[Improving the thermal stability of Bacillus subtilis lipase based on multiple computational design strategies].

Improving the thermal stability of enzymes is a hot and difficult point in the field of biocatalysis. Compared with the traditional directed evolution, computational assisted rational design is more efficient, and is widely used in enzyme engineering. Using Bacillus subtilis LipA as the model protein, the structure cavity of the enzyme was analyzed by Rosetta-VIP design, the mutation which was beneficial to the filling of the structure cavity (ΔΔE<0) was selected, followed by the solvent accessible surface area and evolutionary conservation analysis. The thermal stabilities of six out of sixteen designed single-point mutants were improved, with a maximum ΔTm value of 3.18 °C. These six mutations were further used for iterative combination mutation, the maximum ΔTm of the two-point and three-point combination mutants were 4.04 °C and 5.13 °C, respectively. The Tm of the four-point combination mutant M11 (F17A/L114P/I135V/M137L) was increased by 7.30 °C. The Tm of the six-point combination mutant M10 (F17A/V74I/L114P/I135V/M137A/I157L) was increased by 7.43 °C. The thermal stability of mutation with lower energy value, reduced accessible surface area, while conformed to evolutionary conservatism, was more likely to be improved. Therefore, the multiple virtual screening strategy based on the enzyme structure cavity filling, solvent accessible surface area and amino acid sequence conservation analysis can effectively improve the thermal stability of enzyme.提高酶的热稳定性是生物催化领域的热点和难点，计算机辅助的理性设计相比于传统的定向进化更加高效，在酶工程领域中的应用越来越广泛和深入。文中以枯草芽孢杆菌脂肪酶A 为模式蛋白，首先，利用Rosetta-VIP 计算设计对酶的结构空腔进行分析，选择了16 个有利于结构空腔填充 (ΔΔE<0) 的单点突变，并以突变位点的溶剂可及表面积和进化保守性为二次筛选依据，测定了其热稳定性与酶活性。有6 个单点突变体(F17A、V74I、L114P、I135V、M137A、I157L) 的热稳定性得到了提高，其中Tm 值最大提高3.18 ℃。结果表明，单点突变体满足ΔΔE 越低、蛋白溶剂可及表面积减少且符合序列保守性，则得到保留原有酶活力的正向突变的可能性越大。此外，将热稳定性提高的6 个单点突变进行迭代组合突变，两点组合突变体的Tm 最大提高4.04 ℃，三点组合突变体的Tm最大提高5.13 ℃，四点组合突变体的Tm提高了7.30 ℃，六点组合突变体的Tm提高了7.43 ℃。因此，基于酶的分子结构的空腔分析、溶剂可及表面积及氨基酸序列保守性计算的多重虚拟筛选方法，可有效提高酶的热稳定性。.