Study On Asymmetric Hydrogenation Of System By Carbonyl Reductase From Photosynthetic Bacteria

With the development of asymmetric synthesis compounds, biocatalysis has development the hot spot and focal point of research. It has more specific advantage with the whole cell:mild reaction condition, high yield, few adverse reaction and so on. Meanwhile, it also exists deficiency:low site selectivity, low substrate concentrate and so on. At present, to increase contact with enzyme and substrate, improve optical purity and discover the mechanism of the action catalyzed by enzyme, pure carbonyl reductase asymmetric reduction has been study extensively and is regarded as an important method. One of the purposes of this subject is to purify a carbonyl reductase from photosynthetic bacteria and to study enzymatic characterization. The second of purpose of this subject is to establish and optimize reaction system with asymmetric reduction and to choose the best substrate of the enzyme catalysis. It lays the foundation for enzyme-catalyzed industry in the future.1. An NADPH-dependent carbonyl reductase was purified through cells ultrasonic disrupted, ammonium sulfate precipitation, DEAE-Sephadex A-25 anion exchange. The enzyme specific activity rise from 0.9 U/m to 36 U/mg and purification was about 40 multiple. The enzyme gave a single band on SDS-PAGE. The relative molecular weight of the enzyme was estimated to be 37 kD.2. The enzymatic characterization of pure enzyme was researched by UV detection activity. The results showed the optimal pH and temperature were 8 and 37℃, respectively. The enzyme was stable at pH between 7 and 9, and the temperature stability was low. The apparent Michaelis-Menten constant (Km) and maximum reaction rate (Vmax) for acetophenone were 0.26 mmol/L and 2.4μmol/(min-mg), respectively.3. Coenzyme regeneration system, reduction system and enzyme-catalyzed combining with coenzyme regeneration system were established in the experiment. The conditions with catalysis were optimized. The highest yield and the enantiomeric excess values (ee) of the reduction products (58.5% and 99%) were obtainded with substrate concentration 2.5 mmol/L, reaction temperature 37℃and reaction time 24 h.4. Enzyme reduction aromatic ketones, aliphatic were studied by GC. The result showed that enzyme had high specificity to acetophenone derivate with high electronegativity and pentanone. Enantiomeric excess values of the reduction products were up to 99%.