Rapid and Sensitive Kinetic Assay for Characterization of ω-Transaminases

Abstract

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 100% 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 spectrophotometrically at 245 nm with high sensitivity (epsilon = 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.

Enzymatic Asymmetric Synthesis of Enantiomerically Pure Aliphatic, Aromatic and Arylaliphatic Amines with (R)‐Selective Amine Transaminases

“…[10] The majority of the enzymes show a pH optimum between 8-9, which is also typical for S-ATAs such as the enzyme from Vibrio fluvialis; [11] AspTer and PenChr show a sharp optimum at pH 8.5-9 while AspOry, AspFum and NeoFis have a broader optimal range Table 1. Summary of parameters for the preparation of the lyophilized biocatalysts.…”

“…Summary of parameters for the preparation of the lyophilized biocatalysts. Activities were measured with the acetophenone assay photometrically [10] and protein concentrations were determined with the BC assay according to the supplier's manual.…”

“…Relative activity of the R-ATAs in different buffer systems. The activity towards 7b was measured spectrophotometrically [10] in the different buffers at pH 8 and was normalized to the activity in 50 mM phosphate buffer (set to 100%). All values are means from three individual measurements and standard deviations did not exceed 10%.…”

“…pH profiles of the seven R-ATAs. Activities were measured with the acetophenone assay spectrophotometrically [10] in Davies buffer [12] , which is commonly used for pH profile determinations.…”

Metagenomic discovery of a novel transaminase for valorization of monoaromatic compounds

“…[33][34][35][36][37] These assays employ other enzymes in addition to the TA to transform amination intermediates into products that can be detected using rapid analytical procedures such as spectrophotometry. Enzyme-coupled assays are quite sensitive but typically necessitate extensive and expensive optimization before they can be deployed for metagenomic screening.…”