Purification and Characterization of Elicitor-induced Chitinase from Grape Berries

An induced chitinase was purified from Koshu grapes elicitated with glycolchitin. The protein fraction obtained by salting out with ammonium sulfate (80% saturation) from the extract solution of the grapes was separated by successive chromatographies on Chitopearl BL-3 and Q-Sepharose HP (HiTrap Q) columns. The induced chitinase was homogeneous on polyacrylamide gel electrophoresis. The optimum pH of the induced chitinase was 4.0, and the enzyme was stable under acidic conditions between pH 5.0-6.5. The optimum temperature was 40°C, and the enzyme was stable below 40°C. The N-terminal amino acid sequence of the induced chitinase was NH2-GTITVYXGQNGN, which is highly homologous with that of the class III chitinase of Vitis vinifera. The induced chitinase inhibited the growth of Botrytis cinerea, which causes grey mold disease, strongly suggesting that it plays an important role in the defense mechanism against phytopa-thogens in mature grape.