exiguobacterium sp. gxg2中依赖磷酸烯醇式丙酮酸磷酸转移系统酶ii组分b亚基的功能研究 word格式.docxVIP

AT1b的全基因组上序列的一致性分别为97%和84%；PtsB 在氨基酸水平上与来自Exiguobacteriumsp.MH3的cytochromeCbiogenesisproteinCcmE（GenBank登录号：YP_008802155.1）和来自Exiguobacterium sp.S17的PTS systemlactose/cellobiose-specifictransportersubunitⅡB(GenBank登录号：WP_016510544.1)的一致性最高，分别为99%和93%。利用质粒pET-28a(+)构建含有ptsB基因的重组表达质粒pETPB，导入表达宿主菌株E.coliRosseta(DE3)中构建重组菌株。通过把构建成功的重组菌株Rosseta(DE3)/pETPB接种至柠檬酸铁铵-七叶苷液体培养基进行功能鉴定，证实了重组蛋白PtsB具有水解β-1，4糖苷键的功能。然后对Rosseta(DE3)/pETPB进行诱导表达，利用镍柱亲和层析纯化出重组蛋白PtsB。对PtsB进行生化性质研究，结果显示：在以p-NPG做为底物时，PtsB 的β-葡萄糖苷酶活性的最适反应温度为45℃，最适pH为8.0；在最适反应条件下，PtsB的比活力为1.01U/mg；二价的金属阳离子对PtsB的活性有比较显著的影响，其中Fe2+离子对PtsB的活性起到明显的促进作用，Ca2+、Mn2+和Mg2+也起到一定的促进作用，而Cu2+和Zn2+对PtsB的活性具有明显的抑制作用；金属螯合剂EDTA是PtsB的强抑制剂，0.5mM的ETDA 可以使PtsB失活；动力学参数分析结果显示：PtsB对p-NPG的Km为0.57 mM，转换数kcat 为0.48/s。本研究证实了Exiguobacteriumsp.GXG2中的依赖磷酸烯醇式丙酮酸磷酸转移系统酶Ⅱ的B亚基具有水解β-1,4糖苷键这一兼职功能，拓宽了ⅡB 亚基的功能范围，并为蛋白质的研究提供新的思路。关键词：依赖磷酸烯醇式丙酮酸磷酸转移系统；ⅡB亚基；微小杆菌；兼职功能蛋白THEFUNCTIONALCHARACTERIZATIONOFPHOSPHOENOLPYRUVATE-DEPENDENTPHOSPHOTRANSFERASESYSTEMEⅡBSUBUNITINEXIGUOBACTERIUMSP.GXG2ABSTRACTThephosphoenolpyruvate-dependentphosphotransferasesystem(PTS)isa complexsystemforthesugaruptakeinbacteria,whichhasaccompaniedwith phosphorylation.ThePTSiscomposedof thegeneral proteinsenzymeⅠ(EⅠ), histidinecontainingphosphocarrierprotein(HPr),andthecarbohydrate-specific enzymeIIcomplex(EⅡ).EⅡusuallyisconsistedofthreefunctionalsubunits: twocytoplasmicsubunitsof ⅡAand ⅡB—andatransmembranechannel ofⅡC.Currently,thecombinationofthreedimensionalstructureand molecularexperimentshavebeenclarifiedwiththerelevantmechanismof phosphoryltransferofⅡBsubunit.However,thereisfewresearchreports aboutthe functionofmoonlightingof ⅡBsubunit.Inthepreviouslystudies,DNAlibraryfromExiguobacteriumsp.GXG2 hadbeenconstructedsuccessfully.Oneofthepositivecloneswasnamed pGEMB0,couldalsocontainaβ-glucosidasegene(namedptsB)whichencoding ⅡBsubunitofthephosphoenolpyruvate-dependentphosphotransferase systemes.BioinformaticsanalysisresultdemonstratedthatptsBwascomposed of309basepairs,whichencodeda102-aapeptideswithth