Purification and characterization of a chitinase from Cytophaga sp. HJ isolated from sea sand

Abstract

An extracellular chitinase-producing bacterial strata induced by colloidal chitin was isolated from sea sand and was identified to be a member of the genus Cytophaga. The chitinase was purified successively by 30-60% ammonium sulfate fractionation, and DEAE-BIO gel A column, Octyl-Sepharose CL-4B column, and DEAE-Bio gel A column chromatographies. The enzyme had a molecular mass of 59.75 kDa, and the amino terminal amino acid sequence was ATPNAPVISW MPTDXXLQNXS. The enzyme acted better on colloidal chitin as a substrate than on chitosan. For colloidal chitin and chitosan (Degree of Acetylation, 15-25%), K(cat) values were 0.60 U/mg and 0.08 U/mg, respectively. HPLC analysis of the enzymatic reaction products showed that the chitinase produced mostly N-acetyl-D-glucosamine and di-N- acetylchitobiose. The optimum temperature and pH for the enzyme were 50°C and 4.0, respectively. N-Bromosuccinimide and Hg2+ inhibited the chitinase activity as much as 90%, and Sb3+, diethylpyrocarbonate, and Ag+ inhibited it by 5070%.