反应详情

A transaminase library was purchased from BioCatalytics (Pasadena, Calif.) and the enzymes were tested for production of monatin in coupled reactions using the ProA aldolase from C. testosteroni. The library consisted of: AT-101, a broad range L-aminotransferase; AT-102, a branched chain L-transaminase (i.e., a branched-chain aminotransferase (BCAT, EC 2.6.1.42)); AT-103, a broad range D-transaminase; AT-104, a branched chain L-transaminase (i.e., a branched-chain aminotransferase (BCAT, EC 2.6.1.42)); AT-105, lysine-6-aminotransferase; and AT-106, a broad range L-transaminase. In reactions with AT-103, D-glutamate was used as the amino acid donor. For reactions with AT-105, L-lysine was used as the amino donor. All other reactions utilized L-glutamate as the co-substrate. Enzymes and additional components/substrates were added directly to the reaction buffer provided in the kit, which contained 100 mM potassium phosphate buffer pH 7.5, 100 mM amino donor, and 0.1 mM PLP. To one mL of reaction buffer were added: 4 mg indole-3-pyruvate, 20 mg pyruvate, approximately 50 μg ProA provided in a cellular extract, 1 μL 2 M MgCl2, and 2 mg of aminotransferase enzyme to be tested. All reactions were performed in duplicate, and a negative control reaction was done with no additional aminotransferase added. Background production of monatin is due to native E. coli aminotransferases present in the cellular extract of the recombinant ProA enzyme. The reactions were incubated overnight at 30° C. with gentle shaking (100 rpm). The samples were filtered and submitted for reverse phase LC/MS/MS analysis as described in Example 18. The results are presented below:

1. additionEnzymes and additional components/substrates were added directly to the reaction
2. custombuffer provided in the kit, which
3. customTo one mL of reaction
4. additionbuffer were added