Physical Chemistry I - Biophysical Chemistry, Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Str. 4a, 44227 Dortmund, Germany.
Laboratory of Thermodynamics, Department of Biochemical and Chemical Engineering, TU Dortmund University, Emil-Figge-Str. 70, 44227 Dortmund, Germany.
Colloids Surf B Biointerfaces. 2021 Dec;208:112127. doi: 10.1016/j.colsurfb.2021.112127. Epub 2021 Sep 23.
The application of co-solvents and high pressure has been shown to be an efficient means to modify the kinetics of enzyme-catalyzed reactions without compromising enzyme stability, which is often limited by temperature modulation. In this work, the high-pressure stopped-flow methodology was applied in conjunction with fast UV/Vis detection to investigate kinetic parameters of formate dehydrogenase reaction (FDH), which is used in biotechnology for cofactor recycling systems. Complementary FTIR spectroscopic and differential scanning fluorimetric studies were performed to reveal pressure and temperature effects on the structure and stability of the FDH. In neat buffer solution, the kinetic efficiency increases by one order of magnitude by increasing the temperature from 25° to 45 °C and the pressure from ambient up to the kbar range. The addition of particular co-solvents further doubled the kinetic efficiency of the reaction, in particular the compatible osmolyte trimethylamine-N-oxide and its mixtures with the macromolecular crowding agent dextran. The thermodynamic model PC-SAFT was successfully applied within a simplified activity-based Michaelis-Menten framework to predict the effects of co-solvents on the kinetic efficiency by accounting for interactions involving substrate, co-solvent, water, and FDH. Especially mixtures of the co-solvents at high concentrations were beneficial for the kinetic efficiency and for the unfolding temperature.
共溶剂和高压的应用已被证明是一种有效的方法，可以在不影响酶稳定性的情况下改变酶催化反应的动力学，而酶稳定性通常受到温度调节的限制。在这项工作中，高压停流方法与快速紫外/可见检测相结合，用于研究甲酸脱氢酶反应（FDH）的动力学参数，FDH 用于生物技术中的辅助因子回收系统。进行了补充的傅里叶变换红外光谱和差示扫描荧光研究，以揭示压力和温度对 FDH 结构和稳定性的影响。在纯缓冲溶液中，通过将温度从 25°C 升高到 45°C，将压力从环境升高到千巴范围，酶促效率提高了一个数量级。添加特定的共溶剂进一步使反应的动力学效率提高了一倍，特别是相容的渗透调节剂三甲基氧化胺及其与大分子拥挤剂葡聚糖的混合物。成功地在简化的基于活性的米氏-门捷列夫框架内应用 PC-SAFT 热力学模型，通过考虑涉及底物、共溶剂、水和 FDH 的相互作用来预测共溶剂对动力学效率的影响。特别是在高浓度下共溶剂的混合物有利于动力学效率和展开温度。
