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Engineering Thermostable CYP2D Enzymes for Biocatalysis Using Combinatorial Libraries of Ancestors for Directed Evolution (CLADE)
Author(s) -
Gumulya Yosephine,
Huang Weiliang,
D'Cunha Stephlina A.,
Richards Katelyn E.,
Thomson Raine E. S.,
Hunter Dominic J. B.,
Baek JongMin,
Harris Kurt L.,
Boden M.,
De Voss James J.,
Hayes Martin A.,
Isin Emre M.,
Andersson Shalini,
Jurva Ulrik,
Gillam Elizabeth M. J.
Publication year - 2019
Publication title -
chemcatchem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.497
H-Index - 106
eISSN - 1867-3899
pISSN - 1867-3880
DOI - 10.1002/cctc.201801644
Subject(s) - biocatalysis , directed evolution , dna shuffling , protein engineering , drug discovery , synthetic biology , mutant , computational biology , limiting , chemistry , enzyme , combinatorial chemistry , biology , gene , biochemistry , ionic liquid , catalysis , mechanical engineering , engineering
The structure of metabolites of drug candidates must frequently be characterised during drug discovery and development. However, synthesising metabolites with the correct stereoselective modifications can be challenging for chemically complex parent compounds. Biocatalysis using human drug‐metabolising enzymes, such as cytochrome P450 2D6 (CYP2D6) is an alternative to chemical synthesis. However, most natural enzymes are unstable and have poor efficiency, limiting yields in preparative biotransformations. The aim of this study was to develop a library of robust mutant CYP2D enzymes for biocatalysis. The CLADE (combinatorial libraries of ancestors for directed evolution) approach increased the stability of CYP2D mutants obtained by DNA shuffling using three extant CYP2D forms. The resulting mutants showed divergent profiles of activity towards typical CYP2D substrates and included thermostable forms that may be useful for the further evolution of biocatalysts for specific applications.