Streptomyces lavendulae X33海藻糖合酶基因克隆及酶学特性  

Cloning,Expression and Characterization of a Trehalose Synthase Gene from Streptomyces lavendulae X33

作　　者：陈允妲 李雪亮 赵晓艳[1] 张丽霞 吴晓玉[1,2] 王飞 陈金印[2] CHEN Yunda;LI Xueliang;ZHAO Xiaoyan;ZHANG Lixia;WU Xiaoyu;WANG Fei;CHEN Jinyin(College of Bioscience and Bioengineering,Jiangxi Agriculture University,Nanchang 330045,China;Collaborative Innovation Center of Postharvest Key Technology and Quality Safety of Fruits and Vegetables in Jiangxi Province,Nanchang 330045,China)

机构地区：[1]江西农业大学生物科学与工程学院,江西南昌330045 [2]江西省果蔬保鲜与质量安全创新中心,江西南昌330045

出　　处：《江西农业大学学报》2021年第4期901-909,共9页Acta Agriculturae Universitatis Jiangxiensis

基　　金：国家自然科学基金地区科学项目(31560031);江西省教育厅科学技术研究项目(GJJ160387)。

摘　　要：【目的】海藻糖是一种由2个葡萄糖分子以α,α-1,1-糖苷键连接的非还原性双糖,在生物制品的保护剂方面具有良好的应用前景。海藻糖合酶(Trehalose Synthase,EC.5.4.99.16)可通过转糖苷作用将麦芽糖转化成为海藻糖,该反应仅需一步,且所用原料低廉,在酶法生产海藻糖上显示出一定的应用潜力。对链霉菌Streptomyces lavendulae X33中编码海藻糖合成酶的基因SlTs进行克隆,通过原核表达获得重组海藻糖合成酶,并研究其酶学性质。【方法】以Streptomyces lavendulae X33基因组DNA为模板,使用加尾PCR的方法克隆编码海藻糖合成酶的基因,构建含有组氨酸标签的重组表达载体,并将重组质粒导入大肠杆菌BL21(DE3)中进行原核表达。重组蛋白经Ni-NTA纯化,以麦芽糖为底物研究其酶学性质,包括最适温度、最适pH和pH稳定性及金属离子和抑制剂的影响。【结果】从菌株Streptomyces lavendulae X33基因组中克隆得到了海藻糖合成酶基因片段SlTs GenBank登录号为MW217513。SlTs全长1719 bp,编码一个572个氨基酸的蛋白SlTs,分子量大小为66 ku。氨基酸序列分析表明SlTs与来源于Thermomonospora curvata DSM 43183的海藻糖合酶最高相似性为84%,与其它已报道的海藻糖合酶有较大差异。序列比对的结果显示SlTs与已解析结构的海藻糖合酶TcTs(PDB.5h2t)等具有的基序“DGGYD”“NHTS”“QPDLN”“RXDAVPYL”“LLAEANQ”“LRNHDELTLE”高度保守,具有一个由Asp218-Glu260-Asp330构成的活性中心,表明SlTs归属于糖苷水解酶GH13家族。经异源表达,Ni^(2+)-NTA亲合层析后进行纯化。重组酶rSlTs最适反应温度为20℃,最适反应pH为7.5,在以麦芽糖为底物,最适反应条件下测得的比活力为67.6 U/mg。重组酶rSITs在20℃下处理1 h活力不变,处理9 h时,残留酶活为60%。但在50℃下处理0.5 h活力丧失,表明rSITs对热敏感。重组酶rSITs在pH5.0-8.0有活性,最适反应pH为6.0,在pH6.0-7.0处理12[Objective]Trehalose is a non-reducing disaccharide composed of two glucose molecules which were linked by aα,α-1,1-glycosidic bond.Trehalose plays various biological roles in a variety of prokaryotic and eukaryotic organisms and invertebrates due to its physicochemical properties.Trehalose synthase(EC.5.4.99.16)which converts maltose to trehalose is considered to be a potential biocatalyst for trehalose production.This enzymatic process has the advantage of simple reaction and employs an inexpensive substrate.This study intended to discover and characterize a trehalose synthase from Streptomyces lavendulae X33.[Methods]Genomic DNA extracted from Streptomyces lavendulae X33 was used as the template for gene cloning of the trehalose synthase using PCR reaction.The PCR product was digested with restriction endonucleases Nde I and HindⅢ,then ligated to pET-29a vector.After plasmid was transformed into E.coli BL21(DE3)cells,the recombinant enzyme was expressed by IPTG induction and purified with nickel affinity chromatography(Ni^(2+)-NTA).Characterization of recombinant rSlTs including optimal pH and temperature,metal ions dependency and inhibitor was done using maltose as the substrate.The effects of pH and temperature on the enzyme activity were also measured.[Results]The sequence for the trehalose synthase gene,slTs,isolated from Streptomyces lavendulae X33,was deposited into the GenBank database under the accession number MW217513.The trehalose synthase gene encoded 572 amino acids with a putative molecular size of 66 ku.The amino acid sequence of SlTs shared the highest identity(84%)with the trehalose synthase from Thermomonospora curvata DSM 43183,but it was completely different from other experimentally characterized bacterial trehalose synthases.The results of homology modeling and sequence comparing of SlTs and TcTs(PDB.5h2t)revealed that SlTs possessed the common trehalose synthase motifs such as“DGGYD”,“NHTS”,“QPDLN”,“RXDAVPYL”,“LLAEANQ”and“LRNHDELTLE”.A catalytic triad Asp218-G

关 键 词：Streptomyces lavendulae X33 海藻糖合酶 基因克隆 酶学特性 

分 类 号：Q814[生物学—生物工程]