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 Toyoperal-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.
Gao
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XD
Division of Agriculture and Agricultural Life Sciences, University of Tokyo.
Katsumoto
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T
Onodera
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K
D000581:Amidohydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004187:Disaccharides; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D005658:Fungi / Q000201:enzymology*; D005936:Glucans / Q000378:metabolism; D007700:Kinetics; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity
D004187:Disaccharides; D005936:Glucans; D009844:Oligosaccharides; D010446:Peptide Fragments; C032438:chitobiose; D017886:Durapatite; D000581:Amidohydrolases; C019866:chitin deacetylase