State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China.
Microb Cell Fact. 2012 Aug 16;11:108. doi: 10.1186/1475-2859-11-108.
Biocatalytic asymmetric reductions with whole cells can offer high enantioselectivity, environmentally benign processes and energy-effective operations and thus are of great interest. The application of whole cell-mediated bioreduction is often restricted if substrate and product have low water solubility and/or high toxicity to the biocatalyst. Many studies have shown that a biphasic system is often useful in this instance. Hence, we developed efficient biphasic reaction systems with biocompatible water-immiscible ionic liquids (ILs), to improve the biocatalytic anti-Prelog enantioselective reduction of acetyltrimethylsilane (ATMS) to (R)-1-trimethylsilylethanol {(R)-1-TMSE}, which is key synthon for a large number of silicon-containing drugs, using immobilized Candida parapsilosis CCTCC M203011 cells as the biocatalyst.
It was found that the substrate ATMS and the product 1-TMSE exerted pronounced toxicity to immobilized Candida parapsilosis CCTCC M203011 cells. The biocompatible water-immiscible ILs can be applied as a substrate reservoir and in situ extractant for the product, thus greatly enhancing the efficiency of the biocatalytic process and the operational stability of the cells as compared to the IL-free aqueous system. Various ILs exerted significant but different effects on the bioreduction and the performances of biocatalysts were closely related to the kinds and combination of cation and anion of ILs. Among all the water-immiscible ILs investigated, the best results were observed in 1-butyl-3-methylimidazolium hexafluorophosphate (C(4)mim·PF(6))/buffer biphasic system. Furthermore, it was shown that the optimum substrate concentration, volume ratio of buffer to IL, buffer pH, reaction temperature and shaking rate for the bioreduction were 120 mM, 8/1 (v/v), 6.0, 30°C and 180 r/min, respectively. Under these optimized conditions, the initial reaction rate, the maximum yield and the product e.e. were 8.1 μmol/min g(cwm), 98.6% and >99%, respectively. The efficient whole-cell biocatalytic process was shown to be feasible on a 450-mL scale. Moreover, the immobilized cells remained around 87% of their initial activity even after being used repeatedly for 8 batches in the C(4)mim·PF(6)/buffer biphasic system, exhibiting excellent operational stability.
For the first time, we have successfully utilized immobilized Candida parapsilosis CCTCC M203011 cells, for efficiently catalyzing anti-Prelog enantioselective reduction of ATMS to enantiopure (R)-1-TMSE in the C(4)mim·PF(6)/buffer biphasic system. The substantially improved biocatalytic process appears to be effective and competitive on a preparative scale.
利用全细胞进行生物催化不对称还原可以提供高对映选择性、环境友好的过程和节能操作，因此具有很大的吸引力。如果底物和产物的水溶性低和/或对生物催化剂毒性高，则全细胞介导的生物还原的应用通常受到限制。许多研究表明，两相体系在这种情况下通常很有用。因此，我们开发了高效的两相反应系统，使用生物相容性的疏水性离子液体（ILs），以提高固定化毕赤酵母 CCTCC M203011 细胞作为生物催化剂对乙酰三甲基硅烷（ATMS）的抗普雷洛格对映选择性还原为（R）-1-三甲基硅基乙醇（（R）-1-TMSE）的生物催化效率，（R）-1-TMSE 是许多含硅药物的关键合成子。
发现底物 ATMS 和产物 1-TMSE 对固定化毕赤酵母 CCTCC M203011 细胞表现出明显的毒性。生物相容性的疏水性 IL 可作为底物储库和产物的原位提取剂，与无 IL 的水相体系相比，大大提高了生物催化过程的效率和细胞的操作稳定性。各种 IL 对生物还原有显著但不同的影响，生物催化剂的性能与 IL 的阳离子和阴离子的种类和组合密切相关。在所研究的所有疏水性 IL 中，在 1-丁基-3-甲基咪唑六氟磷酸盐（C（4）mim·PF（6））/缓冲液两相体系中观察到最佳结果。此外，还表明生物还原的最佳底物浓度、缓冲液与 IL 的体积比、缓冲液 pH、反应温度和摇床转速分别为 120mM、8/1（v/v）、6.0、30°C 和 180r/min。在这些优化条件下，初始反应速率、最大产率和产物对映体过量值分别为 8.1μmol/min g（湿重）、98.6%和>99%。高效的全细胞生物催化过程在 450mL 规模上显示出可行性。此外，在 C（4）mim·PF（6）/缓冲液两相体系中，固定化细胞在重复使用 8 批后仍保持初始活性的 87%左右，表现出优异的操作稳定性。
我们首次成功地利用固定化毕赤酵母 CCTCC M203011 细胞，在 C（4）mim·PF（6）/缓冲液两相体系中高效催化 ATMS 的对映选择性反-Prelog 还原为对映纯（R）-1-TMSE。该两相生物催化过程在制备规模上似乎是有效和有竞争力的。
