Cloning and expression of a mannanase gene from Erwinia carotovora CXJZ95-198

Abstract

A gene encoding a mannanase (ManA) was cloned from the genomic library of Erwinia carotovora CXJZ95-198 and expressed in Escherichia coli cells. A 1783 bp DNA fragment containing a manA gene was sequenced. An open reading frame (ORF) of 1137 bp encoded a protein of 378 amino acids. The expressed enzyme had a molecular mass of approximately 42 KD determined by SDS-PAGE. The optimal pH and temperature for the expressed enzyme was 7.5 and 55 °C, respectively. The nucleotide sequence of manA had remarkably low homology with other sequences reported. No typical promoter was found but a palindrome sequence existed downstream of the stop codon. The deduced amino acid sequence from mature ManA showed homology of about 53% with those from Bacillus sp., but much lower homology with those from other strains. The ManA was presumably classified as family 26 of glycosidases. It was also clarified that the 1.3 kb fragment up the locus nt 4449729 of Erwinia carotovora genomic DNA was a mannanase gene.