Isolation of directed evolution mutants of chloroperoxidase resistant to suicide inactivation by primary olefins

Wild-type chloroperoxidase catalyzes the efficient chiral epoxidation of se condary olefins but is rapidly inactivated in a mechanism-based suicide rea ction when incubated with hydrogen peroxide and primary olefins. Directed e volution mutants of chloroperoxidase have now been isolated that are resist ant to suicidal inactivation. Plasmid vectors containing error-prone copies of the chloroperoxidase gene and a hygromycin B resistance marker gene hav e been used to transform Caldariomyces fumago spheroplasts and produce muta nt libraries. The mutant library clones were screened for their ability to resist mechanism-based inactivation by allylbenzene. Four generations of PC R-based random mutagenesis and screening yielded mutants that were complete ly resistant to the suicide-inactivation reaction. Rather surprisingly, the fourth generation mutant developed enhanced epoxidation activity in additi on to resistance to allylbenzene inactivation. These initial results sugges t that the directed evolution technique can be used to produce chloroperoxi dase variants that can further exploit the potential of chloroperoxidase fo r the synthesis of chiral intermediates.