Development of an improved phenylacetaldehyde reductase mutant by an efficient selection procedure
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Chiral alcohols are valuable as diverse chemicals and synthetic intermediate materials. Phenylacetaldehyde reductase (PAR) is an enzyme that converts a wide variety of ketones into chiral alcohols with high optical purity. When an alcohol such as 2-propanol is used as a hydrogen donor, PAR itself will also mediate the regeneration of the coenzyme NADH in situ. Perceiving a capacity for improvement, we sought to develop a PAR that is able to convert higher concentrations of substrates in the presence of high concentrations of 2-propanol. The selection procedure for mutants was re-examined and a procedure able to select an effective amino acid substitution was established. Two advantageous amino acid substitutions were successfully selected using the procedure. When high-concentration substrate conversion reaction was subjected with a mutant that integrated both the two amino acid substitutions, near-complete conversions of m-chlorophenacyl chloride (m-CPC) (2.1 mmol/ml) and ethyl 4-chloro-3-oxobutanoate (ECOB) (1.9 mmol/ml) were achieved.
KeywordsPhenylacetaldehyde reductase (PAR) Rhodococcus sp Optically pure alcohols Asymmetric reduction Coupled NADH regeneration Engineered enzymes 2-Propanol
We thank Sumitomo Chemical for the supply of m-CPC.
Conflict of interest
The authors declare that they have no conflict of interest.
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