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Purification and Properties of Xylanase from Bacillus pumilus
IMR 1211Abstract Xylooligosaccharides were foundto have a
befeficial effect on human intestinal flora. A potent xylanase
(endo-xylanase; EC 3.2.1.8.) producing bacteria,Bacillus pumilus
IMR 1211 was isolated from the soil of Turfan Flaming mountain
in China. This strain produced high activity of xylanase. The
purpose of this study was to explore the purification,
characterization and possible application of xylanase
from Bacillus pumilus 1211.Crude xylanase was prepared from the
fermentation broth of the strain throughsuccessive steps of
centrifugation, ultrafiltration and lyophilization. The crude
xylanase was furhter puripied by sequential steps of
chromatofocusing,ammo-nium sulfate fractionation and Superdex 75
HR gel filtration. By these steps ,the purity of the enzyme
increased by 12.2 fold and the recovery of the enzymeactivity
was 24.2 %. The purified enzyme was almose homogeneous as
analyzed bySDS-PAGE and activity straining.The molecular mass
determined from gelfiltration through Superdex 75 HR was 15 kDa.
SDS-PAGE of the enzyme revealed only one distinct major protein
band with molecularmass of 26.7 kDa. Thus the purified enzyme
was a monomeric protein. The pI was higher than 9.3 as
determined by isoelectric focusing electrophoresis. It was a
basic protein with high pI. The xylanase had an optimasl pH of
6.3, optimal temperture of 50 C, Km value of 0.63 % and
activation energy og 3.88 Kcal/mole for hydrolysis of birchwood
xylan. Mercuric and cupric ions (2.5 mM), N-bromosuccinimide
(0.5 mM) and p-hydroxymercuribenzoic acid (0.5 mM) significantly
inhibited the activity of the enzyme, indicating cysteine and
tryptophan residues were located at or near the active site of
the enzyme. The majoe products for hydrolysis of birchwood xylan
were xylobiose and xylotriose as analyzed by HPLC. The [purified
enzyme belongs to endo-xylanase and could be used for
preparation of xyloologo-saccharides.
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