Cloning, Nucleotide Sequence, and Efficient Expression of the Gene Coding for Thermostable Aldehyde Dehydrogenase from Bacillus stearothermophilus, and Characterization of the Enzyme
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概要
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The aldhT gene coding thermostable aldehyde dehydrogenase (ALDH-T) from Bacillus stearothermophilus SIC1 was cloned by colony hydridization using Escherichia coli JM109 and pUC19 as a host-vector system.Nucleotide sequence analysis showed that the ald T gene was composed of 1,464 bp (488 amino acid residues)which can encode a protein with molecular weight of 52,912. The aldhT gene was highly expressed in E. coli carrying the recombinant plasmid, pUALD27R, which contains a native promoter of the aldhT gene. The enzyme accumulated as a soluble from and not an inclustion body in E. coli, and the concentration was about 10% of total cytoplasmic protein. ALDH-T was purified to homogeneity with a specific activity of 36.3 U/mg protein by heat treatment of the cell extract, ammonium sulfate precipitation, gel filtration and finally ion-exchange chromatography. The molecular weight of the native ALDH-T was estimated to be 220 kDa by gel filtration, whereas subunit molecular weight determined by SDS-PAGE was 53kDa, suggesting a tetrameric enzyme structure. ALDH-T was considerably stable after heating at 65℃ for 30 min, and the optimum temperature for the enzyme reaction was 55 to 60℃. The enzyme was capable of oxidizing several aliphatic aldehydes, particularly C_6-aliphatic aldehyde and hexanal, but did not oxidize benzaldehyde, an aromatic aldehyde. The deduced amino acid sequnce of ALDH-T exhibited 45% homogeneity to that of the human cytoplasmic aldehyde dehydrogenase sequence. Two amino acid residues believed to be implicated in the active site, Cys-289 and Glu-255,were conserved among the different aldehyde dehydrogenases.
- 社団法人日本生物工学会の論文
- 1993-09-25
著者
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MATSUOKA MASAYOSHI
Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo Universi
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Matsuoka Masayoshi
Department Of Biotechnology Faculty Of Engineering Osaka University
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IMANAKA TADAUILO
Department of Biotechnology, Faculty of Engineering, Osaka University
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OHTA TOYOO
Department of Biotechnology, Faculty of Engineering, Osaka University
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SAKODA HISAO
Department of Biotechnology, Faculty of Engineering, Osaka University
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WIDHYASTUTI NUNOK
(Present address)Microbiology Division, R & D Centre for Biology-LIPI
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Matsuoka Masayoshi
Department Of Applied Microbial Technology Faculty Of Biotechnology And Life Science Sojo University
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佐古田 久雄
阪大・工・応生
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Ohta Toyoo
Department Of Biotechnology Faculty Of Engineering Osaka University
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Sakoda H
Osaka Univ. Osaka Jpn
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Sakoda Hisao
Department Of Biotechnology Faculty Of Engineering Osaka University
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Imanaka Tadauilo
Department Of Biotechnology Faculty Of Engineering Osaka University
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Widhyastuti Nunok
(present Address)microbiology Division R & D Centre For Biology-lipi
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