Cloning and Sequencing of Chitinase Gene from Bacillus thuringiensis subsp israelensis

Chitin,a linear homopolymer of N acetyl D glucosamine,is a common constituent of fungal cell walls,exoskeletons of insects,and shells of invertebrates.Thus,chitinase,a chitin hydrolytic enzyme,has become of interest for potential use as biopesticides for controlling insect pests.Using a pair of specific primers,chitinase gene ( ichi ) was amplified by touchdown PCR from Bacillus thuringiensis subsp israelensis chromosomal DNA,and then subcloned into pGEM T easy vector. ichi sequence (GenBank Accession Number:AF526379)with a length of 2570 bp included an open reading frame (ORF) of 2067 bp,which contained 688 amino acids with Mr=75 79 kDa and pI=5 90.The amino acid sequence of ichi gene shows 97 24%,97 18%,97 63% and 63 07% identity to that of Bacillus cereus strain 28 9 chitinase CW, Bacillus cereus CH chitinase B, Bt subsp Mexican chitinase,and Bt subsp pakistani chitinase A71,respectively.It was demonstrated that Ichi contains a signal peptide (46 amino acid residues) and three functional domains:an N terminal catalytic domain (105 amino acid residues),a fibronectin type Ⅲ like domain (74 amino acid residues),and a C terminal chitin binding domain (40 amino acid residues).