Wei Zhen, Chen Jinling, Xu Linxiang, Liu Nannan, Yang Jie, Wang Shujun
Jiangsu Key Laboratory of Marine Bioresources and Environment, Co-Innovation Center of Jiangsu Marine Bio-Industry Technology, Jiangsu Ocean University, Lianyungang, 222005, China.
Jiangsu Institute of Marine Resources Development, Jiangsu Ocean University, Lianyungang, 222005, China.
AMB Express. 2023 Jan 19;13(1):7. doi: 10.1186/s13568-023-01513-2.
As an indispensable enzyme for the hydrolysis of dextran, dextranase has been widely used in the fields of food and medicine. It should be noted that the weak thermostability of dextranase has become a restricted factor for industrial applications. This study aims to improve the thermostability of dextranase AoDex in glycoside hydrolase (GH) family 49 that derived from Arthrobacter oxydans KQ11. Some mutants were predicted and constructed based on B-factor analysis, PoPMuSiC and HotMuSiC algorithms, and four mutants exhibited higher heat resistance. Compared with the wild-type, mutant S357P showed the best improved thermostability with a 5.4-fold increase of half-life at 60 °C, and a 2.1-fold increase of half-life at 65 °C. Furthermore, S357V displayed the most obvious increase in enzymatic activity and thermostability simultaneously. Structural modeling analysis indicated that the improved thermostability of mutants might be attributed to the introduction of proline and hydrophobic effects, which generated the rigid optimization of the structural conformation. These results illustrated that it was effective to improve the thermostability of dextranase AoDex by rational design and site-directed mutagenesis. The thermostable mutant of dextranase AoDex has potential application value, and it can also provide references for engineering other thermostable dextranases of the GH49 family.
作为水解葡聚糖不可或缺的酶，葡聚糖酶已在食品和医药领域得到广泛应用。需要注意的是，葡聚糖酶较弱的热稳定性已成为其工业应用的限制因素。本研究旨在提高来源于氧化节杆菌KQ11的糖苷水解酶（GH）家族49中的葡聚糖酶AoDex的热稳定性。基于B因子分析、PoPMuSiC和HotMuSiC算法预测并构建了一些突变体，其中四个突变体表现出更高的耐热性。与野生型相比，突变体S357P的热稳定性提高最为显著，在60℃下半衰期增加了5.4倍，在65℃下半衰期增加了2.1倍。此外，S357V同时表现出最明显的酶活性和热稳定性增加。结构建模分析表明，突变体热稳定性的提高可能归因于脯氨酸的引入和疏水作用，从而使结构构象产生刚性优化。这些结果表明，通过合理设计和定点诱变提高葡聚糖酶AoDex的热稳定性是有效的。葡聚糖酶AoDex的热稳定突变体具有潜在的应用价值，也可为GH49家族其他热稳定葡聚糖酶的工程改造提供参考。
