Improving the carboligase activity of benzoylformate decarboxylase from Pseudomonas putida by a combination of directed evolution and site-directed mutagenesis

Benzoylformate decarboxylase (BFD) from Pseudomonas putida wassubjected to directed molecular evolution to generate mutantswith increased carboligase activity which is a side reactionof the enzyme. After a single round of random mutagenesis mutantswere isolated which exhibited a 5-fold increased carboligaseactivity in aqueous buffer compared to the wild-type enzymewith a high enantiomeric excess of the product (S)-2-hydroxy-1-phenyl-propanone.From the same library, mutants with enhanced carboligase activityin water-miscible organic solvents have been isolated. The selectedmutants have been characterized by sequencing, revealing thatall mutants carry a mutation at Leu476, which is close to theactive site but does not directly interact with the active center.BFD-L476Q has a 5-fold higher carboligase activity than thewild-type enzyme. L476 was subjected to saturation mutagenesisyielding eight different mutants with up to 5-fold increasedcarboligase activity. Surprisingly, all L476 mutants catalyzethe formation of 2-hydroxy-1-phenyl-propanone with significantlyhigher enantioselectivity than the wild-type enzyme althoughenantioselectivity was not a selection parameter. Leu476 potentiallyplays the role of a gatekeeper of the active site of BFD, possiblyby controlling the release of the product. The biocatalyst couldbe significantly improved for its side reaction, the C–Cbond formation and for application under conditions that arenot optimized in nature.