Journal
BIOCATALYSIS AND BIOTRANSFORMATION
Volume 24, Issue 5, Pages 371-379Publisher
TAYLOR & FRANCIS LTD
DOI: 10.1080/10242420600768771
Keywords
Bacillus pumilus HBP8; cloning; deglycosylation; heterologous expression; Pichia pastoris; xylanase
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The xynHB gene, encoding alkaline xylanase was cloned from Bacillus pumilus by a shot-gun method. The gene was cloned into vector pHBM905A, and expressed in Pichia pastoris GS115. Xylanase-secreting transformants were selected on plates containing RBB-xylan. Enzymatic activity in the culture supernatants was up to 644 U mL(-1) and the optimal secretion time was 4 days at 25 degrees C. SDS-PAGE showed two bands, of 32.2 kDa and 29.6 kDa, both larger than the predicted mass of 22.4 kDa based on its amino acid sequence. Zymogram analysis demonstrated that the enzyme in both bands could hydrolyze xylan. Deglycosylation by endoglycosidase H revealed that both were derived from the same protein but contain different extents of glycosylation (30 and 25%). The optimal pH and temperature of the enzyme was pH6-9 and 50 degrees C, respectively.
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