生物信息学分析葡聚糖蔗糖酶的结构和功能特性  

Analysis of Structure and Functional Properties of Glucansucrase Based on Bioinformatics Method

作　　者：蔡华扬 于连升 李腾鑫 林宜萌 杜仁鹏 CAI Huayang;YU Liansheng;LI Tengxin;LIN Yimeng;Du Renpeng(Engineering Research Center of Agricultural Microbiology Technology,Ministry of Education/Heilongjiang Provincial Key Laboratory of Plant Genetic Engineering and Biological Fermentation Engineering for Cold Region/Key Laboratory of Microbiology,College of Heilongjiang Province/School of Life Sciences,Heilongjiang University,Harbin 150080;Hebei Key Laboratory of Agroecological Safety,Hebei University of Environmental Engineering,Qinhuangdao,Hebei 066102)

机构地区：[1]黑龙江大学,生命科学学院,农业微生物技术教育部工程研究中心,黑龙江省寒区植物基因与生物发酵重点实验室,黑龙江省普通高校微生物重点实验室,哈尔滨150080 [2]河北环境工程学院,河北省农业生态安全重点实验室,河北秦皇岛066102

出　　处：《中国农学通报》2024年第18期105-114,共10页Chinese Agricultural Science Bulletin

基　　金：黑龙江省自然科学基金优秀青年基金项目“乳酸明串珠菌群体感应和第二信使介导右旋糖酐合成的信号通路研究”(YQ2021C030);中国博士后科学基金资助项目“c-di-GMP和c-di-AMP介导乳酸明串珠菌L2胞外多糖合成的分子调控机制研究”(2022MD713755);黑龙江省博士后资助项目“柠檬明串珠菌胞外多糖合成途径中信号网络的作用机制研究”(LBH-Z21082);新时代龙江优秀硕士、博士学位论文项目“肠膜明串珠菌DRP105右旋糖酐生物合成机制的研究”(LJYXL2022-020);河北省农业生态安全重点实验室开放基金课题项目“乳酸菌胞外多糖的分离纯化及构效关系研究”(2023SYSJJ17);黑龙江省省属高等学校基本科研业务费科研项目“c-di-AMP调控肠膜明串珠菌DRP105胞外多糖的生物合成机理”(2022-KYYWF-1075)。

摘　　要：葡聚糖蔗糖酶是一种重要的糖基转移酶(Glucosyltransferase,GTF),对于乳酸菌合成胞外多糖具有关键作用,能够以蔗糖为底物合成葡聚糖或低聚糖,是乳酸菌(Lactic acid bacteria,LAB)合成胞外多糖(Exopolysaccharide,EPS)的关键酶蛋白。然而乳酸菌代谢系统复杂,EPS生物合成机制未得到全面解析,限制了EPS的应用。为了进一步解析乳酸菌EPS的生物合成机制,表征不同来源葡聚糖蔗糖酶的结构,探明酶的催化调控机制是必要手段。本研究以肠膜明串珠菌(Leuconostoc mesenteroides)DRP105的葡聚糖蔗糖酶gtfB基因为目标序列,利用生物信息学技术对葡聚糖蔗糖酶GtfB结构和功能进行了预测分析。研究结果显示:GtfB属于GH70家族,是一种胞外酶,不存在跨膜区,含有7个保守区域和39条重复序列。其理论分子量为308986.21,理论等电点为4.59,属于酸性蛋白质。磷酸化位点含110个Thr、89个Ser、53个Tyr,GtfB含有5个结构域,呈U型折叠,活性中心在A结构域,含有(β/α)8桶状结构。分子对接预测分析表明,蔗糖与GtfB的活性口袋结合紧密。这些结果初步探明了GtfB具有葡聚糖蔗糖酶的特征结构和功能,证明其在乳酸菌EPS合成途径中的关键调控作用,为其在EPS生产过程中的应用提供了理论基础。Glucansucrase is a glycosyltransferase(GTF)that can synthesize dextran or oligosaccharides with sucrose as a substrate,and is a key enzyme protein for the synthesis of exopolysaccharide(EPS)by lactic acid bacteria(LAB).However,the metabolic system of LAB is complex,and the mechanism of EPS biosynthesis has not been elucidated,which limits the development of EPS.In order to further analyze the biosynthetic mechanism of LAB EPS and characterize the structure of dextransucrase from different sources,it is necessary to explore the catalytic regulation mechanism of enzyme.In this study,the structure and function of glucansucrase GtfB from Leuconostoc mesenteroides DRP105 were predicted with bioinformatics techniques.The results showed that GtfB belonged to the GH70 family,which was an extracellular enzyme with no transmembrane region,containing 7 conserved regions and 39 repeats.The theoretical molecular weight of GtfB was 308986.21,and the theoretical isoelectric point was 4.59,which was an acidic protein.The phosphorylation site contained 110 Thr,89 Ser,and 53 Tyr,and GtfB contained 5 domains,which were Ufolded,and the active center was in the A domain,containing(β/α)8 barrel-like structures.Molecular docking prediction analysis showed that sucrose was tightly bound to the active pocket of GtfB.The results of this study preliminarily revealed the characteristic structure and function of GtfB as a glucanase,proving that this enzyme is a key regulatory enzyme in the EPS synthesis pathway of LAB and has potential application value in the EPS production process.

关 键 词：肠膜明串珠菌 葡聚糖蔗糖酶 生物信息学分析 结构预测 分子对接 

分 类 号：S182[农业科学—农业基础科学]