Directed Evolution of Bacterial Alanine Racemases with Higher Expression Level(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
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概要
- 論文の詳細を見る
Bacterial alanine racemase (EC 5.1.1.1) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the interconversion of L-alanine and D-alanine. It can be classified into two groups : biosynthetic enzymes with low catalytic activity and catabolic enzymes with high catalytic activity. It can react with serine to a limited extent. Two biosynthetic alanine racemase genes in Escherichia coli and Salmonella typhimurium were DNA shuffled, and a very diverse chimeric gene library was constructed. An E. coli serine auxotroph was transformed with the shuffled genes, and the recombinant clones were screened on selective media supplemented with 0.5-5mM D-serine as an L-serine supplier. Selected clones were expected to contain racemases exhibiting higher catalytic activities toward alanine as well as serine. Three independent clones that grew on selective media were isolated. The specific activities of crude extracts prepared from cells expressing the chimeric racemases were increased up to approximately three times more than those expressing the parental enzymes. The best chimera Ser15 racemase was expressed at a level approximately twofold higher than the parental alanine racemases. This high protein expression was demonstrated to be post-transcriptionally achieved.
- 社団法人日本生物工学会の論文
- 2005-09-25
著者
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MISONO Haruo
Department of Bioresources Science, Kochi University
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OHNISHI Kouhei
Research Institute of Molecular Genetics, Kochi University
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Meguro H
Tohoku Fukushi University
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Misono H
Kochi Univ. Kochi Jpn
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Misono Haruo
Department Of Bioresources Science Faculty Of Agriculture Kochi University
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Misono Haruo
Department Of Bioresource Science Kochi University
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Misono Haruo
Laboratory Of Applied Microbiology Department Of Agricultural Chemistry Kochi University
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Ohnishi Kouhei
Research Institute Of Molecular Genetics Kochi University
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Ohnishi Kouhei
Res. Inst. Of Molecular Genetics Kochi Univ. Kochi 783-8502 Jpn
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Ju Jiansong
Department of Applied Bioresource Science, The United Graduate School of Agricultural Sciences, Ehim
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Ju Jiansong
Life Coll. Of Sci. And Engineering Shaanxi Univ. Of Sci. And Technol. Xi'an 710021 Chn
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Ju Jiansong
State Key Laboratory Of Microbial Resources Institute Of Microbiology Chinese Academy Of Sciences:li
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MISONO Haruo
Department of Agricultural and Biological Chemistry, Kochi University
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