Secretory expression and characterization of a novel amidase from Kluyvera cryocrescens in Bacillus subtilis
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ORIGINAL RESEARCH PAPER
Secretory expression and characterization of a novel amidase from Kluyvera cryocrescens in Bacillus subtilis Xue-Mei Kang . Xue Cai . Zhi-Qiang Liu
. Yu-Guo Zheng
Received: 1 November 2019 / Accepted: 8 January 2020 Ó Springer Nature B.V. 2020
Abstract Objective Cloning and secretory expression of an amidase from Kluyvera cryocrescens and characterization of its potential in preparation of chiral amino acids. Results An amidase belonging to the Ntn-hydrolase superfamily was identified from Kluyvera cryocrescens ZJB-17005 (Kc-Ami). The maximum activity of Kc-Ami was observed at pH 8.5 and 55 °C. Remarkably, Kc-Ami showed an excellent enantioselectivity (99% ee) using rac-4-(hydroxy(methyl)phosphoryl)2-(2-phenylacetamido) butanoic acid as substrate. KcAmi remained stable at pH 7.0–9.0 and exhibited prominent thermostability with a half-life time of 59.1, 47.4 and 20.4 h at 50, 55 and 60 °C, respectively. Kc-
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10529-020-02959-1) contains supplementary material, which is available to authorized users. X.-M. Kang X. Cai Z.-Q. Liu (&) Y.-G. Zheng The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou 310014, People’s Republic of China e-mail: [email protected] X.-M. Kang X. Cai Z.-Q. Liu Y.-G. Zheng Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People’s Republic of China
Ami could be appllied to synthesize chiral amino acids and its derivatives with excellent enantioselectivity ([ 99% ee). The synthesized chiral amino acids could contain short or long side chain, and further the side chain could be replaced with –OH, –COOH or benzene ring. Conclusions Kc-Ami exhibited remarkable thermostability and excellent enantioselectivity for synthesizing chiral amino acids and its derivatives. This specific characteristic provides great potential for industrial application in preparation of chiral amino acids and its derivatives. Keywords Amidase Ntn-hydrolase superfamily Kluyvera cryocrescens Chiral amino acid Lphosphinothricin
Introduction Chiral amino acids and its derivatives were widely used in pharmaceutical, agricultural, cosmetics, food, and feedstuff industries. The widespread application of chiral amino acids promoted the innovation and development for their synthesis (Xue et al. 2018). Much attention has been paid to the synthetic method of biocatalysis because of the main advantage of strict stereoselectivity, mild reaction conditions, high recovery rate, and avoiding by-product formation. To obtain
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Biotechnol Lett
enzymes which with the highly activity and strictly stereoselectivity were the key challenges for synthesis of chiral amino acid by biocatalysis. Amidase as an enzyme which hydrolyzing nonpeptidic amide bonds (–CO
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