Puri

fication, characterization and mass spectroscopic analysis

of thermo-alkalotolerant

b

-1, 4 endoxylanase from Bacillus sp.

and its potential for dye decolorization

Monika Mishra, Indu Shekhar Thakur

*

School of Environmental Sciences, Jawaharlal Nehru University, JNU Campus, New Delhi 110 067, India

a r t i c l e i n f o

Article history:
Received 12 November 2010
Received in revised form
2 December 2010
Accepted 2 December 2010
Available online 31 December 2010

Keywords:
Alkalotolerant
Bacillus sp.
Decolorization
Dyes
Thermostable
Xylanase

a b s t r a c t

A Bacillus sp., isolated from sludge and sediments of pulp and paper mill, was found to produce xylanase in
a synthetic culture media containing oat spelt xylan (1% w/v) and 10% black liquor as inducers along with
2.5% (w/v) sucrose as additional carbon source. The puri

fied enzyme was highly thermostable with half-life

of 10 min at 90

C and pH 8. The enzyme was stable over a broad range of pH (pH 6

e10) and showed good

thermal stability when incubated at 70

C. Chemicals like EDTA, Hg

2

þ

, Cu

2

þ

and solvents like glycerol and

acetonitrile completely inhibited enzyme activity at high concentration. The molecular weights of the
puri

fied enzyme, determined by matrix-assisted laser desorption/ionization coupled with time-of-flight

mass spectrometry (MALDI-TOF/MS) analysis was analogous to the results obtained from SDS-PAGE, i.e.
55 kDa. Kinetic parameters were determined by using oat spelt xylan as substrate. The K

M

and V

max

values

of the enzyme were 4.4 mg/ml and 287 U/mg respectively. At high xylan concentrations (

>70 mg/ml)

a substrate inhibition phenomenon of the enzyme was observed. In addition, crude xylanase showed
enormous potential for decolorization of various recalcitrant dyes.

Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction

The growing public concern over the environmental impact

of pollutants from paper and pulp industry is the motivating
force behind the search for novel bleaching techniques. Chlorinated
phenolics, chlorinated dehydroabietic acids and polychlorinated
biphenyls are produced from residual lignin during conventional
pulp bleaching (

Mohn and Stewart 1997

). Residual Lignin is very

dark in color due to extensive oxidation and modi

fication. Covalent

interaction of lignin with hemicelluloses and cellulose

fibers makes

it very dif

ficult to be removed from pulp (

Shoham et al. 1992

).

Most of the chloroaromatic compounds released during the pulp
bleaching process e.g. chlorophenols, chlorobiphenyls and other
chlorolignin derivatives such as 2, 3, 7, 8-tetrachlorodibenzo-p-
dioxin, are toxic and accumulate in the biotic and abiotic compo-
nents of the ecosystem (

Bedard et al. 1987; Larsson et al. 1988;

Shoham et al. 1992; Yang et al. 1992; Mohn and Stewart 1997;
Valenzuela et al. 1997; Ali and Sreekrishnan 2001

).

Xylans, with a linear backbone of L-1, 4-linked xylose residues,

form the major group of hemicelluloses present in the wood.

Xylanases are of great importance to pulp and paper industries since
the hydrolysis of xylan facilitates the release of lignin from pulp,
thereby reducing the use of chlorine as a bleaching agent (

Shoham

et al. 1992

). The Kraft pulping process at higher temperatures and

pH ranges necessitates the search for alkalotolerant thermophilic
xylanases.

Jurasek and Paice (1986)

and

Viikari et al. (1986)

were the

first to demonstrate that xylanases could be useful in the paper and
pulp industry.

Due to xylan heterogeneity, the enzymatic hydrolysis of xylan

requires different enzymatic activities. Two major enzymes,

b

-1,4-

endo-xylanase (EC 3.2.1.8) and

b

-xylosidase (EC 3.2.1.37) are

responsible for hydrolysis of the main chain of xylan. The former one
attackes the internal main-chain xylosidic linkages and the later one
releases xylosyl residues by endwise attack on xylooligosaccharides
(

Subramaniyan and Prema 2002

). These two enzymes, produced by

biodegradative microorganisms such as Trichoderma, Aspergillus,
Schizophyllum, Bacillus, Clostridium and Streptomyces sp., are the
major components of xylanolytic systems (

Bedard et al. 1987; Yang

et al. 1992; Valenzuela et al. 1997

). However, for complete hydro-

lysis of the molecule, side-chain cleaving enzyme activities are
also necessary. Since lignocellulose is an abundant and renewable
resource, we have already used it for the production of countless
artifacts. Today, we are trying to exploit microbial capabilities in
biodegradation to expand our uses of this biomass resource. The use

* Corresponding author. Tel.: þ91 11 2670 4321.

E-mail addresses:

[e-mail]

(M. Mishra),

isthakur@hotmail.

com

(I.S. Thakur).

Contents lists available at

ScienceDirect

International Biodeterioration & Biodegradation

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / i b i o d

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e see front matter Ó 2010 Elsevier Ltd. All rights reserved.

doi:

10.1016/j.ibiod.2010.12.002

International Biodeterioration & Biodegradation 65 (2011) 301

e308

of microbial enzymes for the industrial hydrolysis of lignocellulose is
advantageous because of the high speci

ficity of enzyme reactions, the

mildness of the reaction conditions, and the absence of substrate
loss due to chemical modi

fications. Hydrolysis products from ligno-

cellulose may be subsequently converted into liquid fuel, single-cell
protein, solvents, and other chemical products by using selected
fermentative microorganisms.

Xylanases have diverse applications including biodegradation

of lignocelluloses in animal feed, foods, and textiles, Kraft pulp
bleaching and biopulping in the pulp and paper industry (

Schwien

and Schmidt 1982; Viikari et al. 1986; Madlala et al. 2001

). The most

promising application of xylanases is in the prebleaching of pulps,
which could improve pulp

fibrillation and water retention, reduc-

tion of beating times in virgin pulps, restitution of bonding and
increase freeness in recycled

fibers, and removal of xylans from

dissolving pulps (

Beg et al. 2001

).

Although xylanases from various microorganisms have been

reported, few of them are alkalotolerant (

Ohkoshi et al. 1985;

Tsujibo et al. 1990; Dey et al. 1992; Kohli et al. 2001; Mamo et al.
2006

). In this study, we describe the culture conditions necessary

for optimal production of alkaline xylanase(s) from Bacillus sp.
reported earlier (

Mishra and Thakur 2010

). In addition to partial

puri

fication, characterization and MALDI-TOF spectral analysis of

the extracellular bacterial xylanase have also been carried out.
Furthermore, the application of this xylanase in dye degradation
studies also proved it as a promising bioremediation agent for
treatment of colored wastewaters.

2. Material and methods

2.1. Bacteria and culture condition

The bacterial strain, Bacillus sp., used in this study, was taken

from our previous study (

Mishra and Thakur 2010

). Inoculum of

Bacillus sp. was prepared by growing the bacterium overnight in LB
broth. This was inoculated in minimal salt media (MSM) containing
(g/l): Na

2

HPO

4

$2H

2

O, 7.8; KH

2

PO

4

, 6.8; MgSO

4

, 0.2; Fe (CH

3

COO)

3

NH

4

, 0.05; Ca(NO

3

)

2

$4H

2

O, 0.05; NaNO

3

, 0.085; at pH 8

(

Thakur 2004

) and incubated in a rotary shaker (200 rpm) at 35

C

for 48 h. The growth media was optimized by adding sucrose
(2.5% w/v) as additional carbon sources and oat spelt xylan (1%w/v)
along with black liquor (10%v/v) as xylanase inducer to enhance the
production of xylanase. Xylanase activity was determined after 48 h
of incubation. Cell free culture supernatant was obtained after
centrifugation at 10,000

g for 10 min.

2.2. Enzyme assay

Xylanolytic activity was quantitatively determined using 3, 5-

dinitrosalicyclic acid (DNS) method (

Krisana et al. 2005

). The assay

was based on the enzymatic hydrolysis of xylan, and the reaction of
the liberated reducing sugar with DNS. Xylose was used as a reference
reducing sugar for preparing a standard curve. One unit of xylanase
activity was de

fined as the quantity of enzyme that released reducing

sugar xylose at the rate of 1

m

M/min at 70

C and pH 8. Protein

estimation of xylanase solution was performed by the Bradford
method using Bovine Serum Albumin as a standard (

Bradford 1976

).

2.3. Production and puri

fication of enzyme

The protein puri

fication was carried out at 4

C. The culture

supernatant, after centrifugation (10,000

g for 10 min), was

filtered with 0.2

m

size

filter membrane (millipore). This filtrate

was dialyzed against sucrose to obtain concentrated protein. This
concentrated crude extract was applied to DEAE cellulose ion

exchange column equilibrated with the same buffer used in xylanase
assay. Proteins were eluted with a step gradient of Tris

eCl

(0

e1000 mM) at a flow rate of 0.7 ml/min. Fractions having xylanase

activity were pooled and applied to Sephadex-G100 to determine
molecular weight of the protein by using bovine serum albumin
(66 kDa), ovaalbumin (45 kDa), carbonic anhydrae (31 kDa) and
lysozyme (14 kDa) as molecular marker. Protein samples of 5 ml
were collected at

flow rate of 0.4 ml/min and the absorbance

was measured at 280 nm. Fractions with high protein content were
further checked for xylanase activity. SDS-PAGE was performed
by the method of

Laemmli (1970)

with polyacrylamide gel (10%).

The protein bands were stained with Coomassie Brilliant blue R-250
(Sigma) and compared with the molecular weight markers.

2.4. Matrix assisted laser desorption ionization time-of-

flight

mass spectrometry (MALDI-TOF/MS)

The excised protein bands were prepared for MALDI-TOF/MS

analysis by the method of

Kumar et al. (2009)

with some modi

fi-

cations. Excised bands from SDS-PAGE, were digested with trypsin
(200 ng) in 50 mM NH

4

HCO

3

at 37

C for overnight followed by

vacuum drying in a Savant SVC100 Speed Vac and desalting using
C

18

Omix microextraction column tips (Varian, Palo Alto, CA). These

resultant peptides were mixed with matrix solution (

w1.5

m

l)

consisting of 10 mg/ml of alpha-cyano-4-hydroxy-cinnamic acid
(CHCA) in 50% acetonitrile and 0.1% tri

fluoroacetic acid. The samples

were mixed well and spotted onto an Anchor Chip target plate
(Bruker Daltonics Ltd, Coventry, UK) keeping the droplet centered
on the anchor spot (400

m

mol/l target selected). This was kept for

drying, and MALDI mass spectra were obtained using a Bi

flex IV

MALDI-TOF MS (Brucker Daltonics) with a nitrogen laser at 337 nm
following routine calibration. The singly charged peptide

fingerprint

was assigned monoisotopic peptide masses using Biotools software
(Brucker Daltonics). These data were then used to search the NCBI
non-identical protein sequence database using MASCOT software
(Matrix Science), and statistically signi

ficant hits were recorded

together with the number of peptides and percentage coverage of
the protein. Finally, each of the peptides was used to BLAST search
to con

firm that the protein identified by MASCOT was the only

relevant match in the non-redundant protein database for a partic-
ular peptide sequence.

2.5. Characterization and kinetic studies of the enzyme

The puri

fied fractions having xylanase activity were used for

enzyme kinetic studies. Michaelis

eMenten kinetic parameters

(Km and Vmax) were determined using oat spelt xylan as substrate
at concentrations varying from 1.25 mg/ml to 30 mg/ml.

Effect of pH on enzyme stability was estimated by pre-incu-

bating enzyme in buffers at different pH ranging between 4.0 and
10.0. Buffer solutions with different pH values as sodium acetate
(4

e5), sodium phosphate (6e7), TriseHCl (8, 9 and 10) were used.

Half-life of enzyme was calculated by incubating the enzyme along
with assay buffer at different temperatures (50

C

e90

C) for

Table 1
Puri

fication of extracellular xylanase produced by Bacillus sp.

Puri

fication

step

Volume
(ml)

Total
Activity
(U)

Total
Protein
(mg)

Speci

fic

Activity
(U mg

1

)

Yield (%) Fold

Puri

fication

Crude Extract

50

2380

65

37

(100)

(1)

Sucrose Dialysis

30

2118

29

73

89

1.97

DEAE

Chromatography

20

1519

10

152

63.8

4.1

Gel Filteration

10

946

4.3

221

39.7

6.0

M. Mishra, I.S. Thakur / International Biodeterioration & Biodegradation 65 (2011) 301

e308

302

different time intervals (5, 10, 20, 30 and 60 min) to check the
thermo stability of the enzyme. The effects of chemicals like
EDTA, CuSO

4

, HgCl

2

(10

e30 mM) and solvents like acetonitrile and

glycerol (10%, 20%, 30%, 40% and 50%), on the activity of xylanase,
were also determined. Each experiment was repeated twice.

2.6. Application of enzyme in dye decolorization study

While studying the applicability of the enzyme, the cultured

filtrate with higher xylanase activity was used as a source of enzyme
to test its ef

ficiency in decolorization of various synthetic dyes.

The dyes (0.1%w/v), studied for decolorization, were aniline blue
(AB), bromo thymol blue (BTB), crystal violet (CV), brilliant blue R
(BBR), brilliant blue G (BBG), and trypan blue (TB). The enzyme was
incubated with dye in suitable buffer (Tris-Cl) at pH 8 for 2-12 hrs.
in triplicates. The composition of the mixture was 2 ml buffer,
0.5 ml dye solution and 0.5 ml crude enzyme solution, having 2 U
of enzyme in

final volume. The reaction was performed in dark

conditions at 70

C by continuous shaking at 200 rpm. Change in the

spectra (spectral scan from 200 to 800 nm) was observed using Cary
Spectrophotometer. The percentage of dye decolorization achieved
was calculated with reference to the control samples that were not
treated with the enzyme, by measuring the decrease in color at
absorption maxima for particular dyes.

Fig. 1. SDS-PAGE of extracellular Bacillus xylanase puri

fied by anion exchange,

Molecular size markers are indicated in kilodaltons (lane 1), DEAE (lane 2) and
sephadex puri

fied xylanase (lane 3).

Fig. 2. MALDI-TOF-MS analysis of extracellular xylanase puri

fied from Bacillus sp. (a) Peptide sequence coverage of the enzyme found by MS analysis against Bacillus subtilis

b

-1, 4

endoxylanase and matched peptides (in bold letters) are shown (b).

M. Mishra, I.S. Thakur / International Biodeterioration & Biodegradation 65 (2011) 301

e308

303

3. Results and discussion

3.1. Isolation and production of xylanase of Bacillus sp.

Based of the morphological features and DNA sequence of 16S

rDNA, strain was identi

fied as Bacillus sp. and its sequence has been

deposited at Genbank Bethesda, Maryland USA, having accession
number EU741057 (

Mishra and Thakur 2010

).

Xylanase production was enhanced by optimising growth

factors and process parameters of the culture media. Maximum
xylanase production was obtained with MSM containing oat
spelt xylan 1% (w/v), sucrose 2.5% (w/v) and inoculum size 5% (w/v)
along with 10% (v/v) black liquor at pH 8.0. The extracellular
enzyme activity was measurable after 20

e24 h of the onset of

growth in the culture media, having maximum activity at 48 h.

The use of xylanases in pulp and paper industry has increased

appreciably with its discovery by

Viikari et al. (1986)

. The objective

of this study was to isolate, purify and characterize extracellular
xylanase from Bacillus sp. The previously described xylanase
producing bacteria include Bacillus sp. XTR-10, Bacillus amylolique-
faciens, Micrococcus sp., Streptomyces roseiscleroticus NRRL-B-11019,
Cellulomonas uda, Staphylococcus (

Rapp and Wagner 1986; Grabski

and Jeffries 1991; Saxena et al. 1991; Breccia et al. 1998; Gupta
et al. 2000; Saleem et al. 2009

).

The application of various organisms including fungi and bacteria

for degradation and decolorization of pulp and paper mill ef

fluent

has been studied for more than three decades. The possible mecha-
nism behind degradation of polymers in pulp and paper mill ef

fluent

is the production of enzymes (

Thompson et al. 2001

). Some reports

have been cited in which ef

fluents such as black liquor from paper

and pulp mill and molasses spent wash from alcohol distillery are
known to induce xylanase production, when used at a concentration
of 1% (v/v) while decolorizing the ef

fluent (

Raghukumar et al. 2008

).

To our knowledge, this study is the

first report where pulp and

paper mill ef

fluent at a concentration of 10% (v/v), was used to induce

xylanase production by Bacillus sp. in the culture supernatant.

3.2. Puri

fication and MALDI-TOF-MS analysis

of xylanase of Bacillus sp.

Using batch culture of the Bacillus sp. in MSM, we were able to

obtain good xylanase activity, around 47 U/ml. Crude xylanase
solution, obtained after 48 hrs, was puri

fied by sucrose dialysis

followed by DEAE anion exchange chromatography and sephadex-
G100 gel

filtration chromatography. This protocol afforded 6 fold

puri

fication of xylanase from the culture filtrate with a yield of

39.7% and speci

fic activity of 221 U/mg (

Table 1

). Molecular weight

of protein as determined by gel

filtration chromatography was

55 kDa. The puri

fied xylanase produced single band on an SDS-

PAGE gel at a molecular mass of approximately 56 kDa (

Fig. 1

). Most

of the studies had reported the molecular weight of the xylanases
in the range 11

e104 kDa (

Chaudhri et al. 1988

).

The puri

fied band of bacterial xylanase was further studied for

MALDI-TOF/MS analysis to determine the molecular weight of the
puri

fied enzyme. These spectra were obtained using a Perseptive

Biosystems DE-PRO MALDI mass spectrometer equipped with
a TOF analyzer operated in positive ion mode. In MALDI-TOF/MS, only
a

finite number of molecules are actually analyzed by the detector.

The peptide sequence obtained by MALDI-TOF analysis was matched
against NCBI database and FASTA protein sequence database (Mascot
search). Our data indicated that enzyme obtained from Bacillus sp. is
xylanase. MALDI spectra are shown in

Fig. 2

a. The sequence coverage

of the peptide against the Bacillus subtilis

b

- 1, 4 endoxylanase

reached 20% (

Fig. 2

b). Previously some workers have identi

fied

b

- 1,

4 endoxylanase enzyme in B. subtilis and its use in hemicelluloses
degradation (

Kato and Nevins 1984; Yuana et al. 2005

). In this case,

five peptides were found to correspond exactly to internal sequence
of

b

- 1, 4 endoxylanase belonging to B. subtilis (

Table 2

). The nominal

mass of the homologous protein of B. subtilis was 54,561 daltons.
The molecular weights of the protein, determined by gel

filtration

chromatography, SDS-PAGE and MALDI-TOF/MS were almost similar.

3.3. Characterization of xylanase of Bacillus sp.

The kinetic constants of the puri

fied xylanase were determined

using oat spelt xylan as substrate under optimal assay conditions
through Michaelis

eMenten equation and the apparent K

M

and V

max

values of the enzyme were 4.4 mg/ml and 287 U/mg respectively
(

Fig. 3

).

Xylanases are considered to be non-speci

fic for their substrate,

being able to oxidize a wide range of aromatic compounds. In
the present work, oat spelt xylan was used as substrate for
kinetic studies and the enzyme showed typical Michaelis

eMenten

kinetics. The K

M

value for the xylanase is low which shows that

the xylanase has good af

finity with substrate oat spelt xylan. It was

observed that oat spelt xylan at high concentration produces an
inhibitory effect on xylanase activity.

The extracellular xylanase activity was found maximum at pH 8

and it became inactive (20% activity) at pH 4 and 48% active at pH

Table 2
Observed and expected monoisotopic [M

þ H]

þ

masses of selected ions from the tryptic digest of Bacillus sp.

b

-1, 4 endoxylanase.

Expected molecular mass

Observed ion (M/z)

Range

Deviation

Sequence

1262.446

1263.454

434

e446

0.252

TFKANVASALGGK

1300.553

1301.560

121

e133

0.104

WAGASWAPSAAVK

2115.983

2116.990

303

e322

0.005

NPGAFFGGGGNNHHAVFNFR

2457.093

2458.100

451

e474

0.168

LDSANGKLVGTLNVPSTGGTQSWR

3376.752

3377.760

229

e260

0.247

LGPDMTSVAGSASTIDAPFMFEDSGMHKYNGK

r

2

=0.998

Xylan conc.(mg/ml)

0

10

20

30

)

g

m/

ni

m/
M

µ(

yti

vi

tc

a

ci

fi

ce

p

S

0

50

100

150

200

250

300

Vmax(a)=287

Km(b)=4.1

Fig. 3. K

M

and V

max

values of the extracellular xylanase of alkalotolerant Bacillus sp.

isolated from pulp and paper mill ef

fluent and sludge. Data are mean of three inde-

pendent sets of reaction. In

figure K

M

and V

max

values are shown.

M. Mishra, I.S. Thakur / International Biodeterioration & Biodegradation 65 (2011) 301

e308

304

10.0 (

Fig. 4

). The optimum temperature for xylanase activity was

70

C (

Fig. 5

a), however the xylanase was active over broad range

of temperature (50

C

e90

C). The half lives of the enzyme are

49 min, 46 min, 53 min, 24 min and 10 min at 50

C, 60

C, 70

C,

80

C and 90

C respectively (

Fig. 5

b).

This enzyme exhibited high activity and good stability under

alkaline conditions, making it a potent candidate for Kraft pulp
treatment. The use of alkaline active xylanases allows direct enzy-
matic treatment of the alkaline pulp and avoids the cost incurring
and time consuming steps in pH re-adjustment. In particular, alka-
line xylanases which are operationally stable at higher temperature
are more bene

ficial due to savings in cooling cost and time. In

this regard, the present xylanase is expected to be active under
conditions close to those of most mills, i.e. high pH and temperature.
So far, only few xylanases with optimum temperature for activity
exceeding 70

C at or above pH 8 have been reported (

Gessesse 1998;

Gessesse and Mamo 1998; Kohli et al. 2001

). Due to better solubility

of xylan under alkaline conditions, alkaline active xylanases may also
find other potential applications in addition to pulp bleaching.
For example, in waste management programs, xylanases can be used
to hydrolyze xylan in industrial and municipal waste. The optimum
pH for xylan hydrolysis is about 5.0 for most of the fungal xylanases,
which are normally stable at pH 2

e9 (

Silveira et al. 1999;

Subramaniyan and Prema 2002

). Application of bacteria in pulp

and paper industries is advantageous over fungi since the later
require acidic growth media.

pH

3

4

5

6

7

8

9

10

11

yti

vi

tc

A

e

vi

t

al

e

R

0

20

40

60

80

100

120

Fig. 4. Effect of pH on xylanase activity at constant temperature 70

C. The X axis

indicates duration in min and Y axis indicates percentage residual activity (Error bars
are standard deviations).

Temperature(ºC)

20

30

40

50

60

70

80

90

100

yti

vi

tc

A

e

vi

t

al

e

R

0

20

40

60

80

100

120

Time(min)

0

10

20

30

40

50

60

70

yti

vi

tc

A

l

a

u

di

se

R

0

20

40

60

80

100

120

50°C

60°C

70°C

80°C

90°C

a

b

Fig. 5. Effect of temperature at constant pH 8, on xylanase activity (a) and calculation
of half life (b), the X axis indicates duration in min and Y axis indicates residual activity.
(90 U/ml of enzyme activity was taken as 100% activity). Error bars are standard
deviations.

conc(mM)

0

5

10

15

20

25

30

35

n

oi

ti

bi

h

nI
%

0

20

40

60

80

100

EDTA
CuSO

4

HgCl

2

% Concentration

0

10

20

30

40

50

60

n

oi

ti

bi

h

nI
%

30

40

50

60

70

80

90

100

110

Glycerol

Acetonitrile

a

b

Fig. 6. Percentage inhibition of xylanase activity by different concentration of HgCl

2

,

CuSO

4

, EDTA (a) and organic solvents (b). (Error bars are standard deviations).

M. Mishra, I.S. Thakur / International Biodeterioration & Biodegradation 65 (2011) 301

e308

305

The dependence of xylanase on pH usually renders a bell-shaped

pro

file. Although xylanase was active over a wide range of temper-

atures, optimum temperature range for xylanase was 50

C

e80

C.

Very less activity (15%) was detected at 30

C and approximately 79%

of activity was retained at 90

C. The optimal temperature range in

fungal xylanases also lies between 30

C

e60

C (

Subramaniyan and

Prema 2002

). The xylanase from Bacillus sp. was more thermostable

with half-life of 10 min at 90

C.

HgCl

2

and EDTA at 30 mM concentration could inhibit upto 89%

and 69% of the enzyme activity respectively (

Fig. 6

a). In case of

organic solvents, there was complete inhibition by glycerol and
acetonitrile at 50% v/v concentration (

Fig. 6

b).

Chemicals like CuSO

4

, EDTA were less effective in inhibiting the

enzyme activity even at a very high concentration (30 mM). Similar
findings were observed for the xylanase from the fungus Aspergillus
cf. niger BCC14405 (

Krisana et al. 2005

). Acetonitrile had higher

inhibitory effect on xylanase activity as compared to glycerol and
both had strong inhibitory effects with almost complete loss of
enzyme activity at higher concentrations (50%).

3.4. Decolorization of dyes by the enzyme

We selected various synthetic dyes bearing different functional

groups to evaluate decolorization performance of the extracellular
enzyme. The xylanase enzyme showed high decolorization poten-
tial with various dyes at the interval of 2 h, 6 h and 12 h (

Fig. 7

).

Fig. 7. Changes in spectral scan of different dyes after treatment with extracellular xylanase. In this

figure U is untreated; T1 after 2 h; T2 after 12 h and T3 after 24 h.

Table 3
Decolorization of different dyes with extracellular xylanase from Bacillus sp.

Dye

Absorbance maxima (

l

)

% Decolorization

Aniline blue

580

65

1.2

Brilliant blue G (BBG)

582

21

1.8

Brilliant blue R (BBR)

552

75

2.3

Crystal violet

580

48

4.1

Trypan blue

599

77

3.3

Bromo Thymol blue (BTB)

434

70

2.6

M. Mishra, I.S. Thakur / International Biodeterioration & Biodegradation 65 (2011) 301

e308

306

There was almost complete decolorization of Trypan blue and BBR,
showing more than 77% and 75% decolorization respectively. In
contrast, the original color of the BTB dye changed with the addi-
tion of enzyme which showed increased absorption maxima in
visible range (

Table 3

).

Xylanase activity at high temperature may

find potential applica-

tion in treatment of heated industrial ef

fluents. Use of Xylanase

enzyme in decolorization of colored ef

fluent has been studied

previously (

Raghukumar et al. 2008

). In present work, we found easy

decolorization of synthetic dyes by crude extracellular xylanase and
the enzyme showed higher degree of decolorization of trypan
blue and BBR. Decolorization of synthetic dyes was achieved with in
2

e12 h by incubating the extracellular enzyme with the dye and

a major reduction in color was seen in initial 2 h in most of the
cases. Dye decolorization attributed to adsorption of the dye on
microorganism surface or biodegradation by enzyme activity. In our
case, decolorization is due to enzyme activity. Microorganisms, fungi
and bacteria are capable of utilizing a variety of complex compounds
including dyes as sole carbon source but only meager data are avail-
able on bacterial breakdown of azo and other dyes. The degradation of
coloring materials is primarily mediated by peroxidases, oxidases and
hydrolases (

Chang and Lin 2000; Verma and Madamwar 2003

). In this

study xylanase is used for the decolorization of different dyes in order
to

find out an environment friendly and cost competitive alternative

for removal of color and dyes in the environment.

4. Conclusion

A new extracellular bacterial xylanase has been puri

fied and

characterized. Results presented in this work indicated that pulp and
paper mill ef

fluent acted as xylanase inducer when added to the

growing culture of Bacillus sp. in MSM along with carbon sources.
A high activity of xylanase was obtained after 48 h of inoculation.
The enzyme possesses broad range of pH and a

flat optimum

temperature curve, thus bearing good thermo stability properties.

Acknowledgement

This paper is supported by the research grants of Department

of Biotechnology and Council of Scienti

fic and Industrial research

(CSIR), Government of India. Author (MM) thanks CSIR for
providing Junior Research Fellowship. We thank Century Pulp and
Paper mill, Lalkuan, Uttarakhand, India for providing ef

fluent and

sludge/sediments during the course of investigation.

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