Identification of a novel endochitinase from a marine bacterium Vibrio proteolyticus strain No. 442

Abstract

Chitin binding proteins prepared from Vibrio proteolyticus were purified and the N-terminal amino-acid sequence of a protein from a 110-kDa band on SDS-PAGE was found to be 85–90% identical to the 22nd–41st residues of the N-termini of chitinase A precursor proteins from other vibrios. We cloned the corresponding gene, which encodes a putative protein of 850 amino acids containing a 26-residue signal sequence. The chitinase precursor from V. proteolyticus was 78–80% identical to those from Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio carchariae. However, the proteolytic cleavage site for C-terminal processing between R 597 and K 598 in the chitinase precursor of other vibrios was not observed in the amino acid sequence of V. proteolyticus, which instead had the sequence R 600 and A 601. Subsequently, full-length and truncated chitinases were generated in Escherichia coli. The specific activity of full-length chitinase expressed in E. coli was 17- and 20-folds higher for colloidal and α-chitins (insoluble substrate), respectively, than that of the C-terminal truncated enzyme. However, both recombinants showed similar hydrolysis patterns of hexa- N-acetyl-chitohexaose (soluble substrate), producing di- N-acetyl-chitobiose as major product on TLC analysis. We showed that the C-terminus of the V. proteolyticus chitinase A was important for expression of high specific activity against insoluble chitins.