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.

摘要: The keto ester reductase from Candida glabrata, designated as CgKR1, is a highly versatile biocatalyst with broad substrate spectrum. Its preference was altered by rational design of the active pocket for bioreduction aromatic α-keto esters in our previous work. However, its practical application still hindered poor thermostability and high loading. In this work, random mutagenesis followed saturation performed aiming to improve thermostability. Variants M5 (I99Y/D138N/G174A) M6 (D138N) exhibited an obvious increase T50(15) 41.8°C wild-type 53.4°C 48.8°C, respectively, indicating important role residue 138 homologous three-dimensional structures variant revealed that increased might be attributed hydrogen bonds asparagine forms other polar amino acids around it. Combination most thermostable specificity-altered M1 (F92L/F94V) yielded M7 (F92L/F94V/I99Y/D138N/G174A), not only higher activity toward esters, but also value 54.6°C. This corresponds half-life 2.16min 182min at 50°C. methyl ortho-chlorobenzoylformate (CBFM) 0.5M (100g/L) could stoichiometrically converted product 1g/L lyophilized cell M7, 4g/L powder BmGDH cofactor regeneration within 10h, while enzyme gave 84% conversion even reaction time extended 24h.