A fast and sensitive assay for measuring the activity and enantioselectivity of transaminases

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Abstract

Amino transaminases (ATAs) are pyridoxal phosphate dependent enzymes that catalyse the reversible transfer of ammonia from an amine donor to a ketone acceptor. Recently they have attracted considerable interest as biocatalysts for the synthesis of enantiomerically pure chiral primary amines.(1) Particular attention has been paid to (i) overcoming problems of substrate/product inhibition, (ii) solving issues related to poor equilibrium conversions of ketone to amine and (iii) developing ATAs with broader substrate specificity.(2) Equally important is the ability to be able to rapidly screen ATAs for new substrates, with respect to both activity and enantio-selectivity. Previous methods for screening for ATA activity have been based upon monitoring pH changes using the indicator dye phenyl red,(3) formation of coloured Cu-alanine complexes(4) and also an elegant UV spectrophotometric protocol.(5) Herein we describe a simple colorimetric-based method and show that it can be used to determine both the activity and enantioselectivity of a wide range of ATA substrates. Scheme 1 outlines the basis for the new screening method. Amines 1 are treated with an ATA and sodium pyruvate 2. Successful substrates result in conversion of sodium pyruvate

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