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Characterization and Soluble Expression of d-Hydantoinase from Pseudomonas fluorescens for the Synthesis of d-Amino Acids

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Abstract

An active d-hydantoinase from Pseudomonas fluorescens was heterogeneously overexpressed in Escherichia coli BL21(DE3) and designated as d-PfHYD. Sequence and consensus analysis suggests that d-PfHYD belongs to the dihydropyrimidinase/hydantoinase family and possesses catalytic residues for metal ion and hydantoin binding. d-PfHYD was purified to homogeneity by nickel affinity chromatography for characterization. d-PfHYD is a homotetramer with molecular weight of 215 kDa and specific activity of 20.9 U mg−1. d-PfHYD showed the highest activity at pH 9.0 and 60 °C. Metal ions such as Mn2+, Fe2+, and Fe3+ could activate d-PfHYD with 20 % improvement. Substrate specificity analysis revealed that purified d-PfHYD preferred aliphatic to aromatic 5′-monosubstituted hydantoins. Among various strategies tested, chaperone GroES-GroEL was efficient in improving the soluble expression of d-PfHYD. Employing 1.0 g L−1 recombinant E. coli BL21(DE3)-pET28-hyd/pGRO7 dry cells, 100 mM isobutyl hydantoin was converted into d-isoleucine with 98.7 % enantiomeric excess (ee), isolation yield of 78.3 %, and substrate to biocatalyst ratio of 15.6. Our results suggest that recombinant d-PfHYD could be potentially applied in the synthesis of d-amino acids.

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Acknowledgments

We are grateful to the National Natural Science Foundation of China (21276112, 21506073), the Natural Science Foundation of Jiangsu Province (BK20150003), the Fundamental Research Funds for the Central Universities (JUSRP51409B), the Program of Introducing Talents of Discipline to Universities (111-2-06), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions for the financial support of this research.

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  1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, China

    Guo-Chao Xu, Lei Li, Rui-Zhi Han, Jin-Jun Dong & Ye Ni

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Correspondence to Ye Ni.

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All the authors certify that this manuscript is original and has not been published and will not be submitted elsewhere for publication while being considered by Applied Biochemistry and Biotechnology. And the study is not split up into several parts to increase the quantity of submissions and submitted to various journals or to one journal over time. No data have been fabricated or manipulated (including images) to support your conclusions. No data, text, or theories by others are presented as if they were our own. The submission has been received explicitly from all co-authors. And authors whose names appear on the submission have contributed sufficiently to the scientific work and therefore share collective responsibility and accountability for the results. The authors declare that they have no conflict of interest. This article does not contain any studies with human participants or animals performed by any of the authors. Informed consent was obtained from all individual participants included in the study.

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Xu, GC., Li, L., Han, RZ. et al. Characterization and Soluble Expression of d-Hydantoinase from Pseudomonas fluorescens for the Synthesis of d-Amino Acids. Appl Biochem Biotechnol 179, 1–15 (2016). https://doi.org/10.1007/s12010-015-1975-6

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