Development of an improved phenylacetaldehyde reductase mutant by an efficient selection procedure

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Organic Chemistry

Background:

• Chiral alcohols are essential building blocks in chemical synthesis.
• Phenylacetaldehyde reductase (PAR) produces chiral alcohols from ketones with high optical purity.
• Current PAR systems have limitations with high substrate and co-solvent concentrations.

Purpose of the Study:

• To engineer a phenylacetaldehyde reductase (PAR) capable of higher substrate conversion efficiencies.
• To improve PAR performance in the presence of elevated substrate and 2-propanol concentrations.
• To develop a robust enzyme for industrial chiral alcohol production.

Main Methods:

• Re-examination and optimization of the mutant selection procedure for PAR.
• Identification and selection of effective amino acid substitutions through a novel screening method.
• Enzymatic assays to evaluate the performance of engineered PAR mutants with high substrate loads.

Main Results:

• A refined selection procedure successfully identified beneficial amino acid substitutions in PAR.
• Two specific amino acid substitutions were found to significantly enhance enzyme function.
• A double-mutant PAR achieved near-complete conversion of high concentrations of m-chlorophenacyl chloride (m-CPC) and ethyl 4-chloro-3-oxobutanoate (ECOB).

Conclusions:

• The engineered PAR enzyme demonstrates superior activity and stability at high substrate concentrations.
• This advancement offers a more efficient biocatalytic route for producing valuable chiral alcohols.
• The developed methodology can be applied to engineer other enzymes for demanding industrial applications.