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NADPH production from NADP+ using malic enzyme of Achromobacter parvulus IFO-13182
Authors:Shinichiro Suye  Sadaji Yokoyama
Institution:Central Research Laboratory, Takara Shuzo Co. Ltd, 4-1, Seta 3-chome, Ootsu, Shiga 520-21, Japan
Abstract:A sonicate of Achromobacter parvulus IFO-13182 produced NADPH from NADP+by an NADP+-linked malic enzyme l-malate: NAD(P)+oxidoreductase, EC 1.1.1.39–40] reaction in the presence of l-malic acid and divalent metal ions. Malic enzyme of A. parvulus was stabilized by 5% l-malic acid, and activity was maintained at 60°C for 1 h. Contaminating phosphatase (orthophosphoricmonoester phosphohydrolase, EC 3.1.3.1–2) was completely inactivated by this treatment. Among the conditions tested, the optimum NADPH production was done using 36 μmol NADP+, 67 μmol l-malic acid, 63 μmol MgCl2 and 1 unit of the malic enzyme in 3 ml of 55 mm phosphate buffer (pH 7.8). Conversion ratio of NADPH from NADP+ reached 100% after 4 h incubation at 30°C and the amount of NADPH accumulated was ~12 μmol ml?1of the reaction mixture. No dephosphorylation of NADP+to NAD+or of NADPH to NADH was found by high performance liquid chromatography. The NADPH produced by such enzymatic reduction was purified by ethanol precipitation and dried in vacuo in powdered form with 97% purity, judged from the ratio of the absorbances at 340 and 260 nm. The purity of the NADPH produced was determined to be 95% from its coenzyme activity with NAD(P)+-linked glutathione reductase NAD(P)H: oxidized-glutathione oxidoreductase, EC 1.6.4.2].
Keywords:Chemical reactions  enzymatic reduction  NADPH  NADPH production  To whom correspondence should be addressed  
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