PURIFICATION AND CHARACTERIZATION OF CHITIN DEACETYLASE FROM ABSIDIA COERULEA

Chitin deacetylase, which releases the acetyl groups of glycol chitin was purified from a fungus, Absidia coerulea, and characterized, The enzyme was purified 516-fold to homogeneity by means of 65-80% ammonium sulfate precipitation followed by chromatography on Butyl Toyopearl-650M, Gigapite (hydroxyapatite), and DEAE Toyopearl-650M. It had an apparent molecular weight of 75 kDa both on sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration chromatography, indicating that the enzyme exists as a monomer, The amino-terminal sequence was determined to be Gly-Glu-Tyr-Trp-Gln-Ser-Phe-, The enzyme is active on chitooligosaccharides with more than two N-acetylglucosamine residues (chitobiose) and is able to convert the nascent chitin synthesized by chitin synthase to chitosan in vitro, When O-hydroxyethylated chitin (glycol chitin) was used as a substrate, the optimum pH for enzyme activity was 5.0 and the optimum temperature was 50 degrees C, The enzyme was heat-stable and strongly inhibited by Fe3+, Furthermore, chitin deacetylase was demonstrated to be localized near the inner face of the cell wall (periplasmic space) in the mycelia by using immunoelectron microscopy.