4.7 Article

Shifting the pH Optima of (R)-Selective Transaminases by Protein Engineering

Journal

Publisher

MDPI
DOI: 10.3390/ijms232315347

Keywords

amine transaminases; asymmetric synthesis; pH optimum; protein engineering; rational design

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Amine transaminases (ATAs) are efficient biocatalysts for the synthesis of chiral amines, but wild-type ATAs have low catalytic activity under physiological conditions. Researchers have developed a variant, E49Q, of ATA that shows higher catalytic activity at pH 7.5 compared to the wild-type enzyme. This variant has been successfully used for the asymmetric synthesis of (R)-1-phenylethylamine.
Amine transaminases (ATAs) are powerful biocatalysts for the stereoselective synthesis of chiral amines. However, wild-type ATAs usually show pH optima at slightly alkaline values and exhibit low catalytic activity under physiological conditions. For efficient asymmetric synthesis ATAs are commonly used in combination with lactate dehydrogenase (LDH, optimal pH: 7.5) and glucose dehydrogenase (GDH, optimal pH: 7.75) to shift the equilibrium towards the synthesis of the target chiral amine and hence their pH optima should fit to each other. Based on a protein structure alignment, variants of (R)-selective transaminases were rationally designed, produced in E. coli, purified and subjected to biochemical characterization. This resulted in the discovery of the variant E49Q of the ATA from Aspergillus fumigatus, for which the pH optimum was successfully shifted from pH 8.5 to 7.5 and this variant furthermore had a two times higher specific activity than the wild-type protein at pH 7.5. A possible mechanism for this shift of the optimal pH is proposed. Asymmetric synthesis of (R)-1-phenylethylamine from acetophenone in combination with LDH and GDH confirmed that the variant E49Q shows superior performance at pH 7.5 compared to the wild-type enzyme.

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