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  Laboratory Evolution of Enantiocomplementary Candida antarctica Lipase B Mutants with Broad Substrate Scope

Wu, Q., Soni, P., & Reetz, M. T. (2013). Laboratory Evolution of Enantiocomplementary Candida antarctica Lipase B Mutants with Broad Substrate Scope. Journal of the American Chemical Society, 135(5), 1872-1881. doi:10.1021/ja310455t.

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 Creators:
Wu, Qi1, 2, Author              
Soni, Pankaj2, 3, Author              
Reetz, Manfred T.2, 4, Author              
Affiliations:
1Zhejiang Univ, Dept Chem, Hangzhou 310027, Zhejiang, Peoples R China, ou_persistent22              
2Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society, ou_1445588              
3CSIR Inst Microbial Technol, Chandigarh 160036, India, ou_persistent22              
4Univ Marburg, Fachbereich Chemie, D-35032 Marburg, Germany, ou_persistent22              

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 Abstract: Candida antarctica lipase B (CALB) is a robust and easily expressed enzyme used widely in academic and industrial laboratories with many different kinds of applications. In fine chemicals production, examples include acylating kinetic resolution of racemic secondary alcohols and amines as well as desymmetrization of prochiral diols (or the reverse hydrolytic reactions). However, in the case of hydrolytic kinetic resolution of esters or esterifying kinetic resolution of acids in which chirality resides in the carboxylic acid part of the substrate, rate and stereoselectivity are generally poor. In the present study, directed evolution based on iterative saturation mutagenesis was applied to solve the latter problem. Mutants with highly improved activity and enantioselectivity relative to wild-type CALB were evolved for the hydrolytic kinetic resolution of p-nitrophenyl 2-phenylpropanoate, with the selectivity factor increasing from E = 1.2 (S) to E = 72 (S) or reverting to E = 42 (R) on an optional basis. Surprisingly, point mutations both in the acyl and alcohol pockets of CALB proved to be necessary. Some of the evolved CALB mutants are also efficient biocatalysts in the kinetic resolution of other chiral esters without performing new mutagenesis experiments. Another noteworthy result concerns the finding that enantiocomplementary CALB mutants for α-substituted carboxylic acid esters also show stereocomplementarity in the hydrolytic kinetic resolution of esters derived from chiral secondary alcohols. Insight into the source of stereoselectivity was gained by molecular dynamics simulations and docking experiments.

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 Dates: 2013-01-09
 Publication Status: Published in print
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 Rev. Type: Peer
 Identifiers: DOI: 10.1021/ja310455t
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Title: Journal of the American Chemical Society
  Other : J. Am. Chem. Soc.
  Abbreviation : JACS
Source Genre: Journal
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Pages: - Volume / Issue: 135 (5) Sequence Number: - Start / End Page: 1872 - 1881 Identifier: ISSN: 0002-7863
CoNE: https://pure.mpg.de/cone/journals/resource/954925376870