NADPH → Monatin

Participants

Reaction Steps

Production of monatin from indole-3-pyruvate and pyruvate, using BioCatalytics amino acid dehydrogenase enzymes coupled with the C. testosteroni ProA aldolase, was assayed under the following conditions: 6-7 mg/ml dehydrogenase enzyme, 5 mg NADH or NADPH, 50 μg aldolase (unpurified, see Example 7), 3 mM potassium phosphate buffer, 2 mM MgCl2, 4 mg indole-3-pyruvate, and 20 mg pyruvate were added to one mL of AADH reaction buffer (100 mM bicarbonate, pH 9.5, 200 mM NH4Cl). Negative controls contained no amino acid dehydrogenase enzyme. Samples were incubated at 30° C. at 100 rpm overnight. Experiments were performed in duplicate. The dehydrogenases tested were AADH-110, AADH-111, AADH-112, and AADH-113. AADH-110 and 111 have been defined as broad specificity enzymes, while 112 and 113 are glutamate dehydrogenases. AADH-110 produced the most monatin (as measured by LC/MS/MS) in comparison to the negative controls, approximately 0.36 μg/mL. The NADH-utilizing glutamate dehydrogenase, AADH-112, showed higher activity than the NADPH utilizing glutamate dehydrogenase (AADH-113). AADH-111 did not appear to produce more monatin than the negative control under the conditions assayed.

Provenance & Evidence