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Isolation of directed evolution mutants of chloroperoxidase resistant to suicide inactivation by primary olefins
Author(s) -
Rai Gyan P.,
Zong Qin,
Hager Lowell P.
Publication year - 2000
Publication title -
israel journal of chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.908
H-Index - 54
eISSN - 1869-5868
pISSN - 0021-2148
DOI - 10.1560/264g-uh9k-meyu-9yhy
Subject(s) - chemistry , mutant , mutagenesis , directed evolution , hydrogen peroxide , gene , primary (astronomy) , biochemistry , physics , astronomy
Wild‐type chloroperoxidase catalyzes the efficient chiral epoxidation of secondary olefins but is rapidly inactivated in a mechanism‐based suicide reaction when incubated with hydrogen peroxide and primary olefins. Directed evolution mutants of chloroperoxidase have now been isolated that are resistant to suicidal inactivation. Plasmid vectors containing error‐prone copies of the chloroperoxidase gene and a hygromycin B resistance marker gene have been used to transform Caldariomyces fumago spheroplasts and produce mutant libraries. The mutant library clones were screened for their ability to resist mechanism‐based inactivation by allylbenzene. Four generations of PCR‐based random mutagenesis and screening yielded mutants that were completely resistant to the suicide‐inactivation reaction. Rather surprisingly, the fourth generation mutant developed enhanced epoxidation activity in addition to resistance to allylbenzene inactivation. These initial results suggest that the directed evolution technique can be used to produce chloroperoxidase variants that can further exploit the potential of chloroperoxidase for the synthesis of chiral intermediates.