A thermostable quorum-quenching lactonase from Geobacillus kaustophilus (GKL) was used as a template for in vitro directed evolution experiments. Here we describe the overexpression and purification of wild-type GKL, the construction of a quorum-quenching directed evolution platform using bioluminescence as a reporter, and the in vitro kinetic assay for the determination of kinetic parameters of wild-type GKL and its mutants.
Keywords: Bioluminescence; Directed evolution; N-Acyl-homoserine lactonases; N-Acyl-homoserine lactones.