光合细菌羰基还原酶不对称还原苯乙酮的研究

从本实验室筛选得到的类球红杆菌(Rhodobacter sphaeroides)中,通过超声破碎、硫胺沉淀、DEAE-Sepha-dexA-25阴离子交换层析分离得到一种较纯的依赖NADPH的羰基还原酶.对其进行SDS-PAGE电泳分析,显示一条带,测得其相对分子量约为37kD.建立了羰基还原酶催化苯乙酮反应体系并对其进行优化,得出该酶催化苯乙酮的最适反应pH值为8,最适温度为37℃,在pH值为7～9之间比较稳定,其热稳定性较低.该酶对苯乙酮的米氏常数Km和最大反应速率Vmax分别为0.26mmol·L-1和2.4μmol·min-1·mg-1,最佳反应时间为24h·催化苯乙酮的主要产物为(S)-苯乙醇,其产率为58.5%,ee值可达到99%以上,是很有前景的生物催化剂之一.对酶催化与辅酶再生体系相结合进行了初步的研究,提出了氢化酶再生辅酶与菌绿素再生辅酶体系,为今后的酶催化工业生产奠定了基础.

Asymmetric Reduction of Acetophenone by Carbonyl Reductase from Photosynthetic Bacteria

An NADP(H)dependent carbonyl reductase was purified from Rhodobacter sphaeroides in this study. First, cells product was disrupted by ultrasonic treatment at 4 ℃. The enzyme was purified through ammonium sulfate, DEAE-Sephadex A-25 from cell-extract. The enzyme gave a single band on SDS-PAGE. The relative molecular weight of the enzyme was estimated to be 37 kD. Reaction system with asymmetric reduction of ketones was established and optimized. The result shows the optimum pH and temperature for reduction 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 (K_m)and maximum reaction rate (V_(max))for acetophenone were 0.26 mmol/L and 2.4 μmol/(min·mg) respectively. The enzyme can catalyze acetophenone to product corresponding S-alcohols mainly. The yield and the enantiomeric excess values (ee)of the reduction products was 58.5% and up to 99%. So, the enzyme is a potential catalyst. Along with proposing hydrogenase regeneration system and bacteriochlorin regeneration system, further investigation on enzyme-catalyzed combining with coenzyme regeneration lays the foundation for enzyme-catalyzed industry in the future.