APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1995, p. 4468–4470 Vol. 61, No.

12
0099-2240/95/$04.0010
Copyright q 1995, American Society for Microbiology

Purification and Properties of Xylanase A from Alkali-Tolerant

Bacillus sp. Strain BP-23
ANA BLANCO,1 TERESA VIDAL,2 JOSÉ F. COLOM,2 AND F. I. JAVIER PASTOR1*
Department of Microbiology, Faculty of Biology, University of Barcelona,1 and Department of Textile and
Paper Engineering, ETSII Terrassa, Polytechnic University of Catalonia,2 Barcelona, Spain
Received 12 May 1995/Accepted 25 September 1995

Xylanase A from the recently isolated Bacillus sp. strain BP-23 was purified to homogeneity. The enzyme
shows a molecular mass of 32 kDa and an isoelectric point of 9.3. Optimum temperature and pH for xylanase
activity were 50&C and 5.5 respectively. Xylanase A was completely inhibited by N-bromosuccinimide. The
main products of birchwood xylan hydrolysis were xylotetraose and xylobiose. The enzyme was shown to
facilitate chemical bleaching of pulp, generating savings of 38% in terms of chlorine dioxide consumption. The
amino-terminal sequence of xylanase A has a conserved sequence of five amino acids found in xylanases from
family F.

Xylan is an abundant biopolymer found in plant tissues as a
major component of cell walls. It is a complex molecule com-
posed of b-1,4-linked xylose chains with branches containing
arabinose and 4-O-methylglucuronic acid. Biodegradation of
xylan requires the action of several enzymes, among which
xylanases (1,4-b-D-xylan xylanohydrolase [EC 3.2.1.8]) play a
key role. Xylanases have important applications in the pulp
and paper industry (21). They can also be used to increase the
digestibility of animal feed stock and in the baking and brewing
industries (22). In a previous screening, we isolated an alkali-
tolerant strain of Bacillus sp., BP-23, from rice field soil. The
strain secretes a set of multiple xylanases, most of which have
neutral or acidic pI, while the enzyme with the highest level of
activity in isoelectric focusing zymograms has an alkaline pI
(2). In this paper, we report the purification and characteriza-
tion of this enzyme, xylanase A.
Bacillus sp. strain BP-23 was grown in nutrient broth sup-
plemented with 0.2% birchwood xylan at pH 6.8 for 3 days. The
supernatant of the culture was concentrated by ultrafiltration,
adjusted to pH 9.0 with Tris-HCl buffer, and applied to a
carboxymethyl (CM)-Sephadex column. The neutral and acidic
pI xylanases were eluted in a single peak of nonadsorbed ma-
terial, while xylanase A was adsorbed to the gel matrix. Xyla-
nase A was next eluted with a linear gradient of NaCl. Only a
few proteins, at a negligible concentration and without xyla-
nase activity, coeluted with the enzyme. These minor contam-
inants were removed after two chromatography steps with Phe-
nyl Sepharose and CM-Sephadex columns as summarized in
Table 1.
The specific activity of the purified xylanase A, determined
by the method of Nelson and Somogyi (20), was 40.2 U/mg.
The apparent purity of the enzyme was demonstrated by so-
dium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE), which showed that the molecular mass of xylanase A
was 32 kDa (Fig. 1). Zymogram analysis of a gel containing
0.2% birchwood xylan (10) showed a single activity band. The
pI of xylanase A, as determined with isoelectric focusing gels
(Serva, Heidelberg, Germany), was 9.3 (Table 2).
The effect of pH on xylanase activity is shown in Fig. 2.
Optimum pH was 5.5; however, the enzyme also showed a high
level of activity at alkaline pH (72 and 35% activity at pH 9.5
and 11.0, respectively). The enzyme remained stable after in-
cubation at 378C in pH 11.0 buffer for 11 days. The activity and
stability at alkaline pH of xylanase A resemble those of xyla-
nases of several alkalophilic Bacillus species (5, 16, 18). The
optimum temperature for xylanase activity was found to be
508C (Table 2). The study of thermostability showed that the
enzyme was highly stable at temperatures up to 558C. After 40
h of incubation at this temperature and at pH 7 or 8, the
enzyme retained 100% activity. Incubation at higher temper-
atures rapidly inactivated the enzyme; at 598C, the half-life of
the enzyme activity was 1 h.
Xylanase A was completely inhibited by Sn21 at a concen-
tration of 10 mM. Fe31, Ag1, Zn21, and Hg21 also led to
strong inhibition of xylanase A (2, 26, 28, and 44% of the
remaining activity, respectively). Little inhibition was caused
by Mn21, Co21, Ni21, and Cu21. Ca21 and Ba21 did not affect
enzyme activity, while Mg21 had a slight stimulatory effect on
activity. The effect of chemical inhibitors on xylanase activity
was tested. Xylanase A was strongly inhibited by 1 mM N-
bromosuccinimide and 10 mM 2-hydroxy-5-nitrobenzyl bro-
mide (0 and 24% residual activity, respectively). The inhibition
caused by these tryptophan modifiers (13, 19) suggests the
involvement of tryptophan residues in the active site of the
enzyme, similar to what has been reported for several xylanases
(4, 25). N-Acetylimidazole did not affect enzymatic activity,
while p-hydroxymercurybenzoate (10 mM) caused a 12% de-
crease in activity. The kinetic parameters of the enzyme, de-
termined with birchwood xylan as the substrate, are shown in
Table 2.
TABLE 1. Purification of xylanase A from Bacillus sp. strain BP-23
Total
Purification step
Vol
activity
Protein Sp act Yield
(ml) (mg) (U/mg) (%)
(U)

Culture fluid 2,000 2,240 276.0 8.1 100.0

Concentrated culture fluid 55 2,046 117.7 17.4 91.3
* Corresponding author. Mailing address: Departament de Micro- CM-Sephadex 88 700 22.4 31.3 31.2
biologia, Facultat de Biologia, Universitat de Barcelona, Avinguda Phenyl Sepharose 140 459 11.5 40.0 20.5
Diagonal 645, 08028 Barcelona, Spain. Phone: 34-3-4021483. Fax: 34- CM-Sephadex 92 455 11.3 40.2 20.3
3-4110592.

4468
VOL. 61, 1995
FIG. 1. SDS-PAGE of purified xylanase A. Duplicate samples were analyzed
in a 10% polyacrylamide gel containing 0.2% xylan that was split into two parts.
One of them was stained with Coomassie brilliant blue (A), and the other was
developed as a zymogram (B). Lanes 1, concentrated culture fluid; lanes 2,
purified xylanase A. The amounts of purified xylanase loaded in the gel were 2
mg for protein staining and 1 mg for zymogram analysis. The positions of mo-
lecular mass markers are indicated.
The enzyme did not show activity on carboxymethyl cellu-
lose, Avicel, laminarin, lichenan, arabinogalactan, polygalactu-
ronate, starch, p-nitrophenyl-b-D-glucopyranoside, or p-nitro-
phenyl-a-L-arabinofuranoside. Xylans from birchwood, oat
spelt, larchwood, and methylglucuronoxylan were hydrolyzed
by xylanase A. Maximum activity was shown with birchwood
xylan (42.6 U/mg). Xylanase A showed very little activity on
p-nitrophenyl-b-D-xylopyranoside (0.49 U/mg). The products
of birchwood xylan hydrolysis were analyzed with silica gel
thin-layer chromatograms. The main product found in mid- or
long-term incubations was xylotetraose. Xylobiose and small
amounts of xylose were also found, while xylotriose was not
detected. To our knowledge, xylanases with a similar hydrolysis
pattern have not been described, although a fungal xylanase
giving xylotetraose as the main hydrolysis product from xylan
has been reported (6). Oat spelt xylan hydrolysis gave xylo-
biose, xylose, and oligosaccharides with mobilities intermedi-
ate between those of xylotetraose, xylotriose, and xylobiose.
Similar intermediate oligosaccharides have also been de-
scribed with other bacterial xylanases (16) and probably cor-
respond to arabinose- or glucuronic acid-containing small oli-
gosaccharides. Xylanase A did not release arabinose from the
xylans tested. Xylotetraose and xylotriose were hydrolyzed by
the purified enzyme, while xylobiose was not degraded at all.
Our data suggest that xylanase A is an endoxylanase without
debranching activity. The production of small xylo-oligosac-
charides with intermediate mobility seems to indicate that the
enzyme can cleave bonds adjacent to side chains.
The effect of xylanase A on pulp bleaching was studied.
Unbleached pulp from Eucalyptus globulus (kappa number,
8.1; ISO brightness, 50.7%; 0.5% cupriethylenediamine viscos-
ity, 920 cm3/g) was incubated with xylanase A (1 U/g of pulp)
TABLE 2. Properties of purified xylanase A
Property Value
Molecular mass (kDa).................................................................... 32
Isoelectric point (pH)..................................................................... 9.3
Optimum temperature (8C)........................................................... 50
Optimum pH ................................................................................... 5.5
Main products of xylan hydrolysis ................................................ X4, X2
Km (mg of xylan ml21) ................................................................... 10.49
Vmax (mmol of xylose min21 mg21) .............................................. 42.02
NOTES 4469
FIG. 2. Effect of pH on activity of xylanase A. The following buffers were
used: 50 mM sodium citrate (pH 3.0 to 4.0), 50 mM sodium acetate (pH 4.0 to
6.0), 50 mM sodium phosphate (pH 6.0 to 8.0), 50 mM Tris-HCl (pH 8.0 to 9.0),
and 50 mM sodium glycine (pH 9.0 to 11.0).
at 458C for 120 min prior to chemical bleaching, consisting of
two chlorine dioxide treatments separated by an intermediate
alkaline peroxide extraction. Incubation with xylanase A nota-
bly increased the bleachability of pulp. The final brightness of
xylanase-pretreated pulp was higher (88.8%) than that of ref-
erence pulp not pretreated with the enzyme (87.7%). To ob-
tain chemically bleached pulp with brightness similar to that of
the xylanase-pretreated pulp, a 38% increase in chlorine diox-
ide charge was needed. Xylanase A efficiently contributed to
the removal of lignin from pulp. In fact, kappa numbers of
xylanase-pretreated pulp, determined at several stages in the
bleaching sequence, were notably lower than those of control
pulp (22 to 29% decrease). Pulp viscosity was not significantly
affected by the treatment with xylanase A.
The amino acid composition of xylanase A is shown in Table
3. For this analysis, the enzyme was hydrolyzed with HCl as
described by Liu and Chang (14), and amino acids were de-
termined with a Pharmacia LKB Alpha plus series 2 Autoana-
lyzer. The N-terminal sequence of xylanase A was determined
by automated Edman degradation and compared with se-
quences in the PDB, GenBank, and Swissprot databases. Sig-
nificant homology was found to Pseudomonas fluorescens xyla-
TABLE 3. Amino acid composition of xylanase A from
Bacillus sp. strain BP-23
Amino acid
Composition
(mol%)
Asx ............................................................................................. 14.3
Thr ............................................................................................. 4.4
Ser .............................................................................................. 6.0
Glx.............................................................................................. 12.5
Pro.............................................................................................. 4.9
Gly.............................................................................................. 9.7
Ala.............................................................................................. 8.2
Cys.............................................................................................. 0
Val.............................................................................................. 4.9
Met............................................................................................. 1.4
Ile ............................................................................................... 5.3
Leu ............................................................................................. 6.5
Tyr.............................................................................................. 3.5
Phe ............................................................................................. 3.9
Lys.............................................................................................. 4.9
His.............................................................................................. 1.6
Trp ............................................................................................. 4.7
Arg ............................................................................................. 3.3
4470 NOTES
FIG. 3. Alignment of the N-terminal sequence of xylanase A of Bacillus sp.
strain BP-23 (XynA) with the N-terminal sequence of XynB from P. fluorescens.
Identical amino acids are boxed; conservative amino acid replacements are
underlined. The dash represents a space inserted into the XynA sequence for
alignment. The consensus sequence found in the N-terminal region of family F
xylanases is in boldface type.
nase B, a member of the family F b-glycanases according to
Gilkes et al. (9). The sequence of the first 40 amino acid
residues of Bacillus sp. strain BP-23 xylanase A has 48% iden-
tity and 15% conservative changes compared with the N-ter-
minal sequence of the P. fluorescens XynB catalytic domain
(12) (Fig. 3). Little homology was found to other family F
xylanases such as Caldocellum saccharolyticum XynA (15),
Cryptococcus albidus Xyn (3), and Bacillus sp. strain C-125
XynA (11), while homology was not found to family G xyla-
nases. Studies of sequence alignment of family F xylanases
revealed several conserved regions (1, 8). Among them, the
sequence T_EN_MK, is highly conserved in the N-terminal
region of the enzymes of this family. The N terminus of Bacil-
lus sp. strain BP-23 xylanase A contains the sequence
T_EN_TK, which shows identical amino acid residues in four
of the five positions of the proposed consensus sequence. The
pI of Bacillus sp. strain BP-23 xylanase A (9.3) is similar to
those of Bacillus subtilis Xyn (17), Bacillus circulans XlnA (24),
and Bacillus pumilus XynA (7), members of the family G b-gly-
canases. These xylanases have molecular masses of around 22
kDa and pIs of around 9 and are proposed to belong to a class
of low-molecular-mass high-pI xylanases (23). The higher mo-
lecular mass (32 kDa) of Bacillus sp. strain BP-23 xylanase A
does not fit the molecular properties of this class. Xylanases
and cellulases were grouped into families according to the
amino acid sequence homology in their catalytic domain (9).
No information about the catalytic domain sequence in xyla-
nase A is available, but homology to the N-terminal consensus
sequence of family F xylanases suggests that xylanase A be-
longs to this family. Work is in progress to isolate the xynA
gene and to study its similarity to the genes of other known
xylanases.
We acknowledge ENCE and J. Canaval for providing pulp; I. Casals,
P. Fernández, and Servei Cientı́fico-Tècnics of the University of Bar-
celona for support in amino acid analysis; C. Buesa and the Sequence
Service of the University of Barcelona for the N-terminal sequencing
of xylanase A; and A. Juárez and J. Enfedaque for scientific and
technical support. We also thank P. Dı́az and J. Martı́nez for valuable
comments and critical reading of the manuscript.
This work was partially supported by the Scientific and Technolog-
ical Research Council (DGICYT), grant PB91-0561. A. Blanco held a
predoctoral grant (F.P.I.) from the Spanish Ministry of Education and
Science.
REFERENCES
1. Baba, T., R. Shinke, and T. Nanmori. 1994. Identification and characteriza-
tion of clustered genes for thermostable xylan-degrading enzymes, b-xylosi-
APPL. ENVIRON. MICROBIOL.
dase and xylanase, of Bacillus stearothermophilus 21. Appl. Environ. Micro-
biol. 60:2252–2258.
2. Blanco, A., and F. I. J. Pastor. 1993. Characterization of cellulase-free
xylanases from the newly isolated Bacillus sp. strain BP-23. Can. J. Microbiol.
39:1162–1166.
3. Boucher, F., R. Morosoli, and S. Durand. 1988. Complete nucleotide se-
quence of the xylanase gene from the yeast Cryptococcus albidus. Nucleic
Acids Res. 16:9874.
4. Deshpande, V., J. Hinge, and M. Rao. 1990. Chemical modification of xyla-
nases: evidence for essential tryptophan and cysteine residues at the active
site. Biochim. Biophys. Acta 1041:172–177.
5. 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.
6. Fernández-Espinar, M. T., F. Piñaga, P. Sanz, D. Ramón, and S. Vallés.
1993. Purification and characterization of a neutral endoxylanase from As-
pergillus nidulans. FEMS Microbiol. Lett. 113:223–228.
7. Fukusaki, E., W. Panbangred, A. Shinmyo, and H. Okada. 1984. The com-
plete nucleotide sequence of the xylanase gene (xynA) of Bacillus pumilus.
FEBS Lett. 171:197–201.
8. Gat, O., A. Lapidot, I. Alchanati, C. Regueros, and Y. Shoham. 1994. Cloning
and DNA sequence of the gene coding for Bacillus stearothermophilus T-6
xylanase. Appl. Environ. Microbiol. 60:1889–1896.
9. Gilkes, N. R., B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J.
Warren. 1991. Domains in microbial b-1,4-glycanases: sequence conserva-
tion, function, and enzyme families. Microbiol. Rev. 55:303–315.
10. Gosalbes, M. J., J. A. Pérez-González, R. González, and A. Navarro. 1991.
Two b-glycanase genes are clustered in Bacillus polymyxa: molecular cloning,
expression, and sequence analysis of genes encoding a xylanase and endo-
b-(1,3)-(1,4)-glucanase. J. Bacteriol. 173:7705–7710.
11. 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.
12. Kellett, L. E., D. M. Poole, L. M. A. Ferreira, A. J. Durrant, G. P. Hazlewood,
and H. J. Gilbert. 1990. Xylanase B and an arabinofuranosidase from
Pseudomonas fluorescens subsp. cellulosa contain identical cellulose-binding
domains and are encoded by adjacent genes. Biochem. J. 272:369–376.
13. Keskar, S. S., M. C. Srinivasan, and V. V. Deshpande. 1989. Chemical
modification of a xylanase from a thermotolerant Streptomyces. Biochem. J.
261:49–55.
14. Liu, T. Y., and Y. H. Chang. 1971. Hydrolysis of proteins with p-toluenesul-
fonic acid. Determination of tryptophan. J. Biol. Chem. 246:2842–2848.
15. Lüthi, E., D. R. Lowe, J. McAnulty, C. Wallace, P. A. Caughey, D. Saul, and
P. L. Bergquist. 1990. Cloning, sequence analysis, and expression of genes
encoding xylan-degrading enzymes from the thermophile ‘‘Caldocellum sac-
charolyticum.’’ Appl. Environ. Microbiol. 56:1017–1024.
16. Nakamura, S., K. Wakabayashi, R. Nakai, R. Aono, and K. Horikoshi. 1993.
Purification and some properties of an alkaline xylanase from alkaliphilic
Bacillus sp. strain 41M-1. Appl. Environ. Microbiol. 59:2311–2316.
17. Paice, M. G., R. Bourbonnais, M. Desrochers, L. Jurasek, and M. Yaguchi.
1986. A xylanase gene from Bacillus subtilis: nucleotide sequence and com-
parison with B. pumilus gene. Arch. Microbiol. 144:201–206.
18. Rättö, M., K. Poutanen, and L. Viikari. 1992. Production of xylanolytic
enzymes by an alkalitolerant Bacillus circulans strain. Appl. Microbiol. Bio-
technol. 37:470–473.
19. Spande, T. F., and B. Witkop. 1967. Determination of the tryptophan content
of proteins with N-bromosuccinimide. Methods Enzymol. 11:498–506.
20. Spiro, R. G. 1966. The Nelson-Somogyi copper reduction method. Analysis
of sugars found in glycoprotein. Methods Enzymol. 8:3–26.
21. Viikari, L., A. Kantelinen, J. Sundquist, and M. Linko. 1994. Xylanases in
bleaching: from an idea to the industry. FEMS Microbiol. Rev. 13:335–350.
22. Wong, K. K. Y., and J. N. Saddler. 1992. Trichoderma xylanases, their prop-
erties and application. Crit. Rev. Biotechnol. 12:413–435.
23. Wong, K. K. Y., L. U. L. Tan, and J. N. Saddler. 1988. Multiplicity of
b-1,4-xylanase in microorganisms: functions and applications. Microbiol.
Rev. 52:305–317.
24. Yang, R. C. A., R. MacKenzie, D. Bilous, and S. A. Narang. 1989. Hyperex-
pression of a Bacillus circulans xylanase gene in Escherichia coli and charac-
terization of the gene product. Appl. Environ. Microbiol. 55:1192–1195.
25. Zhu, H., F. W. Paradis, P. J. Krell, J. P. Phillips, and C. W. Forsberg. 1994.
Enzymatic specificities and modes of action of the two catalytic domains of
the XynC xylanase from Fibrobacter succinogenes S85. J. Bacteriol. 176:3885–
3894.