Purification and Characterization of a Chitinase from Cytophaga sp. HJ Isolated from Sea Sand

An extracellular chitinase-producing bacterial strain 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 0.60U/mg and 0.08U/mg, respectively. HPLC analysis of the enzymatic reaction products showed that the chitinase produced mostly N-acetyl-D-glucosarnine and di-N-acetylchitobiose. The optimum temperature and pH for the enzyme were $50^{\circ}C$ and 4.0, respectively. N-Bromosuccinimide and $Hg^{2+}$ inhibited the chitinase activity as much as 90%, and $Sb^{3+}$, diethylpyrocarbonate, and $Ag^{+}$ inhibited it by 50-70%.