Dissertation
Dissertation > Biological Sciences > Bioengineering ( Biotechnology ) > Enzyme Engineering > Application of enzymes

Photosynthetic bacteria carbonyl reductase asymmetric hydrogenation catalyst system of

Author YueZhu
Tutor WangMengLiang
School Shanxi University
Course Microbiology
Keywords Rhodobacter bacilli Carbonyl reductase Asymmetric reduction Acetophenone
CLC Q814.9
Type Master's thesis
Year 2010
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As people on the asymmetric synthesis of chiral compounds in-depth research, the development of modern biological research asymmetric hydrogenation of attention and focus. Biocatalysis generally choose the whole microbial cells as a catalyst, because of its mild reaction conditions, high efficiency and relatively simple products, etc., it was found that the enzyme present in the reaction system, site-selective low substrate concentration problems and restrictions. Currently, the researchers used purified carbonyl reductase latent chiral compounds, enzymes can not only increase opportunities for contact with the substrate, increasing the optical purity, and can learn more about the role of the enzyme mechanism, enzymatic reduction set off again at home and abroad New Wave of chiral compounds. This topic hand isolated from photosynthetic bacteria carbonyl reductase purified, and its enzymatic properties of the study; other carbonyl reductase is based catalyst system and optimize its catalytic, selective catalytic optimal substrate for study enzymatic mechanisms and lay the foundation for industrial production. The main contents are as follows: 1. By ultrasonic crushing, ammonium sulfate precipitation, DEAE-Sephadex A-25 anion exchange chromatography-step separation and purification to obtain a more pure coenzyme NADPH-dependent carbonyl reductase. The specific activity of the enzyme from 0.9 U / mg up to 36 U / mg, purification factor of 40. Be analyzed by SDS-PAGE, shows a single band and obtaining measured molecular weight of about 37 kD. 2 UV determination of enzyme activity, the enzyme enzymatic properties were studied. The results showed that: the enzyme optimum pH 8, the optimum temperature is 37 ℃, the pH value between 7-9 is relatively stable, but its low thermal stability. The enzyme Michaelis constant of acetophenone Km and Vmax maximum reaction rate was 0.26 mmol / L and 2.4μmol / (min mg). 3 built coenzyme regeneration system, carbonyl reductase enzyme catalyzed acetophenone system and coupled with coenzyme regeneration system, and catalytic systems for optimization. The results showed that the optimal enzyme conditions Acetophenone: substrate concentration of 2.5 mmol / L, the reaction temperature was 37 ℃, the reaction time was 24 h, the yield of acetophenone time up to 49.3%, ee value was 99%. 4 using gas chromatography methods for the detection, investigation of the enzyme for aromatic ketones, aliphatic ketones restore. The enzyme showed a strong electronegative group in the α-substituted acetophenone derivatives of five-carbon aliphatic ketone and strong specificity;, and with high stereoselectivity, are above 99%.

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