Evolutionary molecular engineering by the combination of random point mutagenesis and random elongation mutagenesis
| Project/Area Number |
10450310
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
生物・生体工学
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| Research Institution | Osaka University |
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Principal Investigator |
URABE Itaru Department of Biotechnology Graduate school of Engineering, Osaka University, Professor, 大学院・工学研究科, 教授 (60029246)
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| Co-Investigator(Kenkyū-buntansha) |
SHIMA Yasufumi Department of Biotechnology Graduate school of Engineering, Osaka University, Assistant Professor, 大学院・工学研究科, 助手 (50187423)
YANO Tetsuya Department of Biotechnology Graduate school of Engineering, Osaka University, Associate Professor, 大学院・工学研究科, 助教授 (00222399)
根来 誠司 姫路工業大学, 工学部, 教授 (90156159)
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| Project Period (FY) |
1998 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥12,200,000 (Direct Cost: ¥12,200,000)
Fiscal Year 2001: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2000: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1999: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1998: ¥4,100,000 (Direct Cost: ¥4,100,000)
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| Keywords | peroxidase / catalase / reaction specificity / random point mutagenesis / random elongation mutagenesis / fitness landscape / ランダム変異 / 非線形性 / 配列空間 / 局所的地形 |
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| Research Abstract |
We describe a new method of random mutagenesis that employs the addition of peptide tails with random sequences to the C-terminal of enzyme molecules. A mutant population of catalase I from Bacillus stearothermophllus prepared by this method has a diversity in thermostability and enzyme activity equal to that obtained after random point mutagenesis. When a triple mutant of catalase I (I108T/D130N/I222T) - the thermostability of which is much lower than that of the wild type - was subjected to random elongation mutagenesis, we generated a mutant population containing only mutants with higher thermostability than the triple mutant. Some had an even higher stability than the wild-type enzyme, whose thermostability is considered to be optimized. These results indicate that peptide addition expands the protein sequence space resulting in a new fitness landscape. The enzyme can then move along the routes of the new landscape until it reaches a new optimum. The combination of random elongation mutagenesis with random point mutagenesis should be a useful approach to the in vitro evolution of proteins with new properties.
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Report
(5 results)
Research Products
(6 results)
