The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
School of Biotechnology, Jiangnan University, Wuxi 214122, China.
J Agric Food Chem. 2022 Jun 15;70(23):7202-7210. doi: 10.1021/acs.jafc.2c01699. Epub 2022 Jun 1.
Fructosyltransferase is a key enzyme in fructo-oligosaccharide production, while the highly demanding conditions of industrial processes may reduce its stability and activity. This study employs sequence alignment and structural analysis to target three potential residues (Gln38, Ile39, and Cys43) around the active center of FruSG from , and mutants with greatly improved activity and stability were obtained through site-directed mutagenesis. The values of C43N and Q38Y were, respectively, reduced to 60.8 and 93.1% compared to those of WT. Meanwhile, the of C43N was increased by 21.2-fold compared to that of WT. These imply that both the affinity and catalytic efficiency of C43N were significantly enhanced compared to WT. The Glide docking score of sucrose inside C43N was calculated to be -5.980, which was lower than that of WT (-4.887). What is more, the proposed general acid/base catalyst Glu273 with a lower p value of C43N calculated by PROPKA might contribute to an easier catalytic reaction compared to that of WT. The thermal stability and pH stability of the mutant C43N were significantly enhanced compared to those of WT, and more hydrogen bonds formed during molecular dynamics simulations might contribute to the improved stability of C43N.
果糖基转移酶是果寡糖生产的关键酶，而工业过程的苛刻条件可能会降低其稳定性和活性。本研究通过序列比对和结构分析，针对 中 FruSG 活性中心周围的三个潜在残基（Q38、I39 和 C43）进行了研究，并通过定点突变获得了活性和稳定性大大提高的突变体。与 WT 相比，C43N 和 Q38Y 的 值分别降低到 60.8%和 93.1%。同时，C43N 的 比 WT 提高了 21.2 倍。这意味着 C43N 的亲和力和催化效率都明显高于 WT。通过 Glide 对接计算，C43N 内部蔗糖的 Glide docking 得分计算为-5.980，低于 WT（-4.887）。此外，通过 PROPKA 计算的 C43N 具有较低 p 值的假定通用酸碱催化剂 Glu273 可能有助于与 WT 相比更容易进行催化反应。与 WT 相比，突变体 C43N 的热稳定性和 pH 稳定性显著提高，分子动力学模拟过程中形成的更多氢键可能有助于提高 C43N 的稳定性。
