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Copyright q 1995, American Society for Microbiology
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 The effect of pH on xylanase activity is shown in Fig. 2.
major component of cell walls. It is a complex molecule com- Optimum pH was 5.5; however, the enzyme also showed a high
posed of b-1,4-linked xylose chains with branches containing level of activity at alkaline pH (72 and 35% activity at pH 9.5
arabinose and 4-O-methylglucuronic acid. Biodegradation of and 11.0, respectively). The enzyme remained stable after in-
xylan requires the action of several enzymes, among which cubation at 378C in pH 11.0 buffer for 11 days. The activity and
xylanases (1,4-b-D-xylan xylanohydrolase [EC 3.2.1.8]) play a stability at alkaline pH of xylanase A resemble those of xyla-
key role. Xylanases have important applications in the pulp nases of several alkalophilic Bacillus species (5, 16, 18). The
and paper industry (21). They can also be used to increase the optimum temperature for xylanase activity was found to be
digestibility of animal feed stock and in the baking and brewing 508C (Table 2). The study of thermostability showed that the
industries (22). In a previous screening, we isolated an alkali- enzyme was highly stable at temperatures up to 558C. After 40
tolerant strain of Bacillus sp., BP-23, from rice field soil. The h of incubation at this temperature and at pH 7 or 8, the
strain secretes a set of multiple xylanases, most of which have enzyme retained 100% activity. Incubation at higher temper-
neutral or acidic pI, while the enzyme with the highest level of atures rapidly inactivated the enzyme; at 598C, the half-life of
activity in isoelectric focusing zymograms has an alkaline pI the enzyme activity was 1 h.
(2). In this paper, we report the purification and characteriza- Xylanase A was completely inhibited by Sn21 at a concen-
tion of this enzyme, xylanase A. tration of 10 mM. Fe31, Ag1, Zn21, and Hg21 also led to
Bacillus sp. strain BP-23 was grown in nutrient broth sup- strong inhibition of xylanase A (2, 26, 28, and 44% of the
plemented with 0.2% birchwood xylan at pH 6.8 for 3 days. The remaining activity, respectively). Little inhibition was caused
supernatant of the culture was concentrated by ultrafiltration, by Mn21, Co21, Ni21, and Cu21. Ca21 and Ba21 did not affect
adjusted to pH 9.0 with Tris-HCl buffer, and applied to a enzyme activity, while Mg21 had a slight stimulatory effect on
carboxymethyl (CM)-Sephadex column. The neutral and acidic activity. The effect of chemical inhibitors on xylanase activity
pI xylanases were eluted in a single peak of nonadsorbed ma- was tested. Xylanase A was strongly inhibited by 1 mM N-
terial, while xylanase A was adsorbed to the gel matrix. Xyla- bromosuccinimide and 10 mM 2-hydroxy-5-nitrobenzyl bro-
nase A was next eluted with a linear gradient of NaCl. Only a mide (0 and 24% residual activity, respectively). The inhibition
few proteins, at a negligible concentration and without xyla- caused by these tryptophan modifiers (13, 19) suggests the
nase activity, coeluted with the enzyme. These minor contam- involvement of tryptophan residues in the active site of the
inants were removed after two chromatography steps with Phe- enzyme, similar to what has been reported for several xylanases
nyl Sepharose and CM-Sephadex columns as summarized in (4, 25). N-Acetylimidazole did not affect enzymatic activity,
Table 1. while p-hydroxymercurybenzoate (10 mM) caused a 12% de-
The specific activity of the purified xylanase A, determined crease in activity. The kinetic parameters of the enzyme, de-
by the method of Nelson and Somogyi (20), was 40.2 U/mg. termined with birchwood xylan as the substrate, are shown in
The apparent purity of the enzyme was demonstrated by so- Table 2.
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 TABLE 1. Purification of xylanase A from Bacillus sp. strain BP-23
0.2% birchwood xylan (10) showed a single activity band. The
Total
pI of xylanase A, as determined with isoelectric focusing gels Purification step
Vol
activity
Protein Sp act Yield
(Serva, Heidelberg, Germany), was 9.3 (Table 2). (ml) (mg) (U/mg) (%)
(U)
4468
VOL. 61, 1995 NOTES 4469