Lysine-Based Site-Directed Mutagenesis Increased Rigid β-Sheet Structure and Thermostability of Mesophilic 1,3-1,4-β-Glucanase
Lysine-Based Site-Directed Mutagenesis Increased Rigid β-Sheet Structure and Thermostability of Mesophilic 1,3-1,4-β-Glucanase
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DOI：
10.1021/acs.jafc.5b00480
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发表时间：
2015-06-03
影响因子：
6.1
通讯作者：
Li, Qi
中科院分区：
文献类型：
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作者：
Niu, Chengtuo;Zhu, Linjiang;Li, Qi
1，3 - 1，4-β-葡聚糖酶在食品工业中有着广泛的应用，但由于其热稳定性差，往往会降低其性能。在先前的研究中，表面赖氨酸残基的化学修饰被证明可以增加β-葡聚糖酶的热稳定性。为了提高嗜温β-葡聚糖酶的热稳定性，通过将12个赖氨酸定点突变为丝氨酸，对来自特奎尔芽孢杆菌的嗜温β-葡聚糖酶进行了合理的工程改造。结果表明，K20 S、K117 S和K165 S突变体均能提高β-葡聚糖酶的比活力和热稳定性。三重突变体（K20 S/K117 S/K165 S）可使最适温度和T-50值分别提高15和14摄氏度。在突变体中观察到5%的结构化残基，其在凹侧形成新的β折叠结构。分子动力学模拟分析表明，突变区域的柔性降低，导致β-葡聚糖酶的整体刚性。因此，基于赖氨酸的定点突变是提高β-葡聚糖酶热稳定性的一种简单有效的方法。
1,3-1,4-beta-Glucanase is widely applied in the food industry, while its low thermostability often reduces its performance. In a previous study, chemical modification of surface lysine residues was proved to increase the thermostability of beta-glucanase. To improve the thermostability, the mesophilic beta-glucanase from Bacillus terquilensis was rationally engineered through site-directed mutagenesis of the 12 lysines into serines. The results showed that the K20S, K117S, and K165S mutants could both enhance the specific activities and thermostability of beta-glucanase. The triple mutant (K20S/K117S/K165S) could increase the optimal temperature and T-50 value by 15 and 14 degrees C, respectively. Five percent more structured residues were observed in the mutant, which formed new beta-sheet structures in the concave side. Molecular dynamics simulation analysis showed that the flexibility in the mutation regions was decreased, which resulted in the overall rigidity of the beta-glucanase. Therefore, the lysine-based site-directed mutagenesis is a simple and effective method for improving the thermostability of beta-glucanase.