Directed evolution of phenylacetone monooxygenase as an active catalyst for the Baeyer-Villiger conversion of cyclohexanone to caprolactone.Revista : Biotechnology and Bioengineering
Volumen : 112
Número : 7
Páginas : 1354-1364
Tipo de publicación : ISI Ir a publicación
Phenylacetone monooxygenase (PAMO) is an exceptionally robust BaeyerVilliger monooxygenase, which makes it ideal for potential industrial applications. However, its substrate scope is limited, unreactive cyclohexanone being a prominent example. Such a limitation is unfortunate, because this particular transformat ion in an ecologically viable manner would be highly desirable, the lactone and the respective lactam being of considerable interest as monomers in polymer science. We have applied directed evolution in search of an active mutant for this valuable C-C activating reaction. Using iterative saturation mutagenesis (ISM), several active mutants were evolved, with only a minimal trade-off in terms of stability. The best mutants allow for quantitative conversion of 2 mM cyclohexanone within 1 h reaction time. In order to circumvent the NADPH regeneration problem, whole E. coli resting cells were successfully applied. Molecular dynamics simulations and induced ﬁt docking throw light on the origin of enhan ed PAMO activity. The PAMO mutants constitute ideal starting points for future directed evolution optimization necessary for an industrial process.