PURIFICATION AND CHARACTERIZATION OF EXTRACELLULAR CHITIN DEACETYLASEFROM COLLETOTRICHUM-LINDEMUTHIANUM

Chitin deacetylase, active in the presence of acetate (96% of the enzy matic activity was retained in the presence of 100 mM sodium acetate), was purified to electrophoretic homogeneity from a culture filtrate o f Colletotrichum lindemuthianum (944-fold with a recovery of 4.05%). T he enzyme was induced in the medium after the eighth day of incubation simultaneously with blackening of the medium. The molecular mass of t he enzyme was 31.5 kDa and 33 kDa as judged by SDS-PAGE and gel filtra tion, respectively, suggesting that the enzyme is a single polypeptide . The optimum temperature was 60 degrees C and the optimum pH was 11.5 -12.0 when glycol chitin was used as substrate. The enzyme was active toward glycol chitin, partially N-deacetylated water soluble chitin, a nd chitin oligomers the degrees of polymerization of which were more t han four, but was less active with chitin trimer adn dimer, and inacti ve with N-acetylglucosamine. The K-m and K-cat for glycol chitin were 2.55 mM and 27.1 s(-1), respectively, and those for chitin pentamer we re 414 mu M and 83.2 s(-1), respectively. The reaction rates of the en zyme toward glycol chitin and chitin oligomers seemed to follow the Mi chaelis-Menten kinetics.