High-throughput selection of (new) enzymes: phage display-mediated isolation of alkyl halide hydrolases from a library of active-site mutated epoxide hydrolases.

Epoxide hydrolase StEH1, from potato, is similar in overall structural fold and catalytic mechanism to haloalkane dehalogenase DhlA from Xanthobacter autotrophicus . StEH1 displays low (promiscuous) hydrolytic activity with (2-chloro)- and (2-bromo)ethanebenzene producing 2-phenylethanol. To investigate possibilities to amplify these very low dehalogenase activities, StEH1 was subjected to targeted randomized mutagenesis at five active-site amino acid residues and the resulting protein library was challenged for reactivity towards a bait chloride substrate. Enzymes catalyzing the first half-reaction of a hydrolytic cycle were isolated following monovalent phage display of the mutated proteins. Several StEH1 derived enzymes were identified with enhanced dehalogenase activities.