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高光学纯度的(S)-4-三甲基硅基-3-丁炔-2-醇是一种重要的药物手性中间体.以4-三甲基硅基-3-丁炔-2-酮作为模型底物,从51株不产氧光合细菌中筛选出一株高效目标功能菌株Thiocapsa roseopersicina SJH001作为生物催化剂进行光动不对称加氢催化反应,在未经优化的反应条件下,其产物(S)-TMSBL的ee值高于99%,产率高达80%以上.从Thiocapsa roseopersicina SJH001分离得到了新的NADPH依赖型氧化还原酶[(S)-氧化还原酶和(R)-氧化还原酶].粗酶经硫酸铵分级沉淀、Q-sepharose阴离子交换层析、Sephacryl S-200丙烯葡聚糖凝胶过滤层析后在SDS-PAGE上显示为单一条带,其酶蛋白的相对分子质量为44.5 kDa,相对酶活为449.8 U/mg,高于文献报道的同类具有对映体选择性氧化还原酶.通过比较光照强度、pH值、反应前对菌体细胞热预处理、底物浓度对Thiocapsa roseopersicina SJH001胞内氧化还原酶的活性和构型产生的影响,进一步在分子水平阐明了光动不对称加氢催化反应的机理.
The high optical purity of (S) -4-trimethylsilyl-3-butyn-2-ol is an important pharmaceutical chiral intermediate.Preparation of 4-trimethylsilyl- -ketone as a model substrate, Thiocapsa roseopersicina SJH001 was screened from 51 non-oxygen-producing photosynthetic bacteria for photo-kinetic asymmetric hydrogenation reaction. Under unoptimized reaction conditions, The ee value of the product (S) -TMSBL was higher than 99% and the yield was over 80% .The new NADPH dependent oxidoreductase [(S) -reductase and (R) -oxidation were isolated from Thiocapsa roseopersicina SJH001 Reductase] .The crude enzyme was precipitated by ammonium sulfate, Q-sepharose anion exchange chromatography, Sephacryl S-200 gel filtration chromatography on SDS-PAGE showed a single band, the enzyme protein relative The molecular mass was 44.5 kDa and the relative activity was 449.8 U / mg, which was higher than that reported in the literature with enantioselective oxidoreductase. By comparing the light intensity and pH value, Effects of different concentrations on the activity and conformation of Thiocapsa roseopersicina SJH001 intracellular oxidoreductase, Further elucidated at the molecular level the mechanism of photodynamic reaction catalyzed asymmetric hydrogenation.