Overexpression of synthesized cephalosporin C acylase containing mutations in the substrate transport tunnel(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
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概要
- 論文の詳細を見る
Cephalosporin C (CPC) acylase converts CPC into 7-aminocephalosporanic acid (7-ACA) by single-step enzymatic catalysis. An optimized CPC acylase gene with substituted codons and a reduced GC content was artificially designed, synthesized and overexpressed in recombinant Escherichia coll. The synthetic CPC acylase (sCPCAcy) exhibited 2.3 times more CPC specific deacylation activity with substrate CPC than with substrate glutaryl-7-ACA (GL-7-ACA). Site-directed mutagenesis of the residues around the active center showed that not only the residues that were adjacent to the CPC D-α-aminoadipyl moiety, but also the residues that were in the substrate transport tunnel (Leu666, Ala675, Leu677), played crucial roles in catalysis as the ones locating in active center. Mutant sCPCAcy^<Leu666Phe> and sCPCAcy^<Leu677Ala> exhibited significantly reduced specific enzymatic activity, while mutant sCPCAcy^<Ala675Gly> demonstrated enhanced activity. The specific activity of purified sCPCAcy and sCPCAcy^<Ala675Gly> was 10.0U/mg and 11.3U/mg, respectively. The optimal CPC acylase productivity of mutant sCPCAcy^<Ala675Gly> reached 5349U/l after 24h in culture, which was a 35% increase over the activity of sCPCAcy.
著者
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Zhang Jing
Institute Of Antibiotics Huashan Hospital Fudan University
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Wang Ying
Institute Of Animal Sciences Chinese Academy Of Agricultural Sciences (caas)
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Luo Hui
Department Of Chemical Engineering Institute Of Biochemical Engineering Tsinghua University
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Yu H
Department Of Chemical Engineering Institute Of Biochemical Engineering Tsinghua University
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Shen Z
Department Of Chemical Engineering Institute Of Biochemical Engineering Tsinghua University
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Yu Huimin
Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University
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Song Wensi
Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University
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An Ming
Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University
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Shen Zhongyao
Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University
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Song Wensi
Institute Of Biochemical Engineering Department Of Chemical Engineering Tsinghua University
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Zhang Jing
Institute Of Biochemical Engineering Department Of Chemical Engineering Tsinghua University
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An Ming
Institute Of Biochemical Engineering Department Of Chemical Engineering Tsinghua University
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Wang Ying
Institute Of Biochemical Engineering Department Of Chemical Engineering Tsinghua University
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LUO Hui
Department of Biological Science and Technology, University of Science and Technology Beijing
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