Purification and Properties of Two Deacetylases Produced byVibrio alginolyticusH-8

Abstract

The Chitinase-producing bacterium Vibrio alginolyticus H-8 isolated from mud of Hamana Lake also produced two deacetylases for (GlcNAc)2 extracellularly. Deacetylases DA1 and DA2 were purified from crude enzyme by column chromatography on Q-Sepharose FF, Phenyl Sepharose HP, Gigapite, and Superdex 200 HR. The final preparation was homogeneous in SDS-PAGE. The molecular weights were 48,000 and 46,000 for deacetylases DA1 and DA2, respectively. The pIs, optimum pHs, and optimum temperatures for deacetylases DA1 and DA2 were as follows; DA1, pI 3.3, optimum pH 8.5–9.0, optimum temperature 45°C, DA2, pI 3.5, optimum pH 8.0–8.5, optimum temperature 40°C. Both deacetylases were stable at pHs between 7.0 and 11.0 and at temperatures below 40°C. The activities of both enzymes were inhibited by Ag+ and Hg2+. 1H-NMR of the reaction product by deacetylase DA1 for (GlcNAc)2 showed that the purified deacetylase selectively hydrolyzed the 2-acetamide group at the reducing end of (GlcNAc)2.