Purification and Properties of a 44 kDa Xylan-Binding Xylanase from ...

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Purification and Binding Properties of Xylan
-
binding
Endoxylanase (48

kDa) from
Bacillus circulans

B6


Patthra Pason,

Rattiya Waeonukul,

Khanok Ratanakhanokchai and Khin
Lay Kyu


Division of Biochemical Technology, School of Bioresources and Technology, K
ing Mongkut’s
Univer
sity

of Technology Thonburi, Bangkok, 10140



When
Bacillus

circulans

B6 was grown in a medium containing xylan as a carbon source, the
bacterium produced multienzyme complex. The crude enzyme showed many protein bands on
SDS
-
PAGE and

at least 1
6

xylanases and
9

carboxymethylcellulases on zymograms.
Purification of xylan
-
binding xylanase was conducted by an affinity adsorption
-
desorption on
insoluble xylan. The molecular mass of purified xylan
-
binding xylanase was estimated at
48,000 d
alton and it acted only on xylan. The enzyme was stable at pH 6.0
-
7.0 and
temperature up to 50

C. The pH and temperature optima were 6.0 and 60

C. The enzymatic
hydrolysis products of xylan were a series of short
-
chain xylooligosaccharides, indicating that

the enzyme was an endoxylanase. The xylan
-
binding endoxylanase could be able to effectively
hydrolyze insoluble xylans of birch wood, larch wood and oat spelt.
The

enzyme

could adsor
p

to insoluble xylan and avicel
.

The adsorption of the enzyme on insolubl
e xylan and avicel

was
affected by pH, sugar
s

and metal ion
s
. High pH and sugar decreased the adsorption of the
purified enzyme to insoluble xylan and avicel. However, CaSO
4
,

FeSO
4
, MgSO
4

and CuSO
4

increased the adsorption of the enzyme to insoluble xylan,

while

CuSO
4

and
FeSO
4

increased
the adsorption of the enzyme to avicel. The purified enzyme can be applied in hydrolysis of
insoluble xylan present in agricultural wastes.


Keywords
: affinity adsorption/

Bacillus circulans
B6/ multienzyme complex/

purifi
cation of enzyme/
xylan
-
binding endoxylanase



2


Introduction

Xylan
, a large proportion of the
hemi
cellulose
, is composed of a backbone of

-
1,4
-
linked
xylose units which are substituted with arabinose and acetate residues (Millard
-
sadler

et al.
,

1994). Comp
lete hydrolysis of heteroxylans requires the synergistic action of many
xylanolytic enzymes (Kelett
et al.
,

1990). These enzymes include endo
-

-
1,4
-
xylanases,

-
xylosidase
s

and enzymes which cleave side chain sugars from the xylan backbone, such as

-
arabi
nofuranosidases and acetyl esterases. Xylan
-
degrading enzymes have attracted much
attention because of their many important practical applications in various industrial
processes, including improving the digestibility of animal
feed stocks

(
Yin

et al.
,

200
1
)
, the
modification of cereal
-
based foodstuffs
(
Law
et al.
,

2002
)
, and the delignification of paper
pulp
(
Guoitz

et al.
,

1997
)
. The noncatalytic polysaccharide
-
binding regions of plant cell wall
hydrolases play an important role in the efficient hydrolysi
s of
insoluble
cellulosic substances
(Karita

et al.
,

1996).
P
lant cell wall hydrolases include noncatalytic domains, carbohydrate
-
binding module
s

(CBMs). The function of the CBMs is largely to attach the enzyme to its
substrate, and therefore enhance the r
ate of catalysis by increasing the probability of
enzyme/substrate interaction (Gil

et al.
,

1999).
CBMs
are

classified
into 42 families
defined as

contiguous amino acid sequence within a carbohydrate
-
active enzyme with a discreet fold
having carbohydrate
-
b
inding activity

(last updated on 2005 Jan 11; http://afmb.cnrs
-
mrs.fr/CAZY/index.html)
.

E
nzyme containing CBMs

was able to purify by affinity adsorption

(Ratanakhanokchai
et al.
, 1999).

Binding is mediated via
several co
-
planar, solvent
-
expo
s
ed
aromatic ri
ngs
, hydrophobic surfaces,

that f
r
o
m stacking interactions with the sugars in the
polysaccharide and also th
r
ough hydrogen bonding

(
Sun
et al.
,

1998)
.

In this study, we
describe the purification and
binding
properties of the xylan
-
binding
endo
xylanase from

Bacillus

circulans

B6.








3

Material
s

and methods

Bacterial strain

and Culture medium


Bacillus
circulans

B6

was isolated from
an
an
a
erobic digester fed with

pineapple
waste

(Kyu
et al.

1994)
.

The culture medium used was Berg’s mineral salt medium

(
Berg
et
al.
,

1972
) supplemented with 0.5% xylan.


Enzyme assays


The xylanase

and CMCase

activity w
ere

assayed
as described previously
(
Ratanakhanokchai

et al.
,
1999)
.


Protein determination


Protein
contents were

determined

by the method of Lowry
et al.

(
1951
)
.



Purification of xylan
-
binding xy
lanase


The culture supernatant was used as a source of xylan
-
binding xylanase. For
purification of xylan
-
binding xylanase, 25 mg of protein from the culture supernatant
was
incubated with

2% insoluble xylan in 10 ml of
100 mM NaCl in 250 mM phosphate buffer
(pH 7.0)

(buffer A)

and
shaken periodically at 4 °C for 30 min.
T
he xylan
-
bound protein
complex was washed four times with the same volume of
buffer A
and then eluted with 1%
triethylamine. The eluted
enzyme

was dialy
zed, assayed for
xylanase activity

(Nelson, 1944)
then

purified second step by

the same method
using

2% insoluble xylan in 10 ml of 50 mM
NaCl in 100 mM phosphate buffer (pH 7.0)

(buffer

B
)

instead
of
buffer A
.
The
unbound
protein and bound protein were co
llected and dialyzed for analysis
.


Gel electrophoresis

and z
ymogram analysis


Sodium dodecyl sulfate
-
polyacrylamide gel electrophoresis (SDS
-
PAGE) was
performed by the method of Laemmli

et al.

(
1970
).
Xylanase

zymogram was a modification
of the published
method
(
Ratanakhanokchai

et al.
,

1999). CMCase zymogram was prepared
in the same manner using carboxymethyl cellulose (CMC) as a substrate.

Hydrolysis of insoluble xylan




4

The insoluble xylan
s

(Irwin
et al.
, 1994)

of birch wood, larch wood, and oat spelt
we
re
hydrolyzed by the purified enzyme.

Each

substance

(2% dry weight)

was hydrolyzed
with 1 U of the
enzyme

at pH 7.0 and 50°C. After
incubation for
5, 10, 15 and 20 min,
samples were removed and the amount of reducing sugars produced was determined.


Analy
sis of xylan hydr
olysis products


The xylan
-
binding xylanase (1 U) was mixed with
2
% xylan in 0.1 M Tris
-
HCl buffer
(pH 7) and incubated at 50°C.
X
ylan hydrolysis products were removed
at 5, 10 and 30 min
and qualitatively determined by thin
-
layer chromato
graphy on silica gel 60 F254 plates
(Merck 1.05554; 20 by 20 cm) with a mixture of
n
-
butanol, acetic acid, distill
ed

water

(
2:1:1
)
as a solvent system

(Kubata

et al.
,

1994)
.

The sugar spots were detected on the plates by
spr
aying them with 4 ml of aniline
(
4 g
)

of
acetone
200 ml of
phosphoric

acid
30 ml
.

0.5%
D
-
Xylose
in 10% isopropanol
was

used as
a standard
.


Ad
sorption assay


Adsorption assays and preparation of insoluble xylan

were performed by the method
of Irwin
et al.

(19994).
The cellulose
-
binding a
ssay was conducted according to the above
procedure using avicel. The purified enzyme was mixed with 50 mg of insoluble substances
with occasional stirring for 30 min at 4

C. Then, the mixture was centrifuged and the amount
of enzyme remained in the supern
atant was determined

by measuring the protein content in
the supernatant. The amount of enzyme bound to the insoluble xylan or avicel estimated from
the difference between the amounts of protein before and after incubation. The ratio (%) of
the protein bou
nd to insoluble xylan or avicel to the total protein added into the assay mixture
was defined as relative adsorption.


Effect of pH on adsorption

of enzyme to insoluble substrate


The adsorption assay was measured under the same conditions as described abo
ve
,

using

var
ious

pH
s
.

The

pH
s were

adjusted with the following buffer systems:

acetate buffer
(pH 4.0 to 6.0),

phosphate buffer (pH 6.0 to 7.0)

and

Tris
-
HCl buffer (pH 7.0 to 9.0).

Effect of sugar on adsorption of enzyme to insoluble substrate




5

The adsor
ption assay was measured under the same conditions as described above
,

using

various sugars (25 mM) such as

xylose, glucose, cellobiose, galactose, arabinose,
mannose and sucrose.


Effect of metal ion on adsorption of enzyme to insoluble substrate


The en
zyme solution was mixed with the final concentration of 10 mM

ethylenediaminetetra
aceticacid (EDTA)
and
dialy
zed with Tris
-
HCl buffer pH 7.0. Then the
EDTA treated
enzyme
solution
was preincubated in a mixture containing
various metal ions
(1

mM) at 30

C f
or 1 hr, after that the adsorption
of enzyme
to insoluble xylan and avicel
was measured under the standard assay condition as described above.


Result
s

and discussion


Enzyme production by Bacillus circulans

B6


When
Bacillus circulans

B6 was grown in xyla
n medium, the extracellular
xylanolytic and cellulolytic enzyme
s

were detected

in the culture supernatant (crude
enzyme)
.
The crude enzyme was found to have binding ability to insoluble substances such
as insoluble xylan and avicel at 77
%

and 68%,

respecti
vely (data not shown).


Purification by affinity chromatography

Xylan
-
binding xylanase
with the molecular weight at

48
,000 Da was purified by
affinity
chromatography
. In
the first

step,
10 ml of 100 mM NaCl in 250 mM phosphate
buffer (pH 7.0) (
buffer A
)

wa
s
use
d

for equilibrat
ing

and washing. Then proteins were eluted
with 1% TEA
and

it showed

2 protein bands on SDS
-
PAGE
with the

molecular
weight
s

of

29
,000 and 48,000 Da (data not showed).
After that

2 proteins

were

separated
by affinity
chromatography

with

10 ml of 50 mM NaCl in 100 mM phosphate buffer (pH 7.0) (
buffer
B
)
, lower hydrophobic
ity

than
buffer A
.
The xylan
-
binding xylanase (48 kDa) appeared in
the bound fraction. It showed single protein band on SDS
-
PAGE (Fig. 1 A, land 2) and only
one band on z
ymogram for xylanase activity
(Fig. 1 B, land 2).
The

purified

enzyme

(48
kDa)
was able to adsorb to insoluble xylan
when

buffer A and B
were present

(high and low


6

hydrophobic condition)

while

another

xylan
-
binding xylanase (29 kDa) had binding capacity
to

insoluble xylan at
only
higher hydrophobic

condition

(Buffer A).

This result indicated that
xylan
-
binding xylanase
with molecular weight at
29 and 48 kDa have differen
t

xylan
-
binding
capacity.
T
he adsorption of the purified enzyme to xylan might be depend
ent on the ionic
strength of the incubation solution

which

play
ed

an important role in the binding of insoluble
polysaccharide

(Sun
et al.
, 1998)
.

High salt condition confirm
ed the

binding criteria of
carbohydrate
-
binding module (CBMs) to insoluble polysac
charide that act by hydrophobic
action (sugar ring)
.

Thus, the purified xylan
-
binding xylanase (48 kDa) was adsorbed to
insoluble xylan with hydrophobic
interaction
.


















A







B

Fig.1 SDS
-
PAGE and zymogram

of

purified enzyme o
f
Bacillus circulans

B6
. (A) SDS
-
PAGE (10% acrylamide gel dyed with Coomassie brilliant blue R
-
250. (B) Zymogram for
xylanase activity (10% acrylamide gel
dyed with Congo red); Lane M = Standard m
olecular
weight, Lane 1 = culture supernatant (crude enzyme)
, Lane 2 = bound enzyme eluted with
1%
TEA








M 1


2



1



2




48 kDa




200




97


66


43



29


20



48 kDa



7

Effect of pH
on activity and stability


The effect of pH on xylanase activity of the purified xylan
-
binding
xy
lanase was
determined. The optimum pH of the enzyme was 6.0. The enzyme showed the pH stabi
lit
y of
6.0 to 7.0 and still 50
% of relative activity at pH
9
.0 (Fig. 2).



Fig. 2 Effect of pH on acti
vity and stability of purified x
ylan
-
binding
x
ylanase of
Bacillus
circulans

B
6. The reaction pH values were adjusted with the following buffer systems:
ac
etate bu
ffer (pH 4.0 to 6.0),

p
hosphate buffer (pH 6.0 to 7.0) and

Tris
-
HCl buffer (pH 7.0
to 9.0).













8

Effect of temper
ature on activity and stability



The optimum temperature of the purified xylan
-
binding
x
ylanase was determined by
varying the re
action temperature at pH 6.0. The enzyme had an optimum temperature of
60

C. The enzyme was stable at temperatures up to 50

C (Fig. 3).



Fig. 3 Effect of temperature on the xylanase activity and stability of purified xylan
-
binding
xy
lanase from
Bacillus c
irculans
B6.













9

Hydrolysis of xylan


The hydrolysis
of

insoluble xylan by the purified xylan
-
binding
x
ylanase
is

show
n

in
Fig. 4. It indicated that the purified enzyme
was
more effective

on
hydroly
sis

of

the insoluble
xylan from
birch

wood

than t
hose of

larch wood

and

oat spelt
.
O
at spelt xylan
is more
substituted
,

compared to
larch wood and
birch wood xylan
s
, respectively

(Li
et al.
,

2001;
Subramanyan
et al.
,

2002).
There
fore, the purified enzyme prefers to hydrol
yz
e linear xylan
more than substi
tuted xylan.
The
purified enzyme was capable of

hyd
rolyz
ing

insoluble xylan
due to

its

xylan
-
binding ability

that is important for

hydrolysis of
insoluble xylan

(Kuno
et
al.
,

1998
;
Black
et al.
,
1997;
Zui
et al.
,

2000
)
.

















Fig. 4 Hydrolysis
of insoluble xylans by purified xylan
-
binding
xylanase
.












10

Thin
-
layer chromatography


When purified enzyme
was incubated
with insoluble oat spelt xylan

for

5, 10 and 30
min
,

short chain
xylo
oligosaccharide
s

w
ere

found

as the product
s

(Fig. 5)
, indic
ating

that the
purified enzyme was
the endo type enzyme (
endoxylanase
)
.












Fig. 5 Thin
-
layer chromatography of
xylan
-
binding
endo
xylanase

with insoluble oat spelt
xylan after incubat
ion for

5, 10 and 30 min.














X
2

X
3

X
4

X
5


Xylose 5 10 30



11

Effect of sugars

on the
adsorption of the xylan
-
binding endoxylanase to insoluble
polysaccharides


The purifi
ed

enzyme
has

two substrate
-
binding
abilities
:

one
is

a xylan
-
binding
ability

and
another
is

a cellulose
-
binding
ability
.

The effect of sugar on the adsorption of enzyme
w
as determined using various sugars (Table 1). The adsorption
decreas
ed by all kinds of
sugar tested, especially xylose and glucose for adsorption of the enzyme to insoluble xylan
and avicel.

Xylose and glucose
a
re hydroly
sis

products from hydrolysis of xyl
an and avicel.
The increase of hydrolyzate sugars decreases the binding of enzyme to substrates

because
s
ugars
a
re competitive to binding
region

of enzyme and reduce binding capacity

to insoluble
substrate

(Sun
et al.
, 1998)
.


Table 1 Effect of sugars on t
he adsorption of
xylan
-
bindings endoxylanase
on insoluble
polysaccharides


Sugar

% Relative adsorption

Xylan

Avicel

Control

100

100

Sucrose

97

78

Mannose

96

81

Arabinose

68

88

Cellobiose

61

65

Galactose

60

88

Glucose

53

49

Xylose

45

59








12

Eff
ect of pH

on the adsorption of the xylan
-
binding en
dox
ylanase to insoluble
polysaccharide


T
he adsorption of the purified enzyme to insoluble xylan and avicel decreased when
pH increased

(Fig. 6).

It was s
imilar
to the

binding of
Trichoderma
reesei
xylanas
es to
polysaccharides
that
was

affected by the pH
(Tenkanen
et al.
, 1995)
.

Most of the e
nzymes
were

strongly bound to
insoluble polysaccharide

when the pH is
lower than

isoelectric point

(
pI
)

and

the binding of enzyme decreased
when

pH
increased
(Jifferies
, 1998).
The

high pH
affected to enzyme conformation and lost binding ability.
It was also found that the
adsorption of enzyme to avicel was higher than insoluble xylan

because t
he enzyme could not
hydrolyze av
icel, t
hus no reducing sugars produced. The in
crease in hydrolyzate sugars
decreased the adsorption

of the purified enzyme

to insoluble xylan

(Sun
et al.
, 1998)
.














Fig.
6

Effect of pH on the adsorption of
xylan
-
bindings endoxylanase

on insoluble
polysaccharides









13

Effect of metal ion on

the adsorption of the xylan
-
binding en
dox
ylanase to insoluble
polysaccharide


T
he adsorptions of purified enzyme to insoluble xylan and avicel were decreased by

10

mM EDTA.
Then, the enzyme was dialyzed and effect of metal ion
(1 mM)
on adsorption
to inso
luble substrate

was determined
.
As showed in Table
2
, the a
dsorption of
xylan
-
binding
endoxylanase

to insoluble xylan was
increase
d

by FeSO
4
, CaSO
4
,
MgSO
4

and
CuSO
4
.

However, the

adsorption

of xylan
-
binding endoxylanase

to avicel was increase
d

by
CuSO
4

and

FeSO
4
.

The
effect

of
m
etal ion
o
n

CBMs
was
found
in the

previous
report

that

Ca
2+

was
essential for adsorption of CBDs to insoluble polysaccharide

(Talabani

et al.
,

2004).


Table 2 Effect of metal ions on the adsorption of
xylan
-
binding endoxylanase

on
in
soluble polysaccharides



















Metal ion

(1 mM)

% Relative adsorption

Xylan

Avicel

None (
-
EDTA)

100

100

None (+EDTA)

7
8

80

FeSO
4

117

10
1

CaSO
4

11
7

100

MgSO
4

112

98

CuSO
4

109

112

ZnSO
4

73

91



14

Conclusion



Due to the presence of xylan
-
bin
ding ability, x
ylan
-
binding endoxylanase
(
48
,000 Da
)

could be

purified by
2 steps
of
affinity chromatography

with

insoluble xylan

and it could

efficiently

hydrolyz
e

the insoluble

xylan
s

from

birch wood, larch wood and oat s
pelt
.

Therefore
, t
he xylan
-
bindin
g endoxylanase can be applied in hydrolysis of insoluble xylan
present in agricultural wastes

and kraft pulps
.


Acknowledgement
s


This work was support
ed

by
t
he
Royal Golden Jubilee
Ph.D. program of the Thailand
Research Fund
and the National Center for Ge
netic Engineering and Biotechnology

(BIOTEC)
grant
.


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