Wang Lu, Gu Jiali, Feng Yinghui, Wang Mingming, Tong Yanjun, Liu Yingjie, Lyu Xiaomei, Yang Ruijin
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
College of Life Sciences, Huzhou University, Huzhou 313000, China.
J Agric Food Chem. 2021 Feb 17;69(6):1907-1915. doi: 10.1021/acs.jafc.0c07073. Epub 2021 Feb 4.
Cellobiose 2-epimerase (CE) offers a promising enzymatic approach to produce lactulose. However, its application is limited by the unsatisfactory isomerization activity and thermostability. Our study attempted to optimize the catalytic performances of CEs by flexible loop exchange, for which four mutants were constructed using CE (CE from ) as a template. As a result, all mutants maintained the same catalytic directions as the templates. Mutant C displayed a 2.2- and 1.34-fold increase in the isomerization activity and catalytic efficiency, respectively. According to the results of molecular dynamics (MD) simulations, it was revealed that the loop exchange in C enlarged the entrance of the active site for substrate binding and benefited proton transfer involved in the isomerization process. Besides, the of mutant C at 70 °C was increased from 29.07 to 38.29 h, owing to the abundance of rigid residues (proline) within the flexible loop of C. Our work demonstrated that the isomerization activity and thermostability of CEs were closely related to the flexible loop surrounding the active site, which provides a new perspective to engineer CEs for higher lactulose production.
纤维二糖2-差向异构酶（CE）为生产乳果糖提供了一种很有前景的酶法途径。然而，其应用受到异构化活性和热稳定性不理想的限制。我们的研究试图通过柔性环交换来优化CE的催化性能，为此以CE（来自 的CE）为模板构建了四个突变体。结果，所有突变体与模板保持相同的催化方向。突变体C的异构化活性和催化效率分别提高了2.2倍和1.34倍。根据分子动力学（MD）模拟结果，发现C中的环交换扩大了底物结合活性位点的入口，并有利于异构化过程中涉及的质子转移。此外，由于C的柔性环内富含刚性残基（脯氨酸），突变体C在70℃下的半衰期从29.07小时增加到38.29小时。我们的工作表明，CE的异构化活性和热稳定性与活性位点周围的柔性环密切相关，这为改造CE以提高乳果糖产量提供了新的视角。
