APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1993, p.

1725-1730

Vol. 59, No. 6

0099-2240/93/061725-06$02.00/0
Copyright ) 1993, American Society for Microbiology

Purification and Characterization of a Thermostable Xylanase

from Bacillus stearothermophilus T-6
ALEXANDER KHASIN, IRIS ALCHANATI, AND YUVAL SHOHAM*
Department of Food Engineering and Biotechnology, The Technion, Technion City, Haifa 32000, Israel
Received 28 December 1992/Accepted 14 March 1993

BaciUlus stearothermophilus T-6 produces an extracellular xylanase that was shown to optimally bleach pulp
enzyme was purified and concentrated in a single adsorption step onto a cation
exchanger and is made of a single polypeptide with an apparent Mr of 43,000 (determined by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis). Xylanase T-6 is an endoxylanase that completely degrades xylan to
xylose and xylobiose. The pls of the purified protein were 9 and 7 under native and denaturing conditions,
respectively. The optimum activity was at pH 6.5; however, 60% of the activity was still retained at pH 10. At
65C and pH 7, the enzyme was stable for more than 10 h; at 65C and pH 9, the half-life of the enzyme was
approximately 6 h. Kinetic experiments at 55C gave V.. and Km values of 288 U/mg and 1.63 mg/ml,
respectively. The enzyme had no apparent requirement for cofactors, and its activity was strongly inhibited by
Zn2+, Cd2 , and Hg2'. Xylan completely protected the protein from inactivation by N-bromosuccinimide. The
N-terminal sequence of the first 45 amino acids of the enzyme showed high homology with the N-terminal region
of xylanase A from the alkalophilic Bacillus sp. strain C-125.
at pH 9 and 65C. The

Xylanases (1,4-P-D-xylan xylanohydrolase; EC 3.2.1.8)

hemicellulases that hydrolyze xylan, which is a major
constituent of the hemicellulose complex (6). Xylan is composed of ,3-1,4-linked xylopyranose units with branches
containing L-arabinofuranosyl and glucopyranosyl residues.
Biotechnological uses and potential applications of xylanases include bioconversion of lignocellulose material to
fermentative products, clarification of juices, and improvements of the consistency of beer and the digestibility of
animal feedstock (45). One of the major potential applications of xylanases involves the pulp and paper industry (44).
In the process of making paper pulp, lignin is removed by
cooking wood chips at a high temperature and a basic pH
(the Kraft process). The residual lignin that remains on the
pulp is dark in color because it has been extensively oxidized
and modified in the cooking process. To obtain high-quality
white paper, all of the lignin must be removed; this is done
traditionally with chlorine-based chemicals (bleaching) (38).
This bleaching process, although highly effective, produces
chlorinated organics and is polluting (40). Today, the pulp
and paper manufacturers are actively seeking new bleaching
procedures that will reduce or even eliminate the need for
chlorine bleaching (36). Several years ago Viikari et al.
demonstrated that hemicellulases can be used to enhance
delignification and bleaching of unbleached pulp (43). The
major effect of the enzymes is due to the hydrolysis of
reprecipitated and readsorbed xylan and xylan-lignin complexes that are separated during the cooking process. A
comparison of different hemicellulases indicated that endo,B-xylanases have a major impact on delignification, even in
softwood pulp, of which mannan is a major component.
Indeed, full-scale mill trials of enzyme prebleaching are
already under way (11, 24, 43). Most of the hemicellulases
studied to date are active at a neutral or acidic pH, and their
optimal activity temperature is below 45C. Hemicellulases
that are active at higher temperatures and pHs are of great

potential since they can be introduced more freely in the

different stages of the bleaching line without the need of
costly changes in temperature and pH. Recently, we have
isolated thermostable alkaline-tolerant xylanases that can
bleach pulp optimally at pH 9 and 65C (16, 35, 37). In this
article, we report the purification and characterization of one
of these enzymes, xylanase T-6.

are

Corresponding author. Electronic mail address: forO610@

technion.

MATERIALS AND METHODS

Organisms and culture conditions. Bacillus stearothennophilus T-6 was isolated by using an enrichment procedure for
bacteria capable of producing extracellular thermostable
xylanases (37). Strain T-6 was identified as B. stearothermophilus by the National Collection of Industrial and Marine
Bacteria (Torry Research Station, Aberdeen, Scotland) and
was designated NCIMB 40221. For xylanase production,
fermentation of the organism was carried out in a 5-liter New
Brunswick Microferm fermentor at 60C and 600 rpm and
with aeration of 8 to 10 liters/min for 12 to 18 h. Growth
medium, XMP, contained the following: vitamin assay
Casamino Acids (Difco), 4.0 g/liter; yeast extract, 0.2 g/liter;
MgSO4, 0.01 g/liter; (NH4)2SO4, 2.0 g/liter; K2HPO4, 0.46
g/liter; KH2PO4, 0.1 g/liter; MOPS (morpholinepropanesulfonic acid), 10.4 g/liter; D-xylose (autoclaved separately),
5.0 g/liter; and trace element solution, 1 ml/liter. The trace
element solution contained the following (in grams per liter):
CaCl2 2H20, 0.39; CuSO4 5H20, 0.62; FeSO4. 7H20,
0.60; MnSO4, 0.59; ZnSO4- 7H20, 0.42; CoCl2 6H20, 0.79;
and Na2MoO4, 0.70. The solution was kept at pH 2 and
added after sterilization of the medium.
Enzyme purification. Enzyme purification was carried out
by a single batch adsorption step onto a cation exchanger
(usually 3 to 5% SE-52; Whatman Ltd., Maidstone, England). The ionic strength of the broth or cell-free broth
(cells were removed by centrifugation at 10,000 x g and 4C
for 10 min) was adjusted to give specific conductivity of less
than 5 mS/cm2 (below 50 mM KCl equivalent), and the
adsorption step was allowed to proceed for at least 30 min.
Elution was carried out with 1 M KCl after the cation
1725

1726

APPL. ENVIRON. MICROBIOL.

KHASIN ET AL.

exchanger had been washed with low-strength buffer (10 mM

phosphate buffer [pH 7]).
Xylanase assay. Appropriately diluted enzyme (250 ,ul) was
mixed with 250 ,ul of 2% oat spelts xylan (Sigma Chemical
Co., St. Louis, Mo.) and 500 ,ul of 0.1 M phosphate buffer
(pH 7.0). (Xylan solution was prepared by sonicating a 2%
xylan solution for 3 min with an Ultrasonic W375, sonicator
at output 7.) Four aliquots of 0.1 ml were taken from this
mixture and placed in four 9-ml glass tubes. Two tubes
served as time-zero controls and were kept at room temperature or on ice; the two other tubes were incubated at 55 or
65C for 10 or 15 min. The reaction was terminated by
placing the tubes in a water bath at room temperature. The
reducing sugar content in the tubes was determined by the
dinitrosalicylic acid (DNS) method (27), with D-xylose as a
standard (0.004% xylose was added to the DNS reagent just
before the color reaction). One unit of xylanase activity was
defined as the amount of enzyme which produces 1 ,umol of
xylose equivalent per min.
Effects of pH and temperature on xylanase activity. The
enzymatic reactions were carried out for 5 min at 55C in
four different buffers (50 mM): citric acid-Na2HPO4 (pH 5 to
6), phosphate buffer (pH 6 to 8), boric acid-NaOH buffer (pH
8 to 9.5), and phosphate-NaOH buffer (pH 9.5 to 11). The
actual pH in the assay mixture was determined at the
reaction temperature. The effect of temperature on the
reaction rate was determined by performing the standard
reaction for 5 min at a temperature range of 55 to 80C.
Thermostability. Eppendorf tubes (1.5 ml) containing 0.2
ml of purified enzyme solution (40 U/ml in 10 mM phosphate
buffer [pH 7.0]) were incubated at 65, 70, and 75C. At
various times, the tubes were removed and placed at -20C.
The residual enzymatic activity in each tube was determined
by the standard assay.
Effect of metals. Various salts (at 0.1, 1.0, and 10 mM)
were added to the standard enzymatic reaction mixtures (to
avoid the formation of insoluble phosphates, 50 mM succinic
acid buffer [pH 7.0] was used instead of the phosphate

buffer).
Protein content. Protein content was determined by the
method of Bradford (5) by using the Bio-Rad protein assay
(Bio-Rad Laboratories, Richmond, Calif.) with bovine albumin fraction V (Sigma) as a standard.
Isoelectric point. Isoelectric focusing of the native protein
was performed by the procedure described by Guilian et al.
(12) with ampholytes of pH 3.5 to 10 (Sigma). Isoelectric
focusing under denaturing conditions was performed by the
procedure of O'Farrell et al. (29). Chromatofocusing was
performed on a fast protein liquid chromatography (FPLC)
Mono P column (Pharmacia, Uppsala, Sweden) with Polybuffer 96 (Pharmacia) as an elution buffer.
Molecular weight. The Mr of the purified enzyme was
estimated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) (25) and by gel filtration on
Sephadex G-75 and Superose 12 FPLC columns (Pharmacia).
Thin-layer chromatography. Xylan digestion products
were analyzed on a silica gel chromatogram. Samples of
xylan digests (-20 ,ug) were applied to silica gel-coated
aluminum foils (Merck 60 254; E. Merck AG, Darmstadt,
Germany). The running solvent consisted of n-butanolacetone-water (4:5:1 [vol/vol/vol]). Xylose and xylobiose
(Sigma) were used as standards. After the run (2 h), the
chromatogram was sprayed with a mixture of 0.5 ml of
anisaldehyde, 50 ml of acetic acid, and 1 ml of H2SO4 and

TABLE 1. Single-step purification of B. stearothermophilus

T-6 xylanase
Fraction
Fraction

Crude broth
Dialyzed broth
CM-52 adsorption and
KCI elution (1 M)

Vol
(Vml)

Total

(MI)

(U)
activity

Sp act
(U/mg)

Yield
(%

100
100
29

145
130
66.7

7.2
7.2
280

100
90
46

heated at 105C for 10 min. Pentoses gave green to gray

spots.
Enzyme modifications. Enzyme (1 p,g) was added to tubes
containing 0.5 ml of 50 mM succinic acid buffer (pH 6.0) with
different amounts of xylan (0 to 5 mg). The modifier,
N-bromosuccinimide at 4 ,uM orp-hydroxymercuribenzoate
at 1 mM (both from Sigma), was added to the tubes, and the
mixtures were incubated for 10 min at 25C. Residual activity of the enzyme was determined in the standard assay by
adding 0.5% xylan to the reaction tubes.
Mode of action. To test whether xylanase T-6 is an
endoxylanase, viscosity and reducing sugar content were
determined in parallel. The enzymatic reaction, carried out
in 50 mM phosphate buffer (pH 7.0) containing xylan (0.4%),
was performed directly in an Ostwald capillary viscometer
(Volac size 50) while the viscosity was being measured (the
buffer value was 184 s at 65C).
Amino acid analyses. Total amino acid analysis was done
on a Dionex amino acid analyzer, by using ion-exchange
chromatography with ninhydrin. In addition, the number of
thiols in the protein was estimated by the Ellman assay after
the enzyme was reduced with dithiothreitol (8). The N-terminal amino acid sequence of xylanase T-6 was determined
with an Applied Biosystems model 475A gas-phase sequencer.
RESULTS AND DISCUSSION

Enzyme production and purification. B. stearothermophilus T-6 was isolated by using an enrichment and screening
procedure for extracellular xylanases that are thermostable
and alkaline tolerant (37). Extracellular xylanase activity
was found to be present in the growth media of B. stearothermophilus T-6 grown in the presence of xylose. In a
typical fermentation on XMP medium, cell turbidity reached
500 to 600 Klett units and the extracellular xylanase activity
was about 2 U/ml. The activity in the broth could be
concentrated either by ammonium sulfate or acetone precipitation. The most efficient way of concentrating the enzyme,
with no loss of activity, was with dialysis tubing placed
against solid polyethylene glycol 20000. The enzyme could
also be concentrated and purified by adsorbing it onto a
cation exchanger such as CM-11, CM-52, or SE-52 (Whatman). It was possible to efficiently adsorb the enzyme
directly from the fermentation broth, provided that the ionic
strength of the broth was sufficiently low (below 50 mM KCI
equivalent). Reduction of the ionic strength was achieved by
either dialyzing the broth against a low-strength buffer or,
alternatively, by omitting the MOPS buffer from the medium
and controlling the pH externally (i.e., via a pH controller).
Typical results of this single-step purification scheme are
shown in Table 1. The total yield was 46%, with a purification factor of about 42. The enzyme obtained in the KCI
eluent was more than 99% pure as judged by FPLC gel

VOL. 59, 1993

XYLANASE FROM B. STEAROTHERMOPHILUS T-6

t*00-0

*,

MW

100

1727

::-4

Y-~

>o

80

00
o

60
1 06,500

4.

*-

40
._i

80,000

"

*"|.|.|.l~~~~~~~~~
m

20

o
49,500

10

11

pH

i.

FIG. 2. Effect of pH on the initial reaction rates of xylanase T-6.

Buffers (50 mM) used include citric acid (0), phosphate (0), boric
acid (-), and phosphate-NaOH (O).
-

32,500

FIG. 1. SDS-PAGE of purified and crude xylanase T-6. Lanes:

A, concentrated crude broth; B, cation exchanger-purified xylanase;
C, FPLC-purified xylanase after a cation exchanger purification.

filtration, N-terminal analysis, and SDS-PAGE (Fig. 1). A

high degree of purification (40- to 50-fold), with a yield of 40
to 50%, was achieved repeatedly in both small- (5-liter) and
large-scale (1,000-liter) purification experiments. This simple
and effective batch adsorption procedure was possible because of (i) the high selectivity of the cation exchanger
adsorption step and (ii) the high partition coefficient of
xylanase T-6 to the adsorbent (37). Purification yields for
other xylanases usually lie in the range of 1 to 30% (1, 3, 10,
15, 30, 46). A similar single-step purification procedure was
reported for a- and ,-galactosidases from Aspergillus niger
(7).
Molecular weight determination. The molecular weight of
the purified xylanase T-6 was estimated by both SDS-PAGE
and by gel filtration on Sephadex G-75 and FPLC Superose
12 HR 10/30 columns (Pharmacia). The SDS-PAGE determination gave a molecular weight of 43,000 (Fig. 1). However, estimation of the molecular weight by gel filtration on
Sephadex G-75 gave varied results, strongly dependent on
the ionic strength of the elution buffer. The retention coefficient of the enzyme decreased with the increase of the
buffer ionic strength (in the range of 20 to 100 mM phosphate
buffer [pH 7]), suggesting that the enzyme interacts with
Sephadex. At buffer concentrations of 100 mM or higher, the
enzyme was eluted as a sharp peak corresponding to a
molecular weight of 31,000. Gel filtration on Superose 12 (the
elution buffer was 100 mM phosphate buffer [pH 7]-100 mM
NaCl-0.02% NaN3) gave retention coefficients of 1.92, 1.76,
and 2.05 for xylanase T-6, bovine serum albumin (molecular
weight, 66,000), and carbonic anhydrase (molecular weight,
29,000), respectively, suggesting a molecular weight of
41,900 for xylanase T-6. The enzyme probably consists of a
single polypeptide chain, since the molecular weight estimations by SDS-PAGE (43,000) and FPLC gel filtration (41,900)
similar.
pl. The isoelectric point of xylanase T-6

are

was

determined

under native and denaturing conditions. The native protein

exhibited a pI of 9.0 on isoelectric focusing gels, and, in
agreement with this result, the protein was eluted at pH 9.0
from a chromatofocusing column. Under denaturing conditions, the pI of the enzyme was 7.0. Under both conditions,
the purified protein gave three minor bands on the isoelectric
focusing gels. These bands probably reflect small changes of
charged groups on the enzyme. The differences in the pl of
the minor components were +0.15 pH unit. The relatively
high pI of the native protein (9.0) explains its positive charge
at neutral pHs and the fact that it can be selectively adsorbed
onto a cation exchanger from the crude supernatant.
Kinetics of xylan degradation. Xylanase T-6 hydrolyzes oat
spelts xylan to release reducing sugars. At 55C, the release
of reducing sugar was linear with time (for at least 20 min)
and proportional to enzyme concentration (for formation of
up to 1.5 ,umol of xylose equivalent in the reaction tubes).
Kinetic experiments at 55C with different xylan concentrations gave a Vm, of 288 U/mg and a Km of 1.63 mg/ml. The
turnover number of the enzyme was 12,382 (moles of reducing ends released per mole of enzyme per minute), calculated
from its Vm. and the estimated molecular weight of 43,000.
Effect of pH. The pH range at which xylanase T-6 is active
was determined in four different buffers covering the range
between pH 5.0 and 11.0. The enzyme was most active in the
neutral pH range, between pH 6.5 and 7.0, but retained 60%
of its activity at pH 10 (Fig. 2). At pHs below 4.5, the
enzyme tended to precipitate in our assay conditions. The
alkaline tolerance of xylanase T-6 is crucial, considering its
potential application in bleaching pulp at high pH values.
Indeed, the enzyme was shown to bleach pulp optimally at
pH 9.0 and 65C (16). Most xylanases known today are
active at acidic (20, 39) or neutral pHs (2, 3, 26, 30).
Recently, however, several alkaline-tolerant xylanases were
characterized (1, 9, 18, 19, 31-33, 41).
Reaction rate at different temperatures. Initial reaction
rates were determined at temperatures between 45 and 85C.
The highest initial reaction rate was obtained at 75C. The
relative activities at the different temperatures were 28, 51,
70, 81, 90, 100, 74, and 40% at 45, 55, 60, 65, 70, 75, 80, and
85C, respectively. The Arrhenius plot-calculated activation

APPL. ENVIRON. MICROBIOL.

KHASIN ET AL.

1728

i
E

80

,60

0)
co
n
n

'

>k

C
0

-._

0*

._

cr

5
40.

5.

* 20

50
100
Time (min)

10

12

Time (hours)
FIG. 3. Thermostability of xylanase T-6 at pH 7.0. Residual
xylanase activity was monitored at various times after incubation at
65C (0), 70C (0), and 75C (-).

energy, 9.3 kcal/mol (ca. 39 kJ/mol), is in the range that is

characteristic of typical enzymatic reactions.
Thermostability. Thermoinactivation experiments for xylanase T-6 were performed by incubating the enzyme solution at different temperatures and determining the residual
activity at various times. The thermoinactivation of the
enzyme at pH 7.0 is shown in Fig. 3. Exposure of the
enzyme for more than 10 h at 65C did not affect the activity.
At 70 and 75C, the half-lives of the enzyme were about 14.5
h and 20 min, respectively. At pH 9.0 and 65C, the half-life
of the enzyme was about 6 h. The activation energy of the
thermoinactivation mechanism can be estimated from the
kinetics of thermoinactivation at pH 7 and is equal to 119
kcal/mol (ca. 498 kJ/mol). This value, which is uncharacteristic of covalent reactions, suggests that thermoinactivation
is controlled by a monomolecular conformational process
(unfolding of the native protein) (22, 23).
Effect of metal ions. The effect of different metals on the
activity of xylanase T-6 is shown in Table 2. The metal ions
TABLE 2. Effect of metal ions on xylanase T-6 activity
Chemical

None
LiCl
NaCl

KCl
MgCl2
CaSO4
BaSO4
FeSO4
Ni(NO3)2

MnCl2
CoCl2
CuS04
Ag(NO3)2
ZnCl2
CdCl2

HgCl2

A1(NO3)3
EDTA

Xylanase activity (%)

10 mM

1 mM

0.1 mM

100
101
110
110
109
88
90
63
51

100
95
94
101
101
102
96
104
98
62
78
48
67
38
47
17
54
100

100

103
104
101
100
98
90
70
91

FIG. 4. Drop in viscosity and formation of reducing power

during the degradation of xylan by xylanase T-6. The reaction was
carried out on 0.4% xylan at 65C. Symbols: 0, relative viscosity;
0, xylose equivalent.

Fe2+, Ni2+, Mn2+, Co2+, Cu2+, Ag2+, and Al` showed

some inhibition of the activity, while the group Ilb metals,
Zn2+, Cd2+, and Hg2+, gave the strongest inhibition. The
group Ilb metals exhibit high affinity for reactive groups. For
example, the affinity of Hg toward reactive groups is SH >
CONH2> NH2> COOH> PO4 (42). Monovalent cations
such as Li+, Na+, and K+ had small stimulating effects on
the activity. The addition of EDTA did not affect the
activity, suggesting that no metals are needed for the enzymatic reaction.
Mode of action. To test whether xylanase T-6 is an
endoxylanase, the decrease of viscosity and formation of
reducing sugar were determined in parallel. The action of
xylanase T-6 on xylan was characterized by a rapid reduction in the viscosity of the substrate and a relatively slow
increase in the concentration of reducing sugar (Fig. 4). The
increase in reducing sugar from 0.4 to 1.7 ,umol of xylose
equivalent per ml (an average of two breakages per molecule) results in a 25% decrease in viscosity. On the basis of
the Einstein equation, which relates molecular weight to
viscosity, it is clear that such reduction in viscosity can
result only from an endo-cleavage of the molecules. To
examine the extent to which xylanase T-6 can degrade xylan,
a complete digestion experiment was performed. The standard enzymatic assay reaction (0.5% xylan) continued for 20
h at 65C, and samples were taken for reducing sugar
analysis and sugar composition analysis on thin-layer chromatography. The enzyme solution (10 ,ul of a 40-U/ml
concentration) was added periodically to the reaction mixture to facilitate the degradation of the polymer. The complete digestion of oat spelts xylan produced about 25 ,umol of
xylose equivalent per ml, compared with the theoretical
maximum value of 37.8 ,umol/ml. The average size of the end
product is about 1.5 xylopyranose units, possibly corresponding to a mixture of xylose and xylobiose. Thin-layer
chromatography of the end products also indicated that
xylose and xylobiose were the only main products after the
20-h digestion (data not shown). Nanmori et al. (28) characterized a xylanase from B. stearothermophilus 21. The
action of this enzyme together with 3-xylosidase on xylan
gave only xylose, suggesting that xylanase 21 and xylanase
T-6 have similar modes of action. The two enzymes, however, have different molecular masses and isoelectric points.
Substrate specificity. The activity of xylanase T-6 was

VOL. 59, 1993

XYLANASE FROM B. STEAROTHERMOPHILUS T-6

TABLE 3. Xylanase T-6 substrate specificity

Substrate"
Xylan (oat spelts)
Guar gum
Locust bean gum
Arabinogalactan
CMC

PNP-P-D-cellobioside
PNP-P-D-xylopyranoside

1,4-D-Xylanase
D-Mannanase
D-Mannanase
D-Galactanase

288
<0.01
<0.01
<0.01
0.03
3.10
0.20
0.04
0.03
0.01
0.02
0.02

Endo-1,4-p-glucanase

Exo-1,4-P-glucanase
P-D-Xylosidase

PN1-ct-L-arabinopyranoside

a-L-Arabinopyranosidase

PNP-a-L-arabinofuranoside

a-L-Arabinofuranosidase
P-D-Galactosidase

PNP-P-D-galactopyranoside
PNP-a-D-glucopyranoside
PNP-P-D-glucopyranoside

(U/ag)b

Related enzyme

a-D-Glucosidase
,-D-Glucosidase

a PNP, p-nitrophenyl; CMC, carboxymethyl cellulose.

b Reactions were
carried out at 55'C with a highly concentrated and purified
enzyme. A unit of activity was defined as the amount of enzyme which
released 1 ,umol of either reducing sugar equivalent orp-nitrophenol per min.
With the following substrates the activity was less than 0.01 U/mg: PNP-13L-arabinopyranoside, PNP-a-L-fucopyranoside, PNP- -L-fucopyranoside,
PNP-a-L-rhamnopyranoside, PNP-3-D-mannopyranoside, PNP-a-D-mannopyranoside.

tested on several cellulose- and hemicellulose-related substrates (Table 3). The enzyme can be classified as a type IIa
endoxylanase, which cannot cleave L-arabinosyl branch
points but does cleave xylooligosaccharides as short as
xylotriose and produces mainly xylose and xylobiose as final
products (34). The very low activity on carboxymethyl
cellulose is an advantage, considering the potential application of the enzyme in biobleaching pulp for high-quality
paper.
Amino acid analyses. The amino acid composition of
xylanase T-6 is given in Table 4. The enzyme is rich in lysine
and probably has no cysteines. No thiol groups were detected with the Ellman assay. The first 45 amino acids from
the N terminus of xylanase T-6 were sequenced. This
sequence was analyzed against the GenBank data bank
libraries with TFasta (GCG software package; Genetics
Computer Group, University of Wisconsin, Madison) and
showed significant homology (45.5% on 33 amino acids
overlap) only with the N-terminal region of xylanase A from
the alkalophilic Bacillus sp. strain C-125 (13, 14, 17, 18) (Fig.
5). The two enzymes have similar molecular weights, exhibit
TABLE 4. Amino acid composition of xylanase T-6

Aspartic acid (Asp + Asn)

Threonine
Serine
Glutamic acid (Glu + Gln)
Proline
Glycine
Alanine
Valine
Methionine
Isoleucine
Leucine

Tyrosine
Phenylalanine
Lysine
Histidine

Arginine

Concn
(nmol)

Residues/

4.71
0.85
0.66
3.64
1.95
1.64
2.44
2.25
0.15
2.06
1.03
1.61
1.27
3.12
0.56
0.90

58
12
10
44
24
20
30
28
2
26
14
22
16
38
6
12

molecule

1729

1
Xylanase T-6
Lys Asn Ala Asp Ser Tyr Ala Lys Lys Pro flia Ile Ser Ala Leu
Val Phe Gly Glu Asn Glu Lys Arg Asn Asp Glan Pro Phe Ala Trp
10
Xylanase A

16

Aan AJA BroQ Gln Leu AS; Lin Arg Tyr Ly- Asn Glu Phe Thr Ile
Gln VYal Ala Ser Leu Ser Glu Arg Tyr Gln Glu GUn Phe Asp Ile
26
31

Gly Ala Ala Val Glu Pro Tyr Gln Leu Gln Asn Glu Lys Asp Val
Gly Ala Ala Val Glu Pro Tyr Gln Leu Glu Gly Arg Gln Ala Gln
41

FIG. 5. Alignment of the N-terminal sequence of xylanase T-6

with the N-terminal sequence of xylanase A from the alkalophilic
Bacillus sp. strain C-125. Identical amino acids are in boldface type;
conservative amino acids replacements are underlined.

broad pH activity curves, and are both active at an alkaline

pH. However, xylanase T-6 is more thermostable and can
hydrolyze xylotriose to xylose and xylobiose, and after
complete hydrolysis of xylan, its end products are only
xylose and xylobiose. It will be interesting to compare the
complete amino acid sequence of the two enzymes and to
identify the regions responsible for their exceptional alkaline
and heat tolerance properties.
Chemical modifications of xylanase T-6. Tryptophan and
cysteine were shown to be involved in the active site of
different xylanases (4, 21, 26). To test whether these residues
are present in the active or binding site of xylanase T-6, we
examined the ability of xylan (the substrate) to protect the
enzyme from tryptophan or cysteine modifiers. Xylanase T-6
(2 p,g/ml) was completely inhibited by 4 ,uM of N-bromosuccinimide (a tryptophan modifier); however, xylan at a concentration as low as 1 mg/ml gave over 95% protection
against this reagent. Only about 15% inhibition was detected
after treatment with 1 mM of p-hydroxymercuribenzoate (a
cysteine modifier), indicating again that cysteine is not
present in the protein. The slight inhibition is probably due
to the reaction of this modifier with other residues.
ACKNOWLEDGMENTS
This work was supported by a grant from Biovik AB and Korsnas
AB. Technical support was provided by the Technion-Otto Meyerhof Biotechnological Laboratories.
We thank Orit Gat, Eugene Rosenberg, Raphael Lamed, and the
Board Paper Pulp-Development group at Korsnfis for helpful comments on the manuscript.

REFERENCES
1. Akiba, T., and K. Horikoshi. 1988. Xylanases of alkalophilic
thermophilic Bacillus. Methods Enzymol. 160:655-659.
2. Berenger, J., F. Chantal, J. Bigliardi, and N. Creuset. 1985.
Production, purification and properties of thermostable xylanase from Clostridium stercorarium. Can. J. Microbiol. 31:635643.
3. Bernier, R., M. Desrochers, L. Jurasek, and M. G. Paice. 1983.
Isolation and characterization of a xylanase from Bacillus
subtilis. Appl. Environ. Microbiol. 46:511-514.
4. Biswas, S. R., S. C. Jana, A. K. Mishra, and G. Nanda. 1990.
Production, purification and characterization of xylanase from a
hyperxylanolytic mutant of Aspergillus ochraceus. Biotechnol.
Bioeng. 35:244-251.
5. Bradford, M. M. 1976. A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal. Biochem. 72:248-254.
6. Browning, B. L. 1963. The composition and chemical reactions
of wood, p. 58-101. In B. L. Browning (ed.), The chemistry of
wood. John Wiley & Sons, Inc. New York.
7. Christakopoulos, P., B. J. Marcris, and D. Kekos. 1990. Excep-

1730

8.
9.

10.
11.

12.
13.

14.
15.
16.

17.
18.
19.
20.

21.
22.
23.
24.

25.

APPL. ENVIRON. MICROBIOL.

KHASIN ET AL.

tionally thermostable a- and P-galactosidases from Aspergillus

niger separated in one step. Proc. Biochem. Int. 210-212.
Creighton, T. E. 1989. Disulphide bonds between cysteine
residues, p. 157. In T. E. Creighton (ed.), Protein structure, a
practical approach. IRL Press, Oxford.
Dey, D., J. Hinge, A. Shendye, and M. Rao. 1991. Purification
and properties of extracellular endoxylanases from alkalophilic
thermophilic Bacillus sp. Can. J. Microbiol. 38:436-442.
Fournier, R. A., M. M. Fredrick, J. R. Fredrick, and P. J. Reilly.
1985. Purification and characterization of endo-xylanase from
Aspergillus niger. Biotechnol. Bioeng. 27:539-546.
Grant, R. 1991. First mill-scale trials get underway. Pulp Paper
Int. 33:61-63.
Guilian, G. G., R. L. Moss, and M. Greaser. 1984. Analytical
isoelectric focusing using a high voltage vertical slab polyacrylamide gel system. Anal. Biochem. 142:421-436.
Hamamoto, T., H. Honda, T. Kudo, and K. Horikoshi. 1987.
Nucleotide sequence of the xylanase A gene of alkalophilic
Bacillus sp. strain C-125. Agric. Biol. Chem. 51:953-955.
Hamamoto, T., and K. Horikoshi. 1987. Alkalophilic Bacillus
xylanase A, a secretable protein through outer membrane of
Escherichia coli. Agric. Biol. Chem. 51:3133-3135.
Hashimoto, S., T. Muramatsu, and M. Funatsu. 1971. Studies on
xylanase from Trichoderma viride. Agric. Biol. Chem. 35:501508.
Hogman, S., H. Joves, E. Rosenberg, and Y. Shoham. 1992.
Bleachability improvement of softwood kraft pulp through treatment with an alkali- and thermostable xylanase, p. 107-113. In
M. Kuwahara and M. Shimada (ed.), Biotechnology in pulp and
paper industry. Uni Publishers, Tokyo.
Honda, H., T. Kudo, and K. Horikoshi. 1985. Molecular cloning
and expression of the xylanase gene of alkalophilic Bacillus sp.
strain C-125 in Escherichia coli. J. Bacteriol. 161:784-785.
Honda, H., T. Kudo, Y. Ikura, and K. Horikoshi. 1985. 'rwo
types of xylanases of alkalophilic Bacillus sp. no. C-125. Can. J.
Microbiol. 31:538-542.
Horikoshi, K., and Y. Atsukawa. 1973. Xylanase produced by
alkalophilic Bacillus no. C-59-2. Agric. Biol. Chem. 37:20932103.
John, M., B. Schmidt, and J. Schmidt. 1979. Purification and
some properties of five endo-1,4-p-D-xylanases and a P-Dxylosidase produced by a strain of Aspergillus niger. Can. J.
Biochem. 57:125-134.
Keskar, S. S., C. Srinivasan, and V. Deshphande. 1989. Chemical modification of a xylanase from thermotolerant Streptomyces. Biochem. J. 261:49-55.
Klibanov, A. M. 1983. Stabilization of enzymes against thermal
inactivation. Adv. Appl. Microbiol. 29:1-24.
Klibanov, A. M., and D. B. Volkin. 1989. Minimizing protein
inactivation, p. 1-24. In T. E. Creighton (ed.), Protein function,
a practical approach. IRL Press, Oxford.
Koponen, R. 1991. Enzyme systems prove their potential. Pulp
Paper Int. 33:81-83.
Laemmli, U. K. 1970. Cleavage of structural proteins during the
assembly of the head of bacteriophage T-4. Nature (London)

227:680-685.
26. Marui, M., K. Nacanishi, and T. Yasui. 1985. Purification and
properties of three types of xylanases induced by methyl-pxyloside from Streptomyces sp. Agric. Biol. Chem. 49:33993407.
27. Miller, G. L. 1959. Use of dinitrosalicylic acid reagent for

determination of reducing sugars. Anal. Chem. 31:426-428.

28. Nanmori, T., T. Watanabe, R. Shinke, A. Kohno, and Y.
Kawamura. 1990. Purification and properties of thermostable
xylanase and 3-xylosidase produced by a newly isolated Bacillus stearothermophilus strain. J. Bacteriol. 172:6669-6672.
29. O'Farrell, P. Z., H. M. Goodman, and P. H. O'Farrell. 1977.
High resolution two-dimensional electrophoresis of basic as
well as acidic proteins. Cell 12:1133-1142.
30. Okada, H., and A. Shinmyo. 1988. Xylanase from Bacillus
pumilus. Methods Enzymol. 160:632-637.
31. Okazaki, W., T. Akiba, K. Horikoshi, and R. Akahoshi. 1984.
Production and properties of two types of xylanases from
alkalophilic thermophilic Bacillus spp. Appl. Microbiol. Biotechnol. 19:335-340.
32. Okazaki, W., T. Akiba, K. Horikoshi, and R. Akahoshi. 1985.
Purification and characterization of xylanases from alkalophilic
thermophilic Bacillus spp. Agri. Biol. Chem. 49:2033-2039.
33. Ratto, M., K. Poutanen, and L. Viikari. 1992. Production of
xylanolytic enzymes by an alkalitolerant Bacillus circulans
strain. Appl. Microbiol. Biotechnol. 37:470-473.
34. Reilly, P. J. 1981. Xylanases: structure and functions, p. 111129. In A. E. Hollaender and R. Robson (ed.), Trends in the
biology of fermentations for fuels and chemicals. Plenum Publishing Corp., New York.
35. Rosenberg, E., and Y. Shoham. August 1991. A preparation
exhibiting enzymatic delignification activity, a method of producing the same, and applications thereof. Sweden patent no.
465 320.
36. Samadni, G. 1991. Pulp bleaching-the race for safer methods.
Chem. Eng. (Int. Ed.) 98:37-43.
37. Shoham, Y., Z. Schwartz, A. Khasin, 0. Gat, Z. Zosim, and E.
Rosenberg. Delignification of wood pulp by a thermostable
xylanase from Bacillus stearothermophilus strain T-6. Biodegradation, in press.
38. Singh, P. S. 1979. Principles of pulp bleaching, p. 15-28. In P. S.
Singh (ed.), The bleaching of pulp, 3rd ed. Tappi Press, Atlanta.
39. Tavobilov, I. M., I. V. Gorbacheva, N. A. Rodionova, and A. M.
Bezborodov. 1981. Purification of endo-1-4-3-xylanase from the
fungus Aspergillus niger strain 15. Appl. Biochem. Microbiol.

17:320-324.
40. Trubacek, I., and A. Wiley. 1979. Bleaching and pollution, p.
423-461. In P. S. Singh (ed.), The bleaching of pulp, 3rd ed.
Tappi Press, Atlanta.
41. Tsujibo, H., T. Sakamoto, N. Nishino, T. Hasegawa, and Y.
Inamori. 1990. Purification and properties of three types of
xylanases produced by an alkalophilic actinomycete. J. Appl.
Bacteriol. 69:398-405.
42. Venugopal, B., and T. D. Luckey. 1978. Metal toxicity in
mammals, p. 68-89. Plenum Press, New York.
43. Viikari, L., A. Kantelinen, M. Ratto, and J. Sundquist. 1991.
Enzymes in pulp and paper processing, p. 12-21. ACS Symp.
Ser. 460 (Enzymes Biomass Conversion). American Chemical
Society, Washington, D.C.
44. Viikari, L., J. Sundquist, and J. Kettunen. 1991. Xylanase
enzymes promote pulp bleaching. Paperi ja Puu - Paper and
Timber 73:384-389.
45. Wong, K. Y., L. U. L. Tan, and J. N. Saddler. 1988. Multiplicity
of 0-1,4-xylanase in microorganisms: functions and applications. Microbiol. Rev. 52:305-317.
46. Yasui, T., M. Marui, I. Kusakabe, and K. Nakanishi. 1988.
Xylanases of Streptomyces. Methods Enzymol. 160:648-654.