title: Gradually accumulating beneficial mutations to improve the thermostability of N-carbamoyl-D-amino acid amidohydrolase by step-wise evolution.
abstract: To further enhance repeated batch reactions with immobilized N-carbamoyl-D-amino acid amidohydrolase (DCase), which can be used for the industrial production of D-amino acids, the stability of high soluble mutant DCase-M3 from Ralstonia pickettii CGMCC1596 was improved by step-wise evolution. In our previous report, six thermostability-related sites were identified by error-prone PCR. Based on the above result, an improved mutant B5 (Q12L/Q23L/H248Q/T262A/T263S) was obtained through two rounds of DNA shuffling, showing a 10°C increase in the T (50) (defined as the temperature at which heat treatment for 15 min reduced the initial activity by 50%) compared with the parental enzyme DCase-M3. Furthermore, several thermostability-related sites (Met(31), Asn(93), Gln(207), Asn(242), Glu(266), Thr(271), Ala(273)) on B5 were identified using amino acid consensus approach based on sequence alignment of homologous DCases. These sites were further investigated by iterative saturation mutagenesis (ISM), and a combinational mutant D1 (Q12L/Q23L/Q207E/N242G/H248Q/T262A/T263S/E266D/T271I/A273P) that enhanced the T(50) by about 16°C over DCase-M3 was obtained. Oxidative stability assay showed that the most heat-resisting mutant displayed only a slight increase in resistance to hydrogen peroxide. Comparative characterization showed that D1 not only maintained its characteristic high solubility but also shared similar k(cat) and K(m) values and optimum reaction pHs with the parental enzyme. The significantly improved mutants in the immobilized form are expected to be applied in the industrial production of D-p-hydroxyphenylglycine.
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Dalong
authors.initials: D
authors.lastname: Zhang
authors.forename: Fuyun
authors.initials: F
authors.lastname: Zhu
authors.forename: Wenchao
authors.initials: W
authors.lastname: Fan
authors.forename: Rongsheng
authors.initials: R
authors.lastname: Tao
authors.forename: Hong
authors.initials: H
authors.lastname: Yu
authors.forename: Yunliu
authors.initials: Y
authors.lastname: Yang
authors.forename: Weihong
authors.initials: W
authors.lastname: Jiang
authors.forename: Sheng
authors.initials: S
authors.lastname: Yang
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D049920:Ralstonia pickettii / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D000581:Amidohydrolases; C080477:N-carbamoyl-D-amino acid amidohydrolase