Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Graz, Austria.
Bioresour Technol. 2012 Mar;108(C):216-23. doi: 10.1016/j.biortech.2012.01.009. Epub 2012 Jan 10.
We report herein on bioprocess development guided by the hydrophobicities of substrate and product. Bioreductions of o-chloroacetophenone are severely limited by instability of the catalyst in the presence of aromatic substrate and (S)-1-(2-chlorophenyl)ethanol. In situ substrate supply and product removal was used to protect the utilized Escherichia coli whole cell catalyst based on Candida tenuis xylose reductase during the reaction. Further engineering at the levels of the catalyst and the reaction media was matched to low substrate concentrations in the aqueous phase. Productivities obtained in aqueous batch reductions were 21-fold improved by addition of 20% (v/v) hexane, NAD(+), expression engineering, cell permeabilization and pH optimization. Reduction of 300 mM substrate was accomplished in 97% yield and use of the co-solvent hexane in subsequent extraction steps led to 88% recovery. Product loss due to high catalyst loading was minimized by using the same extractant in bioreduction and product isolation.
我们在此报告了一种受底物和产物疏水性指导的生物工艺开发方法。在存在芳香族底物和（S）-1-（2-氯苯基）乙醇的情况下，邻氯苯乙酮的生物还原受到催化剂不稳定性的严重限制。在反应过程中，通过原位底物供应和产物去除来保护基于产朊假丝酵母木糖还原酶的利用大肠杆菌全细胞催化剂。在催化剂和反应介质的水平上进行进一步的工程设计，以适应水相中的低底物浓度。通过添加 20%（v/v）己烷、NAD(+)、表达工程、细胞透化和 pH 值优化，在水相分批还原中获得的产物得率提高了 21 倍。在 97%的收率下完成了 300mM 底物的还原，在后续的萃取步骤中使用共溶剂己烷导致 88%的产物回收。通过在生物还原和产物分离中使用相同的萃取剂，最大限度地减少了高催化剂负载导致的产物损失。
