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

• Published in: Protein engineering Vol. 15; no. 7; pp. 585 - 593
• Main Authors: Lingen, B, Grötzinger, J, Kolter, D, Kula, M-R, Pohl, M
• Format: Journal Article
• Language: English
• Published: England Oxford Publishing Limited (England) 01.07.2002
• Subjects: Amino Acid Substitution; Base Pairing - genetics; Carboxy-Lyases - genetics; Carboxy-Lyases - metabolism; Computer Graphics; Enzyme Stability; Evolution, Molecular; Gene Library; Ketones - chemistry; Kinetics; Ligases - metabolism; Models, Molecular; Mutagenesis, Site-Directed; Plasmids; Protein Binding; Pseudomonas putida - enzymology; Stereoisomerism
• ISSN: 0269-2139; 1741-0126; 1741-0134
• DOI: 10.1093/protein/15.7.585

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.