Highly Focused Library‐Based Engineering of Candida antarctica Lipase B with ( S)‐Selectivity Towards sec‐Alcohols

• Published in: Advanced Synthesis & Catalysis
• Main Authors: Cen, Yixin, Li, Danyang, Xu, Jian, Wu, Qiongsi, Wu, Qi, Lin, Xianfu
• Other Authors: National Natural Science Foundation of China
• Format: Article in Journal/Newspaper
• Language: English
• Published: Wiley 2018
• Subjects: Antarc*; Antarctica
• Online Access: http://dx.doi.org/10.1002/adsc.201800711; https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1002%2Fadsc.201800711; https://onlinelibrary.wiley.com/doi/pdf/10.1002/adsc.201800711; https://onlinelibrary.wiley.com/doi/full-xml/10.1002/adsc.201800711

Abstract Candida antarctica lipase B (CALB) is one of the most extensively used biocatalysts in both academia and industry and exhibits remarkable ( R )‐enantioselectivity for various chiral sec ‐alcohols. Considering the significance of tailor‐made stereoselectivity in organic synthesis, a discovery of enantiocomplementary lipase mutants with high ( R )‐ and ( S )‐selectivity is valuable and highly desired. Herein, we report a highly efficient directed evolution strategy, using only 4 representative amino acids, namely, alanine (A), leucine (L), lysine (K), tryptophan (W) at each mutated site to create an extremely small library of CALB variants requiring notably less screening. The obtained best mutant with three mutations W104V/A281L/A282K displayed highly reversed (S)‐selectivity towards a series of sec ‐alcohol with E values up to 115 (conv. 50%, ee 94%). Compared with the previously reported ( S )‐selective CALB variant, W104A, a single mutation provided less selectivity, while the synergistic effects of three mutations in the best variant endow better ( S )‐selectivity and a broader substrate scope than the W104A variant. Structural analysis and molecular dynamics simulation unveiled the source of reversed enantioselectivity. magnified image