title: Significantly improved thermostability of a reductase CgKR1 from Candida glabrata with a key mutation at Asp 138 for enhancing bioreduction of aromatic α-keto esters.
abstract: The keto ester reductase from Candida glabrata, designated as CgKR1, is a highly versatile biocatalyst with a broad substrate spectrum. Its substrate preference was altered by rational design of the active pocket for bioreduction of aromatic α-keto esters in our previous work. However, its practical application is still hindered by its poor thermostability and high biocatalyst loading. In this work, random mutagenesis followed by a saturation mutagenesis was performed aiming to improve its thermostability. Variants M5 (I99Y/D138N/G174A) and M6 (D138N) exhibited an obvious increase in T50(15) from 41.8°C of wild-type to 53.4°C and 48.8°C, respectively, indicating the important role of residue 138 for its thermostability. The homologous three-dimensional structures of the wild-type and variant M6 (D138N) revealed that the increased thermostability might be attributed to the increased hydrogen bonds that asparagine forms with other polar amino acids around it. Combination of the most thermostable variant M5 and the substrate specificity-altered variant M1 (F92L/F94V) yielded variant M7 (F92L/F94V/I99Y/D138N/G174A), with not only higher activity toward aromatic α-keto esters, but also an increased T50(15) value of 54.6°C. This variant M7 corresponds to an obvious increase in half-life from 2.16min of wild-type to 182min at 50°C. The substrate of methyl ortho-chlorobenzoylformate (CBFM) at 0.5M (100g/L) could be stoichiometrically converted to the product by 1g/L lyophilized cell of variant M7, with 4g/L lyophilized powder of BmGDH for cofactor regeneration within 10h, while the wild-type enzyme gave only 84% conversion even the reaction time was extended to 24h.
authors: Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.
authors: Lei
authors: L
authors: Huang
authors: Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.
authors: Jian-He
authors: JH
authors: Xu
authors: Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China. Electronic address: huileiyu@ecust.edu.cn.
authors: Hui-Lei
authors: HL
authors: Yu
mesh_terms: D019943:Amino Acid Substitution; D041221:Candida glabrata / Q000201:enzymology*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D004952:Esters / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism
chemical_list: D004952:Esters; D005656:Fungal Proteins; D010088:Oxidoreductases
keywords: Bioreduction; Directed evolution; Keto ester reductase; Saturation mutagenesis; Thermostability