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Biotechnology and Applied Biochemistry (2000) 32, (197–203) (Printed in Great Britain)

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An Aspergillus chitosanase with potential for large-scale preparation of chitosan oligosaccharides

Chih Yu Cheng and Yaw-Kuen Li¹

Department of Applied Chemistry, National Chiao Tung University, Hsin-Chu, Taiwan 30050

Abbreviations used: CDA, chitosanase-detection agar; DA, degree of deacetylation; DP, degree of polymerization.

¹ To whom correspondence should be addressed (e-mail ykl@cc.nctu.edu.tw)

A chitosan-degrading fungus, designated Aspergillus sp. Y2K, was isolated from soil. The micro-organism was used for producing chitosanase (EC 3.2.1.132) in a minimal medium containing chitosan as the sole carbon source. The induced chitosanase was purified to homogeneity from the culture filtrate by concentration and cationic SP-Sepharose chromatography. The purified enzyme is a monomer with an estimated molecular mass of 25 kDa by SDS/PAGE and of 22 kDa by gel-filtration chromatography. pI, optimum pH and optimum temperature values were 8.4, 6.5 and 65–70 °C, respectively. The chitosanase is stable in the pH range from 4 to 7.5 at 55 °C. Higher deacetylated chitosan is a better substrate. Chitin, xylan, 6-O-sulphated chitosan and O-carboxymethyl chitin were indigestible by the purified enzyme. By endo-splitting activity, the chitosanase hydrolysed chitosan to form chitosan oligomers with chitotriose, chitotetraose and chitopentaose as the major products. The enzyme hydrolyses chitohexaose to form chitotriose, while the chitopentaose and shorter oligomers remain intact. The N-terminal amino acid sequence of the enzyme was determined as YNLPNNLKQIYDDHK, which provides useful information for further gene cloning of this enzyme. A 275 g-scale hydrolysis of chitosan was performed. The product distribution was virtually identical to that of the small-scale reaction. Owing to the simple purification process and high stability of the enzyme, it is potentially valuable for industrial applications.

Received 21 August 2000; accepted 25 September 2000

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