Rapid and Sensitive Kinetic Assay for Characterization of omega-Transaminases

摘要

For the biocatalytic preparation of optically active amines, omega-transaminases (omega-TA) are of special interest since they allow the asymmetric synthesis starting from prostereogenic ketones with 100percent yield. To facilitate the purification and characterization of novel omega-TA, a fast kinetic assay was developed based on the conversion of the widely used model substrate alpha-methylbenzylamine, which is commonly accepted by most of the known omega-TAs. The product from this reaction, acetophenone, can be detected spec-trophotometrically at 245 nm with high sensitivity (epsilon velence 12 mM~(-1) cm~(-1)), since the other reactants show only a low absorbance. Besides the standard substrate pyruvate, all low-absorbing ketones, aldehydes, or keto acids can be used as cosubstrates, and thus the amino acceptor specificity of a given omega-TA can be obtained quickly. Furthermore, the assay allows the fast investigation of enzymatic properties like pH and temperature optimum and stability. This method was used for the characterization of a novel omega-TA cloned from Rhodobacter sphaeroides, and the data obtained were in excellent accordance with a standard capillary electrophoresis assay.