Tataruch Mateusz, Illeová Viera, Miłaczewska Anna, Borowski Tomasz, Mihal' Mario, Polakovič Milan
Institute of Chemical and Environmental Engineering, Slovak Technical University, Radlinského 9, 812 37 Bratislava, Slovakia; Jerzy Haber Institute of Catalysis and Surface Chemistry Polish Academy of Sciences, Niezapominajek 8, PL-30-239 Krakow, Poland.
Institute of Chemical and Environmental Engineering, Slovak Technical University, Radlinského 9, 812 37 Bratislava, Slovakia.
Int J Biol Macromol. 2023 Apr 15;234:123772. doi: 10.1016/j.ijbiomac.2023.123772. Epub 2023 Feb 20.
R-specific 1-(4-hydroxyphenyl)-ethanol dehydrogenase (R-HPED) is a promising biotool for stereoselective synthesis of chiral aromatic alcohols. This work focused on the evaluation of its stability under storage and in-process conditions in the pH range from 5.5 to 8.5. The relationship between the dynamics of aggregation and activity loss under various pH conditions and in the presence of glucose, serving as a stabilizer, was analysed using spectrophotometric techniques and dynamic light scattering. pH 8.5 was indicated as a representative environment in which the enzyme, despite relatively low activity, shows high stability and the highest total product yield. Based on a series of inactivation experiments, the mechanism of thermal inactivation at pH 8.5 was modelled. The irreversible first-order mechanism of R-HPED inactivation in the temperature range of 47.5-60 °C was verified by isothermal and multi-temperature evaluation of data, confirming that in the alkaline pH 8.5, R-HPED aggregation is the secondary process occurring at already inactivated protein molecules. The rate constants were from 0.029 min to 0.380 min for a buffer solution but they decreased to 0.011 min and 0.161 min, respectively, when 1.5 M glucose was added as a stabilizer. The activation energy was however about 200 kJ mol in both cases.
R-特异性1-(4-羟基苯基)乙醇脱氢酶(R-HPED)是一种用于立体选择性合成手性芳香醇的很有前景的生物工具。这项工作着重评估其在pH值5.5至8.5范围内储存和加工条件下的稳定性。使用分光光度技术和动态光散射分析了在各种pH条件下以及在作为稳定剂的葡萄糖存在下聚集动力学与活性丧失之间的关系。pH 8.5被确定为一种典型环境，在该环境中，尽管酶活性相对较低，但显示出高稳定性和最高的总产物产率。基于一系列失活实验，对pH 8.5下的热失活机制进行了建模。通过对数据的等温及多温度评估，验证了47.5-60°C温度范围内R-HPED失活的不可逆一级机制，证实了在碱性pH 8.5条件下，R-HPED聚集是在已经失活的蛋白质分子上发生的二级过程。缓冲溶液的速率常数为0.029 min至0.380 min，但当添加1.5 M葡萄糖作为稳定剂时，速率常数分别降至0.011 min和0.161 min。然而，两种情况下的活化能均约为200 kJ mol。
