| Project/Area Number |
06660104
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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 |
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 Univ.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) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1995: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1994: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | beta-amylase / protein engineering / x-ray crystal analysis |
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| Research Abstract |
For the study of protein engineering of enzymes, X-ray crystallographic analysis and the over-production of the mutated enzymes are inevitable techniques. The author has determined the SH-modified soybean beta-amylases as a model of the mutated enzyme and also developed the over-production system of soybean beta-amylase in Escherichia coli.
1. X-ray crystallography of the SH-modified soybean beta-amylase
Two sulfhydryl groups (Cys95 and Cys343) in soybean beta-amylase exist near the active site of the enzyme. The role of SH (S-S) groups of b-amylase from soybean and Bacillus cereus have been investigated by chemical modification. The three dimensional structures of the complexes of Cys95 and Cys343 blocked soybean b-amylase with maltose or glucose have been determined at around 2.0 A resolution. Two maltose molecules could bind to subsite 1-4 as is the case of the native enzyme complex except for the position of the flexible loop (residues from 96 to 103). In the modified enzyme, the loop position could not be determined owing to high temperature factors, suggesting that the modified side chain of Cys95 inhibit the loop movement from open to closed conformation. The X-ray crystal analysis of Cys343-blocked enzyme/maltose complex showed the normal maltose binding except for the main chain positions of Thr342 and Cys343. It seems that the altered Thr342 residue affect the catalysis of the enzyme by the break of hydrogen bond with catalytic water.
2. Over-production system of soybean beta-amylase
Soybean beta-amylase cDNA has been efficiently expressed in Escherichia coli.with pkk233-2 vector. The purified enzyme from the extract of Escherichia coli.showed the same enzymatic properties as the enzyme from soybean seeds except for the N-terminal blocked residue. The qurified enzyme has been crystallized in the same way as the seed enzyme. The X-ray crystallographic study of the expressed enzyme is now in progress.
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