| Project/Area Number |
10660084
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
MIKAMI Bunzo Kyoto University, Research Institute for Food Science, Associate Professor, 食糧科学研究所, 助教授 (40135611)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 1999: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1998: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Keywords | β-amylase / protein engineering / x-ray crystal analysis / starch binding / starch-binding domain |
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| Research Abstract |
In order to design new functional enzyme, point mutations and domain substitution are important techniques of protein engineering. We have determined the structures of β-amylase from soybean, wheat and Bacillus cereus by X-ray crystal analysis. The bacterial β-amylase has C-terminal starch binding domain and is able to digest low starch granules. Plant and bacterial enzymes have different pH optima which is ascribed to their environment of the catalytic residues. The possibility to design new function of β-amylase was explored by protein engineering and X-ray crystal analysis.
1. X-ray crystallography and design of strach binding domain of Bacillus cereus β-amylase.
The C-terminal starch binding domain of Bacillus cereus β-amylase was expressed in Escherichia coli. and crystallized. The structure determined at 1.95 A resolution by X-ray crystallography revealed that one of the starch binding site out of two does not functional owing to the conformation change of a loop structure. In order to increase the starch binding ability mutations of the loop are now in progress.
2. X-ray crystallography of the mutant soybean β-amylases which have increased pH optimum.
The comparison of active site between plant and bacterial β-amylase suggested that hydrogen bond network exist around one of the catalytic residue (Glu380 in soubean β-amylase) only in the plant β-amylase. The three mutants of soybean enzyme (Met51T, Asn340T, Glu 178Y) expressed in E. coli. had increased pHoptimum about 0.5-1.0 pH unit toward the bacterial enzyme. X-ray crystallographic analysis at 2.0 A resolution demonstrated that all have broken hydrogen bonds with Glu380. We are now trying to make a mutant of B. cereus enxyme which have reduced pH optimum and are useful for industrial application.
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