Pan Kungang, Liu Zhongqi, Zhang Zhengjie, Jin Shanzheng, Yu Zhao, Liu Tianhui, Zhang Tongcun, Zhao Junqi, Li Zhongyuan
State Key Laboratory of Food Nutrition and Safety, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China.
School of Chemical and Biological Engineering, Qilu Institute of Technology, Jinan 250200, China.
Foods. 2022 Aug 16;11(16):2463. doi: 10.3390/foods11162463.
Improving the specific activity and thermostability of psychrophilic xylosidase is important for improving its enzymatic performance and promoting its industrial application. Herein, a psychrophilic xylosidase AX543 exhibited activity in the temperature range between 0 and 35 °C, with optimum activity at 20 °C, which is lower than that of other reported psychrophilic xylosidases. The thermostability, specific activity, and catalytic efficiency of the site-directed variants G110S, Q201R, and L2 were significantly enhanced, without affecting the optimal reaction temperature. Comparative protein structural analysis and molecular dynamics simulation indicated that these improvements might be the result of the increased hydrogen bonds interaction and improved structural rigidity. Furthermore, homologous module substitution with four segments demonstrated that the psychrophilic characteristics of AX543 are the results of the whole protein structure, and the C-terminal segment A4 appears to be more essential in determining psychrophilic characteristics, exhibiting potentiality to produce more psychrophilic xylosidases. This study provides valuable structural information on psychrophilic xylosidases and also offers attractive modification strategies to modify catalytic activity, thermostability, and optimal reaction temperature.
提高嗜冷木糖苷酶的比活性和热稳定性对于改善其酶促性能和促进其工业应用至关重要。在此，一种嗜冷木糖苷酶AX543在0至35°C的温度范围内表现出活性，在20°C时具有最佳活性，这低于其他已报道的嗜冷木糖苷酶。定点变体G110S、Q201R和L2的热稳定性、比活性和催化效率显著提高，而不影响最佳反应温度。比较蛋白质结构分析和分子动力学模拟表明，这些改进可能是氢键相互作用增加和结构刚性提高的结果。此外，用四个片段进行同源模块替换表明，AX543的嗜冷特性是整个蛋白质结构的结果，并且C末端片段A4在决定嗜冷特性方面似乎更为关键，展现出产生更多嗜冷木糖苷酶的潜力。本研究提供了关于嗜冷木糖苷酶的有价值的结构信息，也提供了有吸引力的修饰策略来改变催化活性、热稳定性和最佳反应温度。
