1988 Fiscal Year Final Research Report Summary
Improvement and reconstitution of procymosin by recombinant DNA techniques
| Project/Area Number |
61470130
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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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 | The University of Tokyo |
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Principal Investigator |
BEPPU Teruhiko. The University of Tokyo, Faculty of Agriculture Professor, 農学部, 教授 (80011873)
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| Co-Investigator(Kenkyū-buntansha) |
HORINOUCHI Sueharu. The University of Tokyo, Faculty of Agriculture, Associate Professor, 農学部, 助教授 (80143410)
SHOUN Hirofumi. Tsukuba University, Institute of Applied Biochemistry, Associate Professor, 応用生物化学系, 助教授 (70012036)
UOZUMI Takeshi. The University of Tokyo, Faculty of Agriculture Professor, 農学部, 教授 (40011978)
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| Project Period (FY) |
1986 – 1987
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| Keywords | Prochymosin / Inclusion body / Site-directed mutagenesis / Enzyme activity / 蛋白質工学 |
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| Research Abstract |
We constructed more than 50 kinds of mutated prochymosin by linker insertion and site-directed mutagenesis. Enzymatic properties of the mutated prochymosins were determined after each of the prochymosins had been purified from inclusion bodies in E. coli cells and activated with an acidic treatment. Among these mutated prochymosins,Y^<77> was found to be important for the ratio of clotting activity to protease activity (C/P Calue); I^<77> ,V^<77> or T^<77> showed no enzymatic activity, whereas F^<77> and W^<77> showed weak activities. These results suggest that at the 77th position, the amino acid residues with an aromatic ring are absolutely essential for enzymatic activity. The mutated prochymosin in which K^<221> or F^<40> were changed to other amino acid residues had optimum pHs at around 3.5. In addition, heat-stability of the F^<40> to L^<40> protein significantly decreased. These fundamental data provide useful information for further improvement of chymosin for industrial cheese-making. We are also trying to analyze the reaction rates of each of the mutated chymosins, which may facilitate elusidation of mechanism of reaction.
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