β-葡萄糖苷酶Bgl2A:V224D稳定性的理性改造及其应用

Rational engineering and application of the β-glucosidase Bgl2A:V224D with high stability

摘要:

【背景】β-葡萄糖苷酶(EC 3.2.1.21)在食品、医药领域具有重要的应用价值。海洋微生物来源的β-葡萄糖苷酶Bgl2A:V224D在大豆黄浆水精深加工中有较好的应用潜能，但其较低的稳定性和乙醇耐受性限制了其实际应用。【目的】提升Bgl2A:V224D的应用潜能，对其进行计算辅助的理性设计改造，提升其在应用环境中的稳定性和有机溶剂耐受性。【方法】综合采用3种计算方法设计构建Bgl2A:V224D突变体，利用序列共进化和保守性分析确定候选突变体，再通过湿法实验检测筛选目标突变体。获得稳定性提升的突变体后，通过组合突变进一步提升突变体的稳定性，并对突变体在大豆黄浆水精深加工中转化大豆异黄酮的应用潜能进行评估。【结果】获得多个稳定性提升且酶活维持或提升的单点突变体和组合突变体，其中S360K/E408Y稳定性最高，其在35 ℃、pH 6.5条件下的半衰期提升至出发酶的6.7倍左右。该突变体对乙醇的耐受性也比出发酶更高：前者在35 ℃、20%乙醇条件下孵育20 min，酶活剩余35%左右；而后者在相同条件下孵育10 min，酶活降至5%左右。将S360K/E408Y应用于水解大豆异黄酮制备苷元，其水解效率比出发酶Bgl2A:V224D显著增加，在相同条件下前者可水解的底物量是后者的2倍；用于大豆黄浆水精深加工，转化相同体积黄浆水中的大豆异黄酮，突变体S360K/E408Y所需酶量比出发酶减少1/3左右。【结论】利用提升稳定性的改造方法提升酶对有机溶剂的耐受性的策略适用于β-葡萄糖苷酶，通过这一策略可提升β-葡萄糖苷酶在多领域的应用潜力。

关键词:β-葡萄糖苷酶;稳定性;有机溶剂耐受;理性改造;大豆异黄酮

Abstract:

[Background] β-glucosidase (EC 3.2.1.21) has important application values in food and medicine industries. Bgl2A:V224D, a mutant of a marine microorganism-derived β-glucosidase, is potent in the conversion of soybean isoflavones in the deep processing of soybean yellow slurry water. However, the low stability and low ethanol tolerance limit the application of Bgl2A:V224D. [Objective] To enhance the application potential of Bgl2A:V224D, we adopted a computation-assisted rational design to improve the stability and organic solvent tolerance of this enzyme in the application environment. [Methods] We employed three complementary computational methods to design stable mutants of Bgl2A:V224D by seeking overlapping mutations. Then, sequence coevolution and conservation analyses were performed to determine candidate mutants. Finally, the target mutant enzymes were selected by experimental assays. After the stable mutant enzymes were identified, their stability was further enhanced by combinational mutation. Finally, the application performance of the mutant enzymes for the conversion of soybean isoflavones in the deep processing of soybean yellow slurry water was evaluated. [Results] Several single-point and multi-point mutants with improved stability and maintained or increased enzyme activity were obtained, among which S360K/E408Y had the highest stability, with the half-life at 35 ℃ and pH 6.5 about 6.7 times that of the starting enzyme. Moreover, S360K/E408Y was more tolerant to ethanol than the starting enzyme. The former had the relative activity of about 35% after 20 min of incubation with 20% ethanol at 35 ℃, while the latter had the relative activity of only about 5% after 10 min of incubation under the same conditions. S360K/E408Y was then applied to hydrolyze soybean isoflavones, and its efficiency was significantly increased compared with that of the starting enzyme Bgl2A:V224D. Specifically, the amount of substrate that could be hydrolyzed by the former enzyme was two times that of the latter enzyme under the same conditions. The amount of S360K/E408Y required for converting soybean isoflavones in the deep processing of soybean yellow slurry water was about 1/3 less than that of the starting enzyme. [Conclusion] The stability-enhancing engineering strategy for improving the enzyme tolerance to organic solvents is applicable to β-glucosidases, and the application potential of β-glucosidases in various fields can be enhanced by this strategy.

Key words:β-glucosidase;stability;organic solvent tolerance;rational design;soybean isoflavones