A Research On Enzymetic Property Of An R-mandelate Dehydrogenase And Its Cofactor Regeneration

(S)-mandelic acid (S-MA) is an important intermediate in the field of chiral pharmaceuticals synthesis and fine chemical engineering. S-MA can be obtained through chiral resolution of racemic mandelic acid. The chiral separation by biological enzyme approach has many advantages, such as high specificity, mild reaction condition, less energy consumption, low cost and environmental benign and chiral oxidoreductase is often employed for the chiral resolution of racemic mandelic acid. With nicotinamide adenine dinucleotide (NAD~+) as cofactor, (R)-mandelate dehydrogenase (RMDH) can specificity catalysis R-mandelic acid (R-MA) to benzoylformic acid, and can be used to obtain S-MA through chiral separation of racemic mandelic acid. The aim of this study is to obtain purified RMDH, and to study the enzymatic properties of RMDH, as well as to realize the regeneration of cofactor NAD~+ by enzyme coupling method, which lays the foundation of the production of S-MA with RMDH.A strain of RMDH producing bacteria was screened from soil. According to 16s rDNA sequence analysis and transmission electron microscope observation, the strain was identified as Pseudomonas putida.The strain was rod shaped with polar flagella, approximately 0.6-0.9 μm wide and 1.7-2.3μm long. The bacteria cell which harvested from liquid culture was treated with sonication, ammonium sulfate precipitation and hydrophobic interaction chromatography, and finally obtained RMDH which was purified 11.0 folds with a specific activity of 0.33 U/mg. The purified enzyme was demonstrated as apparent single protein band on SDS-PAGE and its molecular weight was estimated to be 61 kDa. Experiment also studied the enzymatic property of RMDH. The RMDH had relatively higher activity in pH 7.6-pH 9.0, and the optimum pH was 8.5. The RMDH had relatively higher activity in temperature gradient between 20℃ to 40℃, and the optimum temperature was 30℃. At the optimum condition, the Km and　kcat of the RMDH were 2.0×10-2 mM and 0.9 s-1 for (R)-mandelic acid, 1.8×10-2 mM and 0.9 s-1 for NAD~+, as well as 1.5×10-2 mM and 0.3 s-1 for NADP+, respectively. RMDH can use both NAD~+ and NADP+ as cofactor. When NAD~+ was used, the catalytic efficiency was 2.5 times higher than NADP+ was used.A strain of NADH dehydrogenase producing bacteria Escherichia coli JZW506 was screened from soil. The bacteria cell which harvested from liquid culture was treated with sonication, ammonium sulfate precipitation and DEAE-cellulose ion exchange chromatography, and finally NADH dehydrogenase was obtained. The NADH dehydrogenase had relatively higher activity in pH 5.6-pH 8.0, and the optimum pH was 7.6. The RMDH had relatively higher activity in temperature gradient between 25℃ to 60℃, and the optimum temperature was 45℃. At the optimum condition, the Km value of NADH dehydrogenase for NADH was 10.2μM.A bi-enzyme NAD~+ regeneration syetem was established on the basis of coupling the NADH dehydrogenase with laccase, and the system provided a theoretical basis for large-scale application of NAD~+ dependent oxidoreductase. In the bi-enzyme system, on condition that NADH dehydrogenase and laccase have a total activity ratio with 1:1, the optimal catalysis condition of the bi-enzyme system was pH 5.6,30℃. In optimal catalysis condition, which contained 0.5 U NADH dehydrogenase,0.5 U laccase and 0.1mM potassium ferricyanide, about 10 mM (reaction volume 5 mL) of NAD~+was transformed from NADH within 2h.The coupling of RMDH and NADH dehydrogenase was also studied in the experiment. The bi-enzyme system had optimal pH at 7.0 and optimal temperature at 35℃, on condition that the total enzyme activity ratio of RMDH and NADH dehydrogenase was 1:1.