Cloning of β-1,4endo-glucanase gene and expression in Escherichia coli

Bacillus sp.NW-2004a,which can hydrolyze carboxymethyl cellulose (CMC), was obtained from soil sample through selective medium LBCMC,and its genomic library was constructed using shotgun cloning strategy .Two positive clones were obtained from the genomic libray, one of which was chosen to sequence the inserted DNA fragment. An open-reading frame of 2502bp(ORF), coding for 834 amino acids , was found . BLAST analysis of the sequence in NCBI database showed that it has 86% similarity with the glucanase gene of Bacillus sp. KSM-S237, and the edcoded polypeptide shows 87% similarity with the β-1,4-endo-glucanase of Bacillus sp.KSM-64 at amino acid level. It has been accepted by GeneBank with accession number AY663839 .In addition , treated with T4 polymerase, the β-1,4-endo glucanase gene was cloned into the plasmid pHBM625, which was derived from vector pGEX, through restriction endonuclease CpoI and NotI, and expressed in Escherichia coli XL10-Gold.