【作者】 董自星；

【导师】 李华钟；

【作者基本信息】 江南大学 ， 发酵工程， 2013， 博士

【摘要】 胆盐水解酶（Bile salt hydrolase, EC3.5.1.24, BSH）能将结合胆盐水解成游离胆盐和甘氨酸/牛磺酸，这就促使体内由胆固醇从头合成结合胆盐以弥补损失，从而降低了体内的血清胆固醇水平。因此，通过口服产BSH的活细胞、纯酶以及相关制品治疗血清胆固醇过多症（hypercholesterolemia）将具有很大的应用前景。而且BSH成为目前国内外研究的热点之一。然而，关于BSH的降胆固醇机制、底物结合机制以及安全性等还存在争议。本文筛选了一株具有较高胆盐水解酶活性和良好的体外降胆固醇能力的乳酸菌，并利用双精氨酸信号肽将该菌的bsh基因在大肠杆菌（Escherichia coli）中进行了分泌表达，接着对重组酶进行了性质分析和底物特异性改造，最后对其进行了食品级表达。主要研究结果如下：1)通过定性和定量的方法筛选到一株具有较高BSH活性和良好的体外脱除胆固醇能力的乳酸菌，并经过鉴定将其命名为植物乳杆菌BBE7。然后将该菌的bsh基因在E. coli的胞内进行了克隆和表达。通过分析发现该酶是一个由324个氨基酸组成的蛋白质，而且与其它来源的BSH相似度较高，属于N末端亲核水解酶家族。酶活测定的结果显示，重组菌胞内的BSH活性为1.85U mg-1，比野生菌中BSH的比酶活提高了13倍。2)成功利用双精氨酸信号肽将BSH分泌到E. coli的胞外。通过SDS-PAGE分析和酶活测定发现，与Sec途径的PelB信号肽相比，三种双精氨酸途径（Tat）的信号肽都能使BSH在重组菌的胞内和胞外进行表达，尽管大部分BSH都在胞内形成了包涵体，只有少数蛋白被分泌到胞外。由于信号肽DMSA的GRAVY值、稳定性和脂溶指数都高于其它两个信号肽，它对BSH的分泌效率最高。此外，4种宿主对BSH的表达效果差异很大，这是因为双精氨酸信号肽在同种（species）的不同菌株里的作用效果不同，所以可以找到信号肽和宿主的最佳组合使其有效分泌BSH。通过正交试验设计对重组菌的胞外BSH活性进行了优化，使其提高了68.1%。3)依次经过硫酸铵沉淀、透析、Ni²⁺柱和凝胶柱对重组菌发酵上清液中的BSH进行了分离纯化，得到了电泳纯的BSH，纯化倍数为39.34倍，总得率约为12.55%。并通过N端测序证实了胞外的BSH是通过Tat转运系统分泌出去的而不是由细胞渗漏造成的。重组BSH的最适反应pH为5.6-6.4，在pH7.0-8.0的范围内能保留96%的酶活；最适反应温度为37℃，40℃孵育60min能保留83.5%的酶活，而60℃孵育10min后几乎完全失活；10mM的EDTA和DTT分别可以使酶活增加45.1%和86.8%，氧化巯基的试剂Cu²⁺和高碘酸钠能强烈抑制BSH的活性，PMSF和其它金属离子对BSH都有不同程度的抑制作用；对甘氨结合胆盐的水解作用远远高于对牛磺结合胆盐的水解作用；其Vmax，Km，kcat和kcat/Km的值分别为142.8μmol （min mg）-1，2.07mM，88.127s-1和42.57L （s mmol）-1。4)氨基酸序列比对表明，当BSH的65位氨基酸为极性氨基酸时，BSH偏向于水解甘氨结合胆盐；而当其为非极性氨基酸时，BSH偏向于水解牛磺结合胆盐。通过同源结构模拟和叠加也发现，BSH的Tyr65位于底物脱氧胆酸（DCA）的异戊酸侧链附近。而且这个异戊酸侧链离C12位的羟基取代基很近，在这里增加极性的氨基酸会改变酶对相应底物的亲和力。并通过定点突变证实了65位氨基酸的极性的重要性，当它为C和N等极性氨基酸时，BSH就失活了；而当它为A、F和I等非极性氨基酸时，BSH就以活性酶的形式存在。而且突变体Y65A，Y65F和Y65I对牛磺结合胆盐的水解能力提高了（最高倍数达4倍多）。此外，底物的氨基酸部分、类固醇核的C7和C12位的羟基取代基都对酶对相应底物的水解能力有影响。5)使用质粒pNZ8149的筛选标记lacF和质粒pNZ8112的信号肽Usp45等食品级表达元件，实现了BSH在L. lactis NZ3900中的胞内和胞外的食品级表达。根据L. lactis的密码子偏好性，对bsh基因进行了密码子优化，从而使BSH在重组菌胞内和胞外的比酶活分别比原来提高了12倍和9.5%。使用带有两个负电荷的前导肽LEISSTCDA优化了信号肽的c端和成熟蛋白之间的电荷平衡，从而使bsh2编码的BSH的胞外比酶活提高了8.8%。同时使用密码子优化和前导肽LEISSTCDA使胞外BSH的比酶活提高了11.3%。此外，证明了该前导肽的两个负电荷是其发挥促进蛋白分泌的作用所必需的。更多还原

【Abstract】 Bile salt hydrolase （EC3.5.1.24, BSH） catalyzes the hydrolysis of conjugated bilesalts into free bile salts and glycine/turine. This will increase the de novo synthesis ofconjugated bile salts from cholesterol, thus reducing the serum cholesterol level in vivo.Therefore, oral administration of BSH-producing cells，pure enzyme and related productsfor curing hypercholesterolemia has good prospect for applications. Furthermore, BSHbecomes one of the focused researches at home and abroad now. However, there are stillsome controversies about the mechanism of hypocholesterolemic effect by BSH, thesubstrate-binding mechanism and safety of BSH. In this study, a lactic acid bacteriumwith high BSH activity and good cholesterol-lowering effect in vitro was screened.Besides, its bsh gene was extracellularly expressed by twin-arginine signal peptides in E.coli, and the recombinant BSH was then characterized and modified to change thesubstrate specificity. Finally, we achieved the food-grade expression of BSH. The mainresults were as follows:1) A lactic acid bacterium, with high BSH activity and excellent hypocholesterolemic effectin vitro, was screened by qualitative and quantitative methods. This strain was furtheridentified and named as Lactobacillus plantarum BBE7. The bsh gene of this bacteriumwas then cloned and expressed inside the cells of E. coli. As analyzed, BSH, a proteinencoded by324amino acids, showed high similarities to other BSHs and belonged toN-terminal nucleophilic （Ntn） hydrolase family. According to the results of BSH assay, theintracellular BSH activity of recombinant bacterium was1.85U mg-1, which was13timeshigher than that of wide-type strain.2) BSH was successfully secreted outside the cells of E. coli by twin-arginine signalpeptides. As analyzed by SDS-PAGE and BSH assay, these three signal peptides oftwin-arginine translocation pathway allowed both the intracellular and extracellularexpression of BSH in recombinant bacteria compared to signal peptide PelB from Secpathway, although most of the BSH expressed was inclusion bodies inside the cells andonly a few was liberated into the medium. Because signal peptide DMSA had higherGRAVY score, stability and aliphatic index than other two signal peptides, its secretionefficiency was the highest. In addition, the effects of the four hosts on the expression ofBSH differed, which could be attributed to the fact that the utilization of twin-argininesignal peptides varies greatly among different strains of the same species. Thus the bestcombination of signal peptide and host that allows effective secretion of BSH could befound. The extracellular BSH activity of recombinant bacterium was optimized byorthogonal experimental design and increased by68.1%.3) BSH was purified from the supernatant of recombinant bacterium by ammonium sulfate precipitation, dialysis, Ni²⁺colomn and gel colomn. The purification fold and yiled were39.34and12.55%, respectively. N terminal sequencing demonstrated that the extracellularBSH was secreted by Tat pathway but not caused by cell leakage. The optimal pH andtemperature of the purified BSH were5.6-6.4and37°C, respectively. Purified BSH wasstable in pH7.0-8.0, with a more than96%residual activity. After this enzyme wasincubated at40°C for60min, it remained about83.5%residual activity; but a nearlycomplete loss of activity was observed after incubation for10min at60°C. The acitivty ofBSH was increased by45.1%and86.8%by10mM EDTA and DTT, respectively. Butagents that oxidize thiol (Cu²⁺and sodium periodate) strongly inhibited the activity ofBSH. PMSF and other metal ions inhibited BSH activity at different levels. Besides, BSHwas more efficient at hydrolyzing glyco-conjugated bile salts than tauro-conjugated bilesalts. The Vmax, Km, kcatand kcat/Kmof the puribvfied BSH were142.8μmol （min mg）-1,2.07mM,88.127s-1and42.57L （s mmol）-1, respectively.4) As indicated by amino acid sequence alignment, when the amino acid at position65ofBSH was a nonpolar residue, BSH showed preference for tauro-conjugated bile salts, butwhen it was a polar residue, BSH displayed a preferential hydrolysis of glyco-conjugatedbile salts. Homology modeling and superimposition showed that Tyr65located near theisovaleric side chain of deoxycholic acid （DCA） which was near the hydroxyl substituents,and more hydrophilic amino acids added here can alter the affinities toward thecorresponding substrates. The importance of the polarity of residue at position65washighlighted by site-directed mutagenesis and when it was C or N, BSH lost activity; butwhen it was A, F or I, BSH was an active enzyme. Tauro-conjugated bile salts hydrolysiswas all increased by mutants Y65A, Y65F and Y65I （up to4fold）. Besides, amino acidmoieties and hydroxyl substituents at C7and C12of steroid moiety had effects on thehydrolysis of corresponding substrates by BSH.5) BSH was successfully expressed both intracellularly and extracellularly in L. lactisNZ3900, using food-grade selection marker lacF of vector pNZ8149and food-gradesignal peptide Usp45of plasmid pNZ8112. The bsh gene was optimized according to thecodon bias of L. lactis, which increased the intracellular and extracellular BSH activity ofrecombinant bacteria by12fold and9.5%, respectively. Propeptide LEISSTCDA with twonegative charges was used to optimize the charge balance between the c-region of signalpeptide and mature moiety, thus the extracellular activity of BSH encoded by bsh2genewas increased by8.8%. And the extracellular BSH activity was increased by11.3%by thecombination of codon optimization and the acidic propeptide LEISSTCDA. Besides, thetwo negatively charged residues were proved to be necessary for the secretion-enhancingeffect of LEISSTCDA.更多还原