Abstract：
High stability at acidic environment is required for 1,3-1,4-β-glucanase to function in biofuel, brewing and animal feed industries. In this study, a mesophilic β-glucanase from Bacillus terquilensis CGX 5-1 was rationally engineered through sequence alignment and surface charge engineering to improve its acidic resistance ability. Nineteen singly-site variants were constructed and Q1E, I133L and V134A variants showed better acidic stability without the compromise of catalytic property and thermostability. Furthermore, four multi-site variants were constructed and one double-site variant Q1E/I133L with better stability at acidic environment and higher catalytic property was obtained. The fluorescence spectroscopy and structural analysis showed that more surface negative charge, decreased exposure degree of residue No.1, shifted side chain direction of residue No.133 and the lower total and folding free energy might be the reason for the improvement of acidic stability of Q1E/I133L variant. The obtained Q1E/I133L variant has potential applications in industries.
摘要：
1,3-1,4-β-葡聚糖酶在生物燃料中起作用需要在酸性环境下的高稳定性，酿造和动物饲料工业。在这项研究中,通过序列比对和表面电荷工程合理地设计了一种来自南方芽孢杆菌CGX5-1的嗜温β-葡聚糖酶，以提高其抗酸性能力。构建了19个单位点变异体，Q1E，I133L和V134A变体显示出更好的酸性稳定性,而不损害催化性质和热稳定性。此外，构建了四个多位点变体，并获得了一个在酸性环境下具有更好稳定性和更高催化性能的双位点变体Q1E/I133L。荧光光谱和结构分析表明,表面负电荷较多,1号残基的暴露程度降低，133号残基的侧链方向移动以及较低的总自由能和折叠自由能可能是Q1E/I133L变体酸性稳定性提高的原因。获得的Q1E/I133L变体在工业中具有潜在的应用。
