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. Kc-Ami 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-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.
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References
Cai G, Zhu SC, Yang S, Zhao GP, Jiang WL (2004) Cloning, overexpression, and characterization of a novel thermostable penicillin G acylase from Achromobacter xylosoxidans: probing the molecular basis for its high thermostability. Appl Environ Microb 70:2764–2770
Fournand D, Arnaud A (2001) Aliphatic and enantioselective amidases: from hydrolysis to acyl transfer activity. J Appl Microbiol 91:381–393
Grigorenko BL, Khrenova MG, Nilov DK, Nemukhin AV, Svedas VK (2014) Catalytic cycle of penicillin acylase from Escherichia coli: QM/MM modeling of chemical transformations in the enzyme active site upon penicillin G hydrolysis. Acs Catalysis 4:2521–2529
Kang XM, Zhang XJ, Hong LL, Peng F, Liu ZQ, Zheng YG (2019a) Establishment of a novel high-throughput screening method for the detection and quantification of L-phosphinothricin produced by a biosynthesis approach. Process Biochem 76:136–141
Kang XM, Cai X, Liu ZQ, Zheng YG (2019b) Identification and characterization of an amidase from Leclercia adecarboxylata for efficient biosynthesis of L-phosphinothricin. Bioresour Technol 289:121658
Kang XM, Cai X, Huang ZH, Liu ZQ, Zheng YG (2019c) Construction of a highly active secretory expression system in Bacillus subtilis of a recombinant amidase by promoter and signal peptide engineering. Int J Biol Macromol 143:833–841
Lin CP, Wu ZM, Tang XL, Hao CL, Zheng RC, Zheng YG (2018) Continuous production of aprepitant chiral intermediate by immobilized amidase in a packed bed bioreactor. Bioresour Technol 274:371–378
Liu ZQ, Wu L, Zheng L, Wang WZ, Zhang XJ, Jin LQ, Zheng YG (2018) Biosynthesis of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate by carbonyl reductase from Rhodosporidium toruloides in mono and biphasic media. Bioresour Technol 249:161–167
McVey CE, Walsh MA, Dodson GG, Wilson KS, Brannigan JA (2001) Crystal structures of penicillin acylase enzyme-substrate complexes: structural insights into the catalytic mechanism. J Mol Biol 313:139–150
Oinonen C, Rouvinen J (2000) Structural comparison of Ntn-hydrolases. Protein Sci 9:2329–2337
Srirangan K, Orr V, Akawi L, Westbrook A, Moo Young M, Chou CP (2013) Biotechnological advances on penicillin G acylase: pharmaceutical implications, unique expression mechanism and production strategies. Biotechnol Adv 31:1319–1332
Xue YP, Zheng YG, Liu ZQ, Liu X, Huang JF, Shen YC (2014) Efficient synthesis of non-natural L-2-aryl-amino acids by a chemoenzymatic route. ACS Catalysis 4:3051–3058
Xue YP, Cao CH, Zheng YG (2018) Enzymatic asymmetric synthesis of chiral amino acids. Chem Soc Rev 47:1516–1561
Zheng RC, Jin JQ, Wu ZM, Tang XL, Jin LQ, Zheng YG (2018) Biocatalytic hydrolysis of chlorinated nicotinamides by a superior AS family amidase and its application in enzymatic production of 2-chloronicotinic acid. Bioorg Chem 76:81–87
Zhong GN, Zhao QF, Zhang QL, Liu W (2017) 4-alkyl-L-(Dehydro)proline biosynthesis in actinobacteria involves N-terminal nucleophile-hydrolase activity of gamma-glutamyltranspeptidase homolog for C-C bond cleavage. Nat Commun 8:16109
Acknowledgements
This research did not receive any specific grant from funding agencies in the commercial, public, or not-for-profit sectors.
Supporting Information
Supplementary Fig. 1— Neighoubor-joining phylogenetic tree of Kc-Ami and other amidases belonging to the Ntn-hydrolase surperfamily.
Supplementary Fig. 2— Michaelis-Menten kinetic parameters of Kc-Ami.
Supplementary Fig. 3- Fig. 11—The 1H HMR spectra of substrates rac-S1 to rac-S9.
Supplementary Table 1 Primers used in the present study.
Supplementary Table 2— Purification scheme of Kc-Ami from the supernatants of B. subtilis WB800 fermentation mixture.
Supplementary Table 3— Chiral HPLC analysis of the ee of the synthesized amino acids catalyzed by Kc-Ami.
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Kang, XM., Cai, X., Liu, ZQ. et al. Secretory expression and characterization of a novel amidase from Kluyvera cryocrescens in Bacillus subtilis. Biotechnol Lett 42, 2367–2377 (2020). https://doi.org/10.1007/s10529-020-02959-1
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DOI: https://doi.org/10.1007/s10529-020-02959-1