A Thermostable Hydantoinase of Bacillus stearothermophilus NS1122A : Cloning, Sequencing, and High Expression of the Enzyme Gene, and Some Properties of the Expressed Enzyme
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概要
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A DNA fragment containing the gene for a thermostable hydantoinase was cloned from a thermophile, Bacillus stearothermophilus NS1122A in Escherichia coli. Nucleotide sequencing showed that the DNA fragment contains one open reading frame, which is predicted to encode a peptide of 471 amino acids, with a calculated molecular weight of 51,724. When the hydantoinase gene was under the control of both the lpp promoter and the lac promoter-operator, and its expression was induced by isopropyl-1-thio-β-D-galactopyranoside, it was overexpressed in E. coli leading to the formation of an insoluble aggregate. The enzyme was purified to homogeneity from the insoluble aggregate. The molecular mass of the purified active enzyme was approximately 200 kDa by gel filtration. Although the monomer had no activity, the activity was restored by incubation with Mn^<2+> or Co^<2+> at pH 8. 1. These findings suggested that the hydantoinase is a metalloenzyme and the oligomeric structure is required for activity. The oligomeric structure is suggested to contribute to thermostability.
- 社団法人日本農芸化学会の論文
- 1994-09-23
著者
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Nakamura H
Sci. Univ. Tokyo Tokyo Jpn
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NAKAMURA Hidemitsu
Department of Biotechnology, The University of Tokyo
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Ishikawa Tomohiro
Department Of Agricultural Chemistry School Of Agriculture Nagoya University
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Watanabe K
Faculty Of Pharmaceutical Science School Of Medicine Fukuoka University
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Watanabe Katsunori
Department Of Bioresources Chemistry Faculty Of Horticulture Chiba University
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ISHIKAWA Takahiro
Odawara Research Center, Nippon Soda Co., Ltd.
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WATABE Ken
Odawara Research Center, Nippon Soda Co., Ltd.
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MUKOHARA Yukuo
Odawara Research Center, Nippon Soda Co., Ltd.
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NAKAMURA Hiroaki
Odawara Research Center, Nippon Soda Co., Ltd.
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Nakamura H
Department Of Biotechnology The University Of Tokyo
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Mukohara Yukuo
Odawara Research Center Nippon Soda Co. Ltd.
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Watanabe K
Research Institute For Food Science Kyoto University:(present Address)department Of Agricultural Che
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Watanabe K
Department Of Applied Biological Sciences Faculty Of Agriculture Saga University
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Watanabe K
Marine Biotechnol. Inst. Iwate Jpn
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