College of Pharmacy and Bioengineering, Chengdu University, Chengdu, 610106, China.
College of Pharmacy and Bioengineering, Chengdu University, Chengdu, 610106, China.
Int J Biol Macromol. 2019 Jun 1;130:19-23. doi: 10.1016/j.ijbiomac.2019.02.099. Epub 2019 Feb 18.
Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) is a key enzyme in preparing β-substituted alcohols and optically pure epoxides. However, the weak thermostability of HheC greatly limits its industrial application and needs to be improved. In this study, a combinatorial smart library was constructed and screened for enhancing the thermostability of HheC. Mutation sites Lys203 and Lys204 located at the protein surface were selected and simultaneously randomized with charged amino acids (Arg, Asp, Glu, Lys), resulting in a combinatorial smart library. After screening a total of only 48 clones, three positive variants CSL1 (Lys203Arg), CSL2 (Lys204Arg) and CSL3 (Lys203Arg/Lys204Arg) were obtained. Compared to the wild type HheC, variant CSL2 exhibited an increase in thermal stability with a nearly 6.9-fold improvement of half-life (τ) at 55 °C without compromising catalytic activity. In addition, the double mutant CSL3 displayed approximately 3.4-fold higher τ value and 1.8-fold higher k value toward 1,3-dicholoro-2-propanol (1,3-DCP) than that of the wild-type HheC. Homology modeling analysis highlights that the newly gained hydrogen bonds and optimizing charge-charge interactions at the protein surface might contribute to the overall conformational stabilization, leading to improving the thermostability of HheC.
来自根癌农杆菌 AD1（HheC）的卤代醇脱卤酶是制备β-取代醇和光学纯环氧化物的关键酶。然而，HheC 的弱热稳定性极大地限制了其工业应用，需要加以改进。在本研究中，构建并筛选了组合智能文库以提高 HheC 的热稳定性。选择位于蛋白质表面的突变位点 Lys203 和 Lys204，并将其与带电荷的氨基酸（Arg、Asp、Glu、Lys）同时随机化，从而产生组合智能文库。经过总共仅 48 个克隆的筛选，得到了三个阳性变体 CSL1（Lys203Arg）、CSL2（Lys204Arg）和 CSL3（Lys203Arg/Lys204Arg）。与野生型 HheC 相比，变体 CSL2 的热稳定性提高，半衰期（τ）在 55°C 时提高了近 6.9 倍，而催化活性没有降低。此外，双突变体 CSL3 对 1,3-二氯-2-丙醇（1,3-DCP）的τ值约提高了 3.4 倍，k 值提高了 1.8 倍，而野生型 HheC 的τ值和 k 值分别为 1.8 倍。同源建模分析表明，新获得的氢键和优化的蛋白质表面的电荷-电荷相互作用可能有助于整体构象稳定，从而提高 HheC 的热稳定性。
