Purification and characterization of extracellular chitin deacetylase from Colletotrichum lindemuthianum

Chitin deacetylase, active in the presence of acetate (96% of the enzymatic activity was retained in the presence of 100 mM sodium acetate), was purified to electrophoretic homogeneity from a culture filtrate of Colletotrichum lindemuthianum (944-fold with a recovery of 4.05%). The enzyme was induced in the medium after the eighth day of incubation simultaneously with blackening of the medium. The molecular mass of the enzyme was 31.5 kDa and 33 kDa as judged by SDS-PAGE and gel filtration, 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, and chitin oligomers the degrees of polymerization of which were more than four, but was less active with chitin trimer adn dimer, and inactive 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 were 414 mu M and 83.2 s(-1), respectively. The reaction rates of the enzyme toward glycol chitin and chitin oligomers seemed to follow the Michaelis-Menten kinetics.