title: Characterization and Soluble Expression of D-Hydantoinase from Pseudomonas fluorescens for the Synthesis of D-Amino Acids.
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 Mn(2+), Fe(2+), and Fe(3+) 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.
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, China.
authors.forename: Guo-Chao
authors.initials: GC
authors.lastname: Xu
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Li
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, China.
authors.forename: Rui-Zhi
authors.initials: RZ
authors.lastname: Han
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, China.
authors.forename: Jin-Jun
authors.initials: JJ
authors.lastname: Dong
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, China. yni@jiangnan.edu.cn.
authors.forename: Ye
authors.initials: Y
authors.lastname: Ni
mesh_terms: D000581:Amidohydrolases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D000596:Amino Acids / Q000096:biosynthesis* / Q000737:chemistry; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006827:Hydantoins / Q000737:chemistry / Q000378:metabolism; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D006827:Hydantoins; D000581:Amidohydrolases; C020047:dihydropyrimidinase
keywords: Chaperone; D-amino acids; D-hydantoinase; Pseudomonas fluorescens; Soluble expression