Cloning, DNA Sequence, and Expression of Aeromonas caviae WS7b Chitinase Gene

Abstract

A chitinase-producing bacterium, designated WS7b, was isolated from a soil sample obtained from a black-pepper plantation on Bangka Island, Indonesia. Fatty-acid methyl-ester analysis indicated that the isolate was Aeron~onas cnviae. A chitinase gene from WS7b was cloned in a pUC19-based plasmid vector, but without its natural promoter. The complete nucleotide sequence of the gene was determined, and the structural gene consisted of a 2748-bp region encoding 864 amino acids. DNA sequence analysis indicated that the gene had been cloned without its promoter, and this was confirmed by chitinase-plate assay of the truncated version of the gene in Escherichici coli. The chitinase gene product shgwed amino-acid sequencc similarity to chiA from A. caviae. Chitinase enzyme activity was determined spectrophotonietrically, using colloidal chitin azure as substrate for extracellular and intracellular fractions. The ability of the ,:.;:;.,:r cloned in E. coli to hydrolyze chitin was less than that of the enzyme in its indigenous host. Index Entries: Aero17zolzns caviae; chitinase gene; DNA sequence; chitinolytic activity.