Rapid and sensitive kinetic assay for characterization of omega-transaminases.
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.
Schätzle
Sebastian
S
Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix Hausdorff-Str. 4, 17487 Greifswald, Germany.
Höhne
Matthias
M
Redestad
Erik
E
Robins
Karen
K
Bornscheuer
Uwe T
UT
D000098:Acetophenones / Q000032:analysis*; D001596:Benzylamines / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019289:Pyruvic Acid / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013696:Temperature; D000637:Transaminases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D000098:Acetophenones; D001596:Benzylamines; D011994:Recombinant Proteins; D019289:Pyruvic Acid; C030796:benzylamine; D000637:Transaminases; C038699:acetophenone