β-葡萄糖苷酶的糖耐受和促活性质研究  

Study and rational engineering of the glucose tolerance and promotion ofβ-glucosidase

作　　者：李璇 刘帅峰 孟春雨 程尧 房伟[1,2,3,4] 张寅良 张学成 肖亚中[1,2,3,4] LI Xuan;LIU Shuaifeng;MENG Chunyu;CHENG Yao;FANG Wei;ZHANG Yinliang;ZHANG Xuecheng;XIAO Yazhong(School of Life Sciences,Anhui University,Hefei 230601,Anhui,China;Anhui Key Laboratory of Modern Biomanufacturing,Hefei 230601,Anhui,China;Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis,Hefei 230601,Anhui,China;Anhui University Green Industry Innovation Research Institute,Hefei 230000,Anhui,China)

机构地区：[1]安徽大学生命科学学院,安徽合肥230601 [2]现代生物制造安徽省重点实验室,安徽合肥230601 [3]安徽省微生物与生物催化工程技术研究中心,安徽合肥230601 [4]安徽大学绿色产业创新研究院,安徽合肥230000

出　　处：《微生物学报》2023年第5期2007-2019,共13页Acta Microbiologica Sinica

基　　金：安徽省科技攻关重大项目(202003a06020015);国家自然科学基金(31870056);国家重点研发计划(2018YFC0311106)。

摘　　要：【目的】以葡萄糖耐受并促活的β-葡萄糖苷酶Bgl2A为出发材料,寻找与β-葡萄糖苷酶的葡萄糖耐受和促活性质相关的重要氨基酸残基位点并对其进行突变;对突变酶性质进行检测,结合分子对接,探究突变对酶的糖耐受和促活性质的影响及机制;进而对葡萄糖不耐受的Bgl3A(Bgl2A:A22S/V224S)进行分子改造,以获得应用潜能更好的突变酶。【方法】通过序列和结构比对、统计耦联分析和结构分析,选取Bgl2A底物通道口、蛋白质表面以及活性中心附近可能间接影响葡萄糖耐受和促活性质的残基作为突变位点,构建了多个突变酶,并对其酶学性质进行检测。【结果】以Bgl2A为出发酶,D322I、W325A、W126Y、F172N、C173I和N226V的糖耐受和促活性质显著提升。分子对接提示,这些突变可能是通过变构效应影响活性中心与葡萄糖结合的自由能,从而改变酶葡萄糖耐受和促活性质。据此,在Bgl3A分子上对应构建多个突变体,筛选获得了较出发酶在糖耐受和促活性质提升的同时保持较高酶活和稳定性的突变酶N226V和F172N。【结论】除了酶与葡萄糖直接结合的位点,不与葡萄糖直接相互作用的位点也可通过远程作用间接影响β-葡萄糖苷酶的葡萄糖耐受和促活性质,这为β-葡萄糖苷酶的相关理性改造提供了新思路。[Objective]In this study,we identified possibly critical sites for the higher glucose tolerance and promotion of Bgl2A through three ways,and checked their significance by mutation and characterized the mutants.Further,we investigated the mechanism of the critical sites affecting the glucose tolerance and promotion by molecular docking.Further,Bgl3A(Bgl2A:A22S/V224S),with low glucose tolerance,was rationally engineered to obtain mutants with better application potential.[Methods]Through sequence and structure comparison,statistical coupling analysis,and structural analysis,we selected the residues in the substrate tunnel and near the active center that may indirectly affect the glucose tolerance and promotion for mutations to different type amino acids,then prepared the mutants and tested their enzymatic properties.[Results]Some mutants with higher glucose tolerance and promotion,such as D322I,W325A,W126Y,F172N,C173I and N226V were obtained.Molecular docking showed that these mutations may change the energy of glucose binding to the active center by allosteric effect,thus influence the glucose tolerance and promotion.Based on the above results,corresponding mutants were then made for Bgl3A.Some mutants,such as N226V and F172N,with high glucose tolerance and promotion while considerable activity and stability remained,have higher application potential.[Conclusion]The results demonstrated that in addition to the sites where glucose directly binds to,there are other sites that do not interact directly with glucose can indirectly influence the glucose tolerance and promotion ofβ-glucosidase through long range effect,which provides a new clue for rational engineeringβ-glucosidases.

关 键 词：Β-葡萄糖苷酶 葡萄糖耐受与促活 理性改造 分子对接 变构效应 

分 类 号：Q55[生物学—生物化学]