Cloning and characterization of the constitutively expressed chitinase C gene from a marine bacterium, Salinivibrio costicola strain 5SM-1.

The chitinase C gene (chiC) encoding chitinase C (ChiC) from Salinivibrio costicola 5SM-1 was cloned and the nucleotide sequence was determined. S. costicola ChiC was expressed constitutively and repressed by glucose. A single operon composed of two complete open reading frames organized in the order of chiB, chiC and one partial open reading frame of chiA was found in the same transcriptional direction. chiC was composed of 2610 bp encoding for 870 amino acids with a calculated molecular mass of 94 kDa including a signal peptide. Analysis of the deduced amino acid sequence alignment revealed a domain structure consisting of an N-terminal catalytic domain, followed by a putative cadherin-like domain and two type 3 chitin-binding domains located at the C terminus. Mutation of three highly conserved amino acid residues, two aspartic acids (Asp-313 and Asp-315) and one glutamic acid (Glu-317) resulted in a complete loss of chitinase activity against colloidal chitin substrate. This suggests that these amino acid residues which reside in the putative catalytic domain play an important role in catalysis. chiB classified as a chitin-binding protein with C-terminal type 3 chitin-binding domain was composed of 390 amino acids with the molecular mass of 43 kDa and does not have any detectable chitinase activity. Chitinase C was identified as an exo-type chitinase releasing chitobiose as a major product from colloidal chitin hydrolysis.