Domain engineering of β-amylase
| Project/Area Number |
12660077
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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, Graduate School of Agriculture Associate Professor, 農学研究科, 助教授 (40135611)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2001: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2000: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | β-amylase / protein engineering / X-ray crystallography / optimum pH / starch binding domain / 至適pHの変換 |
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| Research Abstract |
In order to design and produce a new functional β-amylase for industrial use, we have analyzed the domain structures of β-amylases from soybean, barley and Bacillus cereus by X-ray crystallography. We have designed the mutants of Bacillus cereus β-amylase that have shifted optimum pH to acidic side and have improved ability of raw-starch digestion, and examined their properties and structures by X-ray crystallographic analysis.
1. We could be succeeded to design mutants of soybean β-amylase that have shifted optimum pH to alkaline pH (bacterial type) in previous studies. In the present study we tried to convert the optimum pH of Bacillus cereus β-amylase to acidic side (plant type) in order to clarify the factors which control the optimum pH in the active site of β-amylase. Two mutants of Bacillus cereus β-amylase, Y164E, Y164Q and T328N, showed the decreased optimum pH by more than 1 pH unit in spite of 1/2, 1/5 and 1/10 reduced specific activity of the wild-type enzyme, respectively.
… More
The X-ray crystallographic analysis at around 2 Å showed that the side chain of E367, catalytic residue, had altered conformation in the complex of T328N/maltose, suggesting the strongly reduced specific activity. No direct hydrogen bonds were found from the mutated side chains to E367, which were expected from the analysis of the mutants of soybean β-amylase. These results show that not only hydrogen bonds but also nonpolar environment is important to control the pKa of E367.
2. Comparison of the C-terminal starch-binding domain (SBD) of Bacillus cereus β-amylase with that of glucoamylase indicated that one of the starch-binding sites of Bacillus cereus β-amylase is not functional. We have replaced 6 residues of this binding site of SBD in order to increase the affinity toward raw-starch. We failed to express the resultant mutant in E. coli owing to the incomplete protein folding. We are now trying to get the mutants by enzyme evolutional system.
3. In order design the enzyme with high specific activity, it is inevitable to get the fine structure of the protein including the positions of hydrogen atoms. We have started the preliminary study of X-ray crystallography β-amylase at atomic resolution. We could refine the structure of soybean β-amylase/maltose complex at 1.25 Å by improving the conditions of crystallization. Less
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Report
(3 results)
Research Products
(13 results)
