1988 Fiscal Year Final Research Report Summary
Reaction mechanisms of chitinase and chitosanase produced by bacteria, and application of the enzymes to protoplast fusion of fungi
| Project/Area Number |
62560094
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
発酵・醸造
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| Research Institution | Department of Agricultural Chemistry, Faculty of Horticulture, Chiba University |
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Principal Investigator |
FUJII Takaaki Faculty of Horticulture, Chiba University, Professor, 園芸学部, 教授 (50125952)
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| Co-Investigator(Kenkyū-buntansha) |
ANDO Akikazu Faculty of Horticulture, Chiba University, Assistant professor, 園芸学部, 助教授 (80125898)
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| Project Period (FY) |
1987 – 1988
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| Keywords | Chitinase / Chitosanase / Protoplasts of fungi / エンド型キトサナーゼ |
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| Research Abstract |
A chitinase produced in the culture broth by Aeromonas hydrophila subsp. anaerogenes A52 was purified to homogeneity by affinity abso-rption with chitin and ion-exchange chromatography with CM-Sephadex C-50. The isoelectric point of the enzyme was determined to be 4.60. The enzyme showed a molecular weight of about 105,000.
A chitosanase was purified from the culture broth of Bacillus circulans MH-Kl to homogeneity by CM-cellulose and gel permeation chromatography. The enzyme has a molecular weight of about 30,000,its Km is 0.63 mg chitosan/ml and its pi is 9.2. The enzyme showed an endo-splitting type of activity, and the end product of chitosan deg-radation contained a mixture of the dimer and trimer of glucosamine. The smallest of the substrate was a tetramer of glucosamine. The N-terminal amino acid sequence of the enzyme was determined to be Ala-Ser-Pro-Asp-Asp-Asn-Phe-Ser-Pro-Glu-Thr-Leu-Gln-Phe-Leu-Arg-Asn-Asn-Cys-Gly-Leu-Asp-. A methionine residue was not found in the enzyme.
Protoplasts of aspergillus usami and Aspergillus awamori were efficiently produced by treatment of cells with the above chitinase. The chitosanase was available for preparetion of protoplasts from Mucor javanicus and Rhizopus hangchaoo.
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