Improving the carboligase activity of benzoylformate decarboxylase from Pseudomonas putida by a combination of directed evolution and site-directed mutagenesis.
TL;DR: 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.
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Abstract: 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.
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Citations
Semi-rational approaches to engineering enzyme activity: combining the benefits of directed evolution and rational design.
TL;DR: Efficient sampling of mutations likely to affect enzyme function has been conducted both experimentally and computationally, with remarkable improvements in substrate selectivity and specificity and in the de novo design of enzyme activities within scaffolds of known structure.
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Directed evolution of enzymes for applied biocatalysis
TL;DR: Directed evolution has rapidly emerged as a powerful new strategy for improving the characteristics of enzymes in a targeted manner, but further work is required to broaden the range of screens that can be used, particularly in terms of reaction type, and functional characteristics, such as enantioselectivity and regioselectivities.
204
Directed evolution: an approach to engineer enzymes
Jasjeet Kaur,Rohit Sharma +1 more
TL;DR: By providing a simple and reliable route to enzyme improvement, directed evolution has emerged as a key technology for enzyme engineering and biocatalysis.
Optimising enzyme function by directed evolution.
TL;DR: Recent advances in 'smart' library design and computational screening are now permitting much deeper searches of sequence space, which potentially increases the extent to which enzyme function can be modified.
152
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