β-葡萄糖苷酶是纤维素降解的限速酶，对其活性调控机制的研究具有重要意义。以往研究主要关注酶和底物之间的相互作用，而水分子是影响其活性的三个关键因素之一，其活性调控机制尚未明晰。我们前期改造Ks5A7，发现两个突变体活性显著提高，同源建模和分子对接显示，突变并未改变蛋白整体结构和底物结合模式，但通道分析提示酶中可能存在连接活性中心与外部环境的水通道，且突变残基位于通道瓶颈位置。我们推测：该通道具有运输水分子功能，突变拓宽了通道瓶颈半径，使更多水分子运输到活性中心，从而提高酶活性。据此，本项目拟在解析Ks5A7晶体结构基础上，鉴定所推测通道是否为水通道；通过更多突变实验和酶学性质分析，验证该通道调控其活性的有效性；结合分子动力学模拟和氢氘交换质谱，获得水通道介导水分子运输的可靠证据。所得结果有望阐明水通道介导水分子调控Ks5A7活性的新机制，为获得活性更高的β-葡萄糖苷酶提供更充分的科学依据。

β-Glucosidase (BGL) is the rate-limiting enzyme for efficient degradation of cellulose. Therefore，it is of great importance to study its activity regulation mechanism. Enzyme, substrate and water molecule are three key factors determining its hydrolysis activity. Previous studies have mainly focused on the interactions between enzymes and substrates, while the regulation mechanism of water molecules on the activity of BGL is not clear. Recently, we obtained two mutants with significantly improved cellobiose activities in the directed evolution of BGL Ks5A7. However, homologous modeling and molecular docking studies showed that the overall structure of the protein and the substrate binding mode were not changed by the mutations. Further analysis identified a potential water channel in the enzyme that connects the active center to the external solvent environment, and the mutations are located at the bottleneck of this channel. We hypothesized that this channel has the ability to transport water molecules and the mutations widens the radius of its bottleneck, allowing more water molecules to be transported into the active center, thus increasing the enzyme activity. Hereby, this project intends to: 1) identify whether the putative channel is a true water channel by solving the crystal structure of Ks5A7 and channel analysis; 2) verify the validity of this channel in regulating the activity of the enzyme by more mutational experiments and activity analysis; 3) obtain reliable evidence of the channel-mediated water transportation by combining the results of molecular dynamics simulation and hydrogen deuterium exchange mass spectrometry. The results obtained in this project are expected to elucidate a new activity regulation mechanism of BGL by channel-mediated water molecules, and provide a more solid scientific basis for obtaining more efficient β-glucosidase through protein engineering.