CLONING, CHARACTERIZATION, AND EXPRESSION OF XYLANASE GENE FROM BACILLUS LYTICUS IN ESCHERICHIA COLI AND BACILLUS SUBTILIS

Abstract

A genomic library of Bacillus lyticus was constructed in λ GEM 11 vector and screened for the xylanase gene using Congo red plate assay. A 16-kb fragment containing the xylanase gene was obtained which was further subcloned using Mbo I partial digestion in an E.coli pUC 19 vector. A 1.3-kb sub-fragment was obtained which coded for a xylanase gene of M_r23,650 Da. This fragment was sequenced and the homology was checked with known xylanases. The maximum homology was 97%, which was obtained with an endo xylanase gene from Bacillus species at the DNA level, while the translated sequence showed only one amino acid change from alanine to serine at position number 102. Expression was checked in E.coli, using the native promoter, and an extracellular activity of 5.25 U/mL was obtained. Cloning of the gene was done in Bacillus subtilis using a shuttle vector pHB 201, which resulted in increasing the basal level xylanase activity from 14.02 to 22.01 U/mL.

ACKNOWLEDGMENTS

We acknowledge Dr. Vijay Chaudhari for providing automatic sequencing of the xylanase gene. We also thank Franklin J. Sah for his valuable help and suggestions.

This work was supported by a grant from the Department of Biotechnology, Govt. of India.