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

Journal of Biotechnology
Lei HuangHui-Lei Yu

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 vari...Continue Reading

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Citations

Jun 22, 2016·Applied Microbiology and Biotechnology·Xudong FengChun Li
Apr 9, 2017·Applied and Environmental Microbiology·Min LiJian-He Xu
Mar 12, 2020·Biotechnology and Bioengineering·Misha AliQayyum Husain

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