Nucleotide Sequence of the Neutral Protease Gene (nprL) from Lactobacillus sp. and Characterization of the Enzyme
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概要
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The neutral protease gene (nprL) from Lactobacillus sp. no. 1 was cloned and sequenced. Cloning was performed with Bacillus subtilis DB104 [his nprR2 nprE18 ΔaprA3] as a host and pISA^<ts>412 as a vector plasmid. The protease-productive clone harbored a plasmid with a 4.5kb fragment insert. Nucleotide sequencing revealed that the nprL gene was composed of 1,698 bases (566 amino acids, M.W. 60,961Da). However, the molecular weight of the purified extracellular NprL was 37,000 by SDS-PAGE. The amino acid sequence of the first 9 residues of the purified protease completely matched that deduced from the DNA sequence starting from GTA (Val), 747 bases (249 amino acids) downstream from the iniation codon, ATG. These results indicated that NprL was initially translated as a pre-pro structure. NprL could not degrade gramicidin S, unlike the previously reported protease from the same strain. The optimal temperature and pH for NprL activity were 60℃ and 8.0,respectively. About 70% of the activity was retained after treatment at 60℃ for 30min. The protease activity was inhibited by EDTA, but not by phenylmethylsulfonyl fluoride (PMSF). The amino acid sequence of the secreted form of the neutral protease (317 amino acids) was homologous to the neutral protease from Bacillus cereus (95%) and thermolysin (72%). Accordingly, NprL was suggested to be a kind of metal-chelator-sensitive neutral protease. Analyses of hydrolysates of oxidized insulin A and B chains and of synthetic dipeptides by NprL revealed that the protease preferentially cleaves the peptide bonds at the amino side of bulky and/or hydrophobic residues such as Lue and Phe, in a similar but more limited fashion to that of thermolysin.
- 社団法人日本生物工学会の論文
- 1994-04-25
著者
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TAKAGI Mutsumi
International Center for Biotechnology, Osaka University
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IMANAKA Tadayuki
Department of Synthetic and Biological Chemistry, Graduate School of Engineering, Kyoto University
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TAKAGI MASAHIRO
Department of Biotechnology, Graduate School of Engineering, Osaka University
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MAEDA Takuya
Department of Tropical Medicine and Parasitology, School of Medicine, Keio University
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Takagi M
Asahi Chemical Industry Co. Ltd. Shizuoka Jpn
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Maeda Takuya
Department Of Biotechnology Faculty Of Eigineering Osaka University
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Maeda Takuya
Department Of Biological Science And Technology Faculty Of Engineering The University Of Tokushima
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Imanaka Tadayuki
Department Of Applied Biotechnology Faculty Of Engineering Osaka University
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Takagi M
Japan Advanced Inst. Sci. And Technol. Ishikawa Jpn
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Kawano S
Kaneka Corp. Hyogo Jpn
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Kawano Shigeru
Department Of Biotechnology Faculty Of Eigineering Osaka University
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Kawano Shigeru
Fine Chemicals Research Laboratories Kaneka Corporation
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Takagi Masahiro
Department Of Biotechnology Faculty Of Engineering Osaka University
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KAWANO Shigeru
Frontier Biochemical and Medical Research Laboratories, Kaneka Corporation
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Maeda Takuya
Department of Agricultural Chemistry, University of Osaka Prefecture
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