Purification and hydrolytic action of a chitosanase from Nocardia orientalis.
Chitosanase from the culture filtrate of Nocardia orientalis was purified to apparent homogeneity by precipitation with ammonium sulfate followed by CM-Sephadex chromatography, biospecific affinity chromatography on a Sepharose CL-4B with immobilized chitotriose and by gel filtration on Sephadex G-75. The enzyme specifically acted on chitooligosaccharides and chitosan to yield chitobiose and chitotriose as final products. The mode of action of the chitosanase on chitooligosaccharides and their corresponding alcohols suggests that the enzyme requires substrates with four or more glucosamine residues for the expression of activity and its shows maximum activity on chitohexaose and chitoheptaose. In the hydrolysis of chitosans of varying N-acetyl content, the enzyme cleaved about 30% acetylated chitosan with maximum activity and the enzyme activity decreased with increasing the degree of deacetylation of chitosans tested. The analysis of products formed from 33% acetylated chitosan shows the chitosanase is capable of cleaving between glucosamine and glucosamine or N-acetylglucosamine, but not cleaving between N-acetylglucosamine and glucosamine. On the basis of the results, the whole pathway of enymatic degradation of partially acetylated chitosan by a combination of chitosanase, exo-beta-D-glucosaminidase and beta-N-acetylhexosaminidase is proposed.
Sakai
K
K
Research and Development Laboratories, Yaizu Suisan Kagaku Industry Co., Ltd., Shizuoka Japan.
Katsumi
R
R
Isobe
A
A
Nanjo
F
F
D000117:Acetylglucosamine / Q000378:metabolism; D002853:Chromatography, Liquid; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D005944:Glucosamine / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D006868:Hydrolysis; D009615:Nocardia / Q000201:enzymology*; D013379:Substrate Specificity
D006026:Glycoside Hydrolases; C019868:chitosanase; D005944:Glucosamine; D000117:Acetylglucosamine