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 was subjected to directed molecular evolution to generate mutants with increased carboligase activity which is a side reaction of the enzyme. After a single round of random mutagenesis mutants were isolated which exhibited a 5-fold increased carboligase activity in aqueous buffer compared to the wild-type enzyme with a high enantiomeric excess of the product (S)-2-hydroxy-1-phenyl-propanone. From the same library, mutants with enhanced carboligase activity in water-miscible organic solvents have been isolated. The selected mutants have been characterized by sequencing, revealing that all mutants carry a mutation at Leu476, which is close to the active site but does not directly interact with the active center. BFD-L476Q has a 5-fold higher carboligase activity than the wild-type enzyme. L476 was subjected to saturation mutagenesis yielding eight different mutants with up to 5-fold increased carboligase activity. Surprisingly, all L476 mutants catalyze the formation of 2-hydroxy-1-phenyl-propanone with significantly higher enantioselectivity than the wild-type enzyme although enantioselectivity was not a selection parameter. Leu476 potentially plays the role of a gatekeeper of the active site of BFD, possibly by controlling the release of the product. The biocatalyst could be significantly improved for its side reaction, the C-C bond formation and for application under conditions that are not optimized in nature.
Lingen
B
B
Institut für Enzymtechnologie der Heinrich-Heine-Universität Düsseldorf, im Forschungszentrum Jülich, D-52426 Jülich, Germany. b.ligen@fz-juelich.de
Grötzinger
J
J
Kolter
D
D
Kula
M-R
MR
Pohl
M
M
D019943:Amino Acid Substitution; D020029:Base Pairing / Q000235:genetics; D002262:Carboxy-Lyases / Q000235:genetics* / Q000378:metabolism*; D003196:Computer Graphics; D004795:Enzyme Stability; D019143:Evolution, Molecular; D015723:Gene Library; D007659:Ketones / Q000737:chemistry; D007700:Kinetics; D008025:Ligases / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011485:Protein Binding; D016958:Pseudomonas putida / Q000201:enzymology*; D013237:Stereoisomerism
D007659:Ketones; D002262:Carboxy-Lyases; C019778:benzoylformate decarboxylase; D008025:Ligases