect of
enzymatic hydrolysis on reducing sugar production from differ-
ently pretreated sorghum straw. The 3% alkaline hydrogen per-
oxide pretreated sorghum straw yielded maximum reducing sugar
(34.94 mg/g) after 36 h of enzymatic hydrolysis. Whereas, alkali
pretreated, acid pretreated and untreated biomass yielded 29.56,
23.81 and 2.58 mg/g reducing sugar after 48 h. In 3% alkaline
hydrogen peroxide pretreated sorghum straw after an initial phase
of rapid sugar formation during 36 h the rate of hydrolysis
decreased which could be due to enzyme inactivation or depletion
of an easily hydrolysable fraction of hemicellulose. As compared to
alkali and acid pretreatments, alkaline hydrogen peroxide treat-
ment is more effective in lignin solubilisation (Chen et al., 2008)
and improving of crop residue digestibility (Talebnia et al., 2010)
(Fig. 14).
4. Conclusion
Due to the increasing economic relevance of xylanases present
study was designed and attempt was made to optimize different
process parameters. The detailed optimization study resulted in a
3.74-fold enhancement in xylanase production as compared to
that of the initial conditions. The cellulase-free and thermo-alkali-
solvent stable xylanase produced by the newly isolated B. altitu-
dinis DHN8 using least reported sorghum straw as substrate is one
of the rare xylanases because of its stability at extreme process
conditions prevailing in the paper industry. The xylanase was able
to release reducing sugars mainly xylose from alkaline hydrogen
peroxide treated sorghum straw biomass which could be further
fermented to biofuel.
Acknowledgement
Authors are greatly acknowledges the nancial assistance from
Department of Science and Technology (DST), Ministry of Science
and Technology, Govt. of India, for giving INSPIRE Fellowship to
Mr. Dharmesh N. Adhyaru, under INSPIRE Program.
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0
5
10
15
20
25
30
35
40
0 12 24 36 48
R
e
d
u
c
i
n
g

s
u
g
a
r

(
m
g
/
g
)

Time (h)
Untreated Acid treated Alkali treated Alkaline peroxide treated
Fig. 14. Enzymatic hydrolysis of chemically pretreated sorghum straw using crude
xylanase from B. altitudinis DHN8. Saccharication was carried out at 50 1C,
100 rpm for 48 h. Aliquots were taken at regular interval of 12 h and analysed for
reducing sugar production.
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