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通过耦合透性化博伊丁假丝酵母(Candida boidinii,C.boidinii)加强了近平滑假丝酵母(Candida parapsilosis,C.parapsilosis)内羰基还原酶(Carbonyl reductase,CR)催化反应中的辅酶循环与反复利用,构建了有效的手性(R)-苯基乙醇透性化细胞耦合制备体系。实验结果如下:透性化的C.parapsilosis细胞底产物渗透效果增强,底物转化率由33.5%提升到43.8%;进一步外添加辅酶后,底物转化率由43.8%提高到了52.1%;耦合透性化C.boidinii细胞并同时外添加辅酶可大大增强辅酶循环效率,底物转化率可提高至71.8%。优化透性化细胞耦合体系反应条件,当以0.2 g近平滑假丝酵母(C.parapsilosis)为基准时,在C.parapsilosis与C.boidinii细胞质量比为1.5∶1,底物浓度为10 mmol/L,辅酶浓度为0.1 mmol/L时,底物转化率可达78.2%。离心法收集细胞进行多批次反应,当反应12批次时,耦合体系仍然保持有55%底物转化率,此时总的底物转化率达70.3%,产物经高效液相色谱测定为(R)-苯基乙醇,对映体过量值(e.e.值)达98.2%。
The coenzyme cycle in the catalytic reaction of Carbonyl reductase (CR) in Candida parapsilosis (C. parapsilosis) is enhanced by the coupling of Candida boidinii (C. boidinii) And repeatedly used to construct an effective chiral (R) -phenylethanol permeabilized cell-coupled preparation system. The experimental results are as follows: Permeabilization of permeabilized C. parapsilosis cells enhanced the substrate conversion rate from 33.5% to 43.8%; further addition of coenzyme increased substrate conversion from 43.8% to 52.1% Catabolization of C.boidinii cells with simultaneous addition of coenzyme greatly enhanced the efficiency of coenzyme cycling and the substrate conversion rate was increased to 71.8%. Optimization of the reaction conditions of the permeabilized cell-coupled system, when 0.2 g C. parrapsilosis was used as a reference, at a mass ratio of C. parapsilosis to C. boidinii cells of 1.5: 1 and a substrate concentration of 10 mmol / L, when the coenzyme concentration was 0.1 mmol / L, the substrate conversion rate could reach 78.2%. The cells were collected by centrifugation and subjected to batch reactions. When the reaction was performed for 12 batches, the coupling system still maintained 55% substrate conversion, at which point the total substrate conversion was 70.3% and the product was analyzed by high performance liquid chromatography ( R) -phenylethanol, the enantiomeric excess (ee value) was 98.2%.