1997 Fiscal Year Final Research Report Summary
Protein engineering of beta-amylase
| Project/Area Number |
08660109
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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) |
1996 – 1997
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| Keywords | beta-amylase / protein engineering / X-ray crystal analysis / thermostability |
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| Research Abstract |
In order to elucidate the relationship between structure and function of beta-amylases and to perform the structure based protein engineering of beta-amylase, the three-dimensional structures of the enzymes from soybean, barley and Bacillus cereus has been determined by X-ray crystallography. beta-Amylases from soybean and B.cereus has been produced in E.coli for mutation experiments.
1. X-ray crystal analysis of Bacillus cereus beta-amylase. Bacillus cereus beta-amylase expressed in E.coli.has been purified and crystallized. The structure has been determined by X-ray crystallography at 2.1* resolution by multiple isomorphous replacement method. The enzyme has a core and a C-terminal starch binding domain instead of the C-terminal loop found in plant beta-amylases. The structure of the enzyme complex with maltose clealy showed that maltose binds to three different sites, the active site in core, C-terminal starch binding domain and a maltose binding site in core. These results explain t
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he ability of raw starch digestion found in only bacterial beta-amylase.
2. X-ray crystal analysis of a mutant barley beta-amylase. The structure of a seven-fold mutant barley beta-amylase with increased thermostability has been determined by X-ray crystallography at 2.5* resolution. The comparison of the structure with that of soybean enzyme elucidated the individual mechanisms of the increased thermostabilty at four mutated amino acid residues.
3. Mutation and X-ray crystal analysis of soybean beta-amylase. Mutants of soybean beta-amylase of which two catalytic residues(E 178 and E 380)were mutated to glutamine were produced and crystallized. The complex of E 380 Q with maltose clearly showed that maltose bind to subsites 1-2 and 4-5 in contrast to subsites 1-2 and 3-4 in the case of the wild type enzyme. This strongly suggested the activation mechanism of a catalytic water by E 380 in the catalytic step. The complex of the wild type enzyme with beta-cyclodextrin showed the importance of subsite 4 for the multiple attack mechanism of the enzyme. Less
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Research Products
(5 results)
