Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization.

We describe the selection of a phosphotriesterase with a very fast k(cat) (over 10(5) s(-1)), 63 times higher than the already very efficient wild-type enzyme. The enzyme was selected from a library of 3.4 x 10(7) mutated phosphotriesterase genes using a novel strategy based on linking genotype and phenotype by in vitro compartmentalization (IVC) using water-in-oil emulsions. First, microbeads, each displaying a single gene and multiple copies of the encoded protein, are formed by compartmentalized in vitro translation. These microbeads can then be selected for catalysis or binding. To select for catalysis the microbeads are re-emulsified in a reaction buffer of choice with a soluble substrate. The product and any unreacted substrate are coupled to the beads when the reaction is finished. Product-coated beads, displaying active enzymes and the genes that encode them, are detected with anti-product antibodies and selected using flow cytometry. This completely in vitro process selects for all enzymatic features simultaneously (substrate recognition, product formation, rate acceleration and turnover) and single enzyme molecules can be detected.