Directed Evolution of Chloroperoxidase for Improved Epoxidation and Chlorination Catalysis

Abstract

Chloroperoxidase (CPO) from the fungus Caldariomyces fumago is undoubtedly the most versatile member of the heme protein family. In addition to functioning as a halogenating enzyme and a classical peroxidase, CPO catalyzes the dismutation of peroxides in a catalase-type reaction and carries out cytochrome P450 oxygen insertion reactions. From the viewpoint of biocatalysis the most important CPO reactions are chiral epoxidations, hydroxylations, and sulfoxidations. CPO catalyzes a variety of chiral epoxidation reactions with high yields and high enantioselectivities. However, the industrial use of native CPO for the synthesis of chiral epoxides is limited because of its relatively low epoxidation rates in comparison to its high catalase activity, which robs the epoxidation reaction of oxidant. The use of CPO is also restricted by its poor reactivities in organic solvents. Directed evolution technology has been used to address these problems. After three rounds of PCR-based random mutagenesis, we have isolated mutants of chloroperoxidase having greatly enhanced epoxidation activity compared to the wild-type enzyme. In addition, in the screening of a first generation library of random mutation transformants, we have isolated three CPO mutant clones having improved chlorination activity and enhanced stability in a ternary solvent microemulsion comprised of toluene, isopropanol and water. Surprisingly, all three recombinant variants carry a single mutation in the cysteine residue that functions as the proximal heme ligand in the native enzyme. Two of these mutant clones are identical, having the proximal cysteine heme-ligand replaced with a tyrosine residue. The third mutant has the cysteine-29 replaced with a histidine residue. The cysteine mutation in the three mutants is the only amino acid replacement. All other mutations in the three clones were silent mutations. These data suggest that ‘‘directed evolution’’ can be successfully applied to the engineering of chloroperoxidase in the quest for a better industrial biocatalyst.