Streptomyces griseus胰蛋白酶的分子改造

Molecular Engineering of Streptomyces Griseus Trypsin

作者: 专业:发酵工程 导师:堵国成 年度:2013 学位:博士 

关键词
链霉菌胰蛋白酶 前导肽 理性设计 自活化 高效表达

Keywords
Streptomyces trypsin, propeptide, Rational Design, Auto-activation, Overexpression
        链霉菌胰蛋白酶(Streptpmyces trypsin, SGT, EC3.4.21.4)是由灰色链霉菌(Streptomyces griseus)所产生的丝氨酸蛋白酶中的一种重要类型。胰蛋白酶的专一催化水解特性使其在皮革加工、医药、食品加工中存在广泛应用。链霉菌胰蛋白酶与哺乳动物胰蛋白酶在结构与功能上高度相似,并且由于其微生物来源的特性,从而避免了哺乳动物胰蛋白酶的免疫原性,且利用微生物产胰蛋白酶有利于酶品质的控制。目前对胰蛋白酶的需求主要依赖哺乳动物胰蛋白酶,而利用链霉菌发酵产胰蛋白酶发酵周期长,产量低,因此利用基因工程技术产重组链霉菌胰蛋白酶,并通过蛋白质工程手段对重组酶进行改造将有助于链霉菌胰蛋白酶的开发利用。本研究将链霉菌胰蛋白酶在P. pastoris系统中进行表达,分别就其前导肽进行理性设计,对重组酶酶学性质进行比较分析,对其自降解loop环中关键氨基酸R145进行定点突变,以及重新设计链霉菌胰蛋白酶自活化融合突变体提高重组酶产量,主要研究结论如下:(1)链霉菌胰蛋白酶在毕赤酵母中的表达及初步优化将链霉菌胰蛋白酶编码基因mt搭配不同启动子pGAP,pAOX1,pFLD在不同的毕赤酵母宿主(P. pastoris GS115,P. pastoris SMD1168,P. pastoris X33,P. pastoris KM71)中进行异源表达,确定最佳启动子和宿主搭配为:pAOX1和P. pastoris GS115。确定重组菌P. pastoris GS115/pAOX1/mt摇瓶发酵产重组酶的适宜条件为:诱导剂甲醇浓度为20g·L–1,诱导温度为30°C,最佳辅助碳源为4g·L–1甘油,经过挡板摇瓶发酵5天,重组链霉菌胰蛋白酶表达水平为9.6U·mL–1。采用高密度发酵技术,以4g·L–1甘油为诱导阶段辅助碳源,在3L发酵罐水平,重组菌P. pastoris GS115/pAOX1/mt产重组链霉菌胰蛋白酶表达水平为15.6U·mL–1。(2)基于链霉菌胰蛋白酶前导肽功能分析新型胰蛋白酶的设计通过链霉菌胰蛋白酶原及其缺失突变体的表达,证明在链霉菌胰蛋白酶原前导肽中,抑制胰蛋白酶成熟酶活性的主要是靠近成熟酶N端P1位的脯氨酸残基,并且发现链霉菌胰蛋白酶维持正确构象的作用力主要包括:a,二硫键(C42-C58,C168-C182,C191-C220)所形成的盐键作用力;b,N端与催化区域的氢键作用力(V16-loopD189-D194);c,维持活性中心刚性的氢键作用力(H57-D102,H57-S195)。经过对前导肽的理性设计,获得新型链霉菌胰蛋白酶突变体Exmt(YVEFmt)和IVEFmt,对酰胺键的比酶活由852U·mg–1降低至242U·mg–1,因此,Exmt是在链霉菌胰蛋白酶N端残留有前导肽的改良突变体。(3)重组链霉菌胰蛋白酶Exmt的酶学性质比较及分析将重组链霉菌胰蛋白酶Exmt与野生链霉菌胰蛋白酶wSGT和牛胰蛋白酶BT进行酶学性质比较和分析,Exmt和野生wSGT的最适反应pH为8.0,与牛胰蛋白酶BT的最适反应pH为10,Exmt N端残留前导肽YVEF改善了链霉菌胰蛋白酶的pH稳定性;wSGT和Exmt最适反应温度为50°C,BT的最适反应温度为60°C。Exmt在40°C,50°C,60°C下,其半衰期分别提高了1.8倍,2.5倍,31.3倍。Exmt N端前导肽增加了链霉菌胰蛋白酶对金属离子抑制或促进作用的敏感性,减弱了链霉菌胰蛋白酶的部分有机溶剂抗性,而机溶剂对Exmt的酯酶活力起到了明显的促进作用。由于其N端前导肽YVEF的存在,Exmt对BAPNA和BAEE底物的催化效率分别提高3.1和5.5倍。由于YVEF的存在使得链霉菌胰蛋白酶的蛋白质结构的柔性增加及分子内氢键的增加,从而使得其催化效率提高,pH耐受性和热稳定性得以改善。(4)自降解位点R145突变对链霉菌胰蛋白酶催化特性及表达的影响通过对Exmt中R145进行突变,在摇瓶发酵水平,各突变体的重组链霉菌胰蛋白酶产量均增加。其中Exmt(R145I)突变体的酰胺酶比酶活为1242.85±99.15U·mg–1,比未突变的重组酶和野生酶分别提高0.5倍和2.6倍;Exmt(R145I)突变体的酯酶比酶活为101491.58±1225.56U·mg–1,比未突变的重组酶和野生酶分别提高0.3倍和2.9倍。Exmt(R145I)对酰胺键底物催化效率提高0.2倍;其对酯键底物亲和力提高0.3倍。Exmt(R145I)的抗自降解性能亦有所提高。采用高密度发酵技术,以4g·L–1甘油为诱导阶段辅助碳源,在3L发酵罐水平,重组菌P. pastoris GS115/pAOX1/Exmt(R145I)产重组链霉菌胰蛋白酶表达水平为18.7U·mL–1。(5)自活化融合突变体高效表达重组链霉菌胰蛋白酶杂交链霉菌胰蛋白酶原VD4Kmt由于前导肽VD4K包埋于蛋白质N端,无法实现表达时的自活化,需经过体外肠激酶进行活化。而自活化融合突变体TLmt(D4K)经摇瓶发酵5天,胞外酰胺酶产量可达5.8U·mL–1,与单独表达链霉菌胰蛋白酶时的摇瓶发酵产量相近。其自活化方式是在表达过程中,通过有活性的链霉菌胰蛋白酶部分将融合蛋白TrxA连同linker部分切除,从而得到自活化后的链霉菌胰蛋白酶。采用高密度发酵技术,以4g·L–1甘油为诱导阶段辅助碳源,在3L发酵罐水平,重组菌P. pastoris GS115/pAOX1/TLmt(D4K)产重组链霉菌胰蛋白酶表达水平为19.85U·mL–1。
    Bacterial Streptomyces trypsin (SGT, EC3.4.21.4) is one of the serine proteinases inStreptomyces griseus and acts as a key mediator during microorganism growth and cellulardifferentiation. It is also a sort of serine protease which has potential applications in leatherbating, food processing, pharmacy, clinical diagnoses and biochemical tests. S. griseus trypsinis highly identical to bovine trypsin with respect to the structure and function. Although theproduction process was easily controlled and natural SGT was immunogenicity to humanbeing, commercial trypsin is mainly from mammal production. For S. griseus fermentationperiod was long and its production level was low. As a result, heterologous production ofStreptomyces trypsin was an attractive alternative for its protein engineering and application.This study was involved in heterologous expression of Streptomyces trypsin, especiallyfocused on rational design of its propeptide, analysis and optimization of its enzymecharacters and production by site-directed mutagenesis of the R145and construction ofauto-activated Streptomyces trypsin. Major results were listed below:(1) After comparatively investigated different heterologous expression systems pGAP,pAOX1and pFLD in different hosts P. pastoris GS115, P. pastoris SMD1168, P. pastoris X33and P. pastoris KM71). pAOX1and P. pastoris GS115were identified as the bestcombination. The highest recombinant Streptomyces trypsin production9.6U·mL–1wasinduced with20g·L–1methanol for5days at30°C co-feeded with4g·L–1glycerol in shakeflask fermentation. The recombinant P. pastoris GS115/pAOX1/mt had the highest trypsinexpression level of15.6U·mL–1under the optimized condition with the high cell densityfermentation in3L fermentor.(2) Through the heterologous expression of Streptomyces trypsinogen and its propeptidedeteletion mutants, the residue at P1site next to the N terminus of the mature trypsin wasproved to be the most necessary amino acid for the inhibition effect of the propeptide.Moreover, the major features of native trypsin were characterized. First, three disulfide bondsbetween residues C168-C182, C191-C220, and C42-C58held the substrate binding pocketrigid and the correct fold was observed. Second, three Hydrogen (H) bonds among thecatalytic triad (H57, D102and S195) maintained the accurate conformation of the catalyticcenter. Third, one H-bond had formed between V16and loopD189-D194and this interactionstabilized the structure of trypsin. Novel Streptomyces trypsin mutant Exmt (YVEFmt) andIVEFmt were obtained by the rational design of the propeptide. The specific activity towardsamidase substrate BAPNA of Exmt (YVEFmt) is852U·mg–1, yet the specific activitytowards amidase substrate BAPNA of IVEFmt decreased to242U·mg–1. Finally, through therational design of propeptide, the optimized recombinant Exmt was obtained. (3) Compared the enzyme characteristics of recombinant Streptomyces trypsin (Exmt)with wild Streptomyces trypsin (wSGT) and Bovine trypsin (BT). The optimized catalytic pHof Exmt, wSGT and BT were8.0,8.0and10.0respectively. The optimized catalytictemperature of Exmt, wSGT and BT were50°C,50°C and60°C respectively. The Nterminal propeptide YVEF of Exmt improved its pH tolerance and thermostability. The Exmtshowed significant increase of the thermostability, which values of t1/2were1.8-fold,2.5-foldand31.2-fold of that of the SGT at40°C,50°C,60°C respectively. Furtherly, the N terminalpropeptide YVEF of Exmt increase its resitance to metal ions. Its tolerance to organic solventswere decreased, but its esterase activity was increased by the organic solvents. Moreover, thecatalytic efficiency (representing as specificity constant, kcat/Km) of Exmt was also improvedby3.1-fold and5.5-fold towards BAPNA and BAEE respectively, because of the increase ofthe kcatand decrease of the Km. In summary, these improvements were mainly because of theincrease of protein structure flexibility and inner molecular hydrogen bonds.(4) Through the site-directed mutagenesis of the R145of Exmt, all mutants showedincrease of recombinant trypsin expression level in shake flask fermentation. Furtherly,Exmt(R145I) was obtained of the specific activity1242.85±99.15U·mg–1towards BPANA.Compared with Exmt and wSGT, it increased1.46-fold and3.61-fold. Moreover, And it hasthe specific activity101491.58±1225.56U·mg–1towards BAEE. Compared to Exmt andwSGT, it has increased0.3-fold and2.9-fold. What’s more, the catalytic efficiency(representing as specificity constant, kcat/Km) of Exmt(R145I) was also improved by0.2-foldand0.3-fold towards BAPNA and BAEE respectively. The resitance to the auto hydrolysis ofExmt(R145I) was also improved. Finally, the recombinant P. pastorisGS115/pAOX1/Exmt(R145I) have the highest trypsin production of18.7U·mL–1under theoptimized condition with the high cell density fermentation in3L fermentor.(5) The non-autoactivated hybrid Streptomyces trypsinogen VD4Kmt was activated byenterokinase. It was mainly because of the propeptide VD4K buried into the inner space ofthe trypsin structure. The autoactivated fusion mutant TLmt(D4K) can be acitivated by itselfand obtained the recombinant trypsin production of5.8U·mL–1in shake flask. Furtherly, wehave proved that the autoactivation process of TLmt(D4K). Firstly, the fusion part TrxAdoesn’t inhibit the trypsin activity during expression; Secondly, the active trypsin part cleavedat K site in the linker part to remove the fusion part TL(D4K). Finally, the recombinant P.pastoris GS115/pAOX1/TLmt(D4K) had the highest trypsin expression level of19.85U·mL–1with the optimized condition by the high cell density fermentation method in3L fermentor.
        

Streptomyces griseus胰蛋白酶的分子改造

摘要3-5
Abstract5-6
目录7-11
第一章 绪论11-23
    1.1 链霉菌胰蛋白酶的概述11-15
        1.1.1 胰蛋白酶的来源及其催化特性11
        1.1.2 胰蛋白酶的功能和应用11-12
        1.1.3 胰蛋白酶的生产12
        1.1.4 链霉菌源胰蛋白酶的产生及结构12-13
        1.1.5 链霉菌源胰蛋白酶的催化机理13-14
        1.1.6 胰蛋白酶活性的定量分析方法14-15
    1.2 胰蛋白酶的异源表达研究进展15-19
        1.2.1 动物胰蛋白酶的生产及其异源表达研究15-16
        1.2.2 微生物胰蛋白酶的异源表达研究16-18
        1.2.3 胰蛋白酶异源表达的影响因素18-19
    1.3 链霉菌胰蛋白酶工程19-21
        1.3.1 前导肽工程19-20
        1.3.2 链霉菌胰蛋白酶分子改造策略20-21
    1.4 本课题的立题依据、研究意义及主要研究内容21-23
        1.4.1 立题依据和研究意义21-22
        1.4.2 主要研究内容22-23
第二章 链霉菌胰蛋白酶在毕赤酵母中的表达及优化23-42
    2.1 前言23
    2.2 材料与方法23-31
        2.2.1 菌株和质粒23
        2.2.2 试剂和仪器23-25
        2.2.3 培养基及培养条件25
        2.2.4 DNA 操作25-26
        2.2.5 毕赤酵母不同启动子搭配不同重组菌的构建26-28
        2.2.6 胰蛋白酶酶活力测定28-29
        2.2.7 毕赤酵母重组菌的摇瓶发酵及优化29-30
        2.2.8 P. pastoris 重组菌 3 L 罐高密度发酵30-31
    2.3 结果与讨论31-40
        2.3.1 P. pastoris 重组菌的构建及验证31-32
        2.3.2 启动子和宿主对 P. pastoris 重组胰蛋白酶的影响32-34
        2.3.3 基因拷贝数及αFactor 信号肽对 P. pastoris 重组菌产胰蛋白酶的影响34-36
        2.3.4 甲醇浓度和诱导温度对 P. pastoris 重组菌产胰蛋白酶的影响36-37
        2.3.5 双碳源混合添加对 P. pastoris 重组菌产胰蛋白酶的影响37-39
        2.3.6 流加甲醇及甲醇-甘油混合物对 P. pastoris 重组菌高密度发酵影响39-40
    2.4 本章小结40-42
第三章 链霉菌胰蛋白酶前导肽功能解析及新型胰蛋白酶的设计42-55
    3.1 前言42-43
    3.2 材料与方法43-46
        3.2.1 菌株和质粒43
        3.2.2 试剂和仪器43
        3.2.3 培养基及培养条件43
        3.2.4 DNA 操作43-44
        3.2.5 链霉菌胰蛋白酶突变体的构建44-45
        3.2.6 胰蛋白酶体外活力测定方法45
        3.2.7 链霉菌胰蛋白酶突变体的纯化45-46
        3.2.8 SDS-PAGE 电泳分析及蛋白浓度测定46
        3.2.9 链霉菌胰蛋白酶突变体蛋白质结构的分子模拟46
        3.2.10 链霉菌胰蛋白酶突变体蛋白质结构分析46
    3.3 结果与讨论46-53
        3.3.1 链霉菌胰蛋白酶原前导肽缺失突变体的验证46-48
        3.3.2 链霉菌胰蛋白酶原前导肽缺失突变体的分子模拟及分析48-50
        3.3.3 链霉菌胰蛋白酶前导肽突变体的分子模拟及分析50-51
        3.3.4 链霉菌胰蛋白酶前导肽突变体的验证51-53
    3.4 本章小结53-55
第四章 重组链霉菌胰蛋白酶的酶学性质分析55-69
    4.1 前言55
    4.2 材料与方法55-57
        4.2.1 菌株55
        4.2.2 试剂和仪器55
        4.2.3 培养基及培养条件55-56
        4.2.4 重组链霉菌胰蛋白酶的纯化56
        4.2.5 SDS-PAGE 电泳分析及蛋白浓度测定56
        4.2.6 重组链霉菌胰蛋白酶的最适 pH 及 pH 稳定性56
        4.2.7 重组链霉菌胰蛋白酶的最适温度及温度稳定性56
        4.2.8 金属离子对重组链霉菌胰蛋白酶的影响56-57
        4.2.9 抑制剂和有机溶剂对重组链霉菌胰蛋白酶的影响57
        4.2.10 重组链霉菌胰蛋白酶的酶反应动力学参数57
        4.2.11 重组链霉菌胰蛋白酶蛋白质结构的模拟及分析57
    4.3 结果与讨论57-68
        4.3.1 不同来源的胰蛋白酶的分离纯化及 SDS-PAGE 分析57-58
        4.3.2 pH 对重组链霉菌胰蛋白酶的酶活及稳定性的影响58-60
        4.3.3 温度对重组链霉菌胰蛋白酶的酶活及稳定性的影响60-61
        4.3.4 金属离子对重组链霉菌胰蛋白酶的酶活的影响61-62
        4.3.5 抑制剂和有机溶剂对重组链霉菌胰蛋白酶的酶活的影响62-63
        4.3.6 重组链霉菌胰蛋白酶的酶反应动力学参数63-64
        4.3.7 牛胰蛋白酶和野生链霉菌胰蛋白酶性质差异比较分析64-66
        4.3.8 野生和重组链霉菌胰蛋白酶性质差异比较分析66-68
    4.4 本章小结68-69
第五章 自降解位点 R145 突变体的构建及其催化特性和表达分析69-79
    5.1 前言69
    5.2 材料与方法69-71
        5.2.1 菌株69
        5.2.2 试剂和仪器69
        5.2.3 培养基及培养条件69-70
        5.2.4 重组链霉菌胰蛋白酶 R145 突变体酶活测定70
        5.2.5 重组链霉菌胰蛋白酶 R145 突变体的纯化70
        5.2.6 SDS-PAGE 电泳分析及蛋白浓度测定70
        5.2.7 重组链霉菌胰蛋白酶 R145 突变体的酶反应动力学参数70
        5.2.8 重组链霉菌胰蛋白酶 R145 突变体的抗自降解验证70
        5.2.9 重组链霉菌胰蛋白酶 R145 突变体蛋白质结构的模拟及分析70
        5.2.10 链霉菌胰蛋白酶 R145 突变体的高密度发酵70-71
    5.3 结果与讨论71-78
        5.3.1 重组链霉菌胰蛋白酶 R145 突变菌株的获得及验证71
        5.3.2 野生及重组链霉菌胰蛋白酶菌株的摇瓶发酵比较71-72
        5.3.3 野生及重组链霉菌胰蛋白酶的纯化和 SDS-PAGE 分析72-73
        5.3.4 野生及重组链霉菌胰蛋白酶的酰胺酶和酯酶比酶活的比较73-74
        5.3.5 野生及重组链霉菌胰蛋白酶的酶反应动力学参数的比较74
        5.3.6 野生及重组链霉菌胰蛋白酶的抗自降解比较74-75
        5.3.7 野生及链霉菌胰蛋白酶 Exmt(R145I)的模拟结构比较75-76
        5.3.8 链霉菌胰蛋白酶 Exmt(R145I)重组菌的 3 L 罐高密度发酵76-78
    5.4 本章小结78-79
第六章 链霉菌胰蛋白酶自活化融合突变体的构建及其表达分析79-91
    6.1 前言79-80
    6.2 材料与方法80-81
        6.2.1 菌株80
        6.2.2 试剂和仪器80
        6.2.3 培养基及培养条件80
        6.2.4 胰蛋白酶酶活的测定80
        6.2.5 链霉菌胰蛋白酶突变体的 SDS-PAGE 分析80
        6.2.6 链霉菌胰蛋白酶突变体 VD4Kmt 的体外激活80
        6.2.7 链霉菌胰蛋白酶突变体蛋白质结构的模拟及分析80
        6.2.8 链霉菌胰蛋白酶自活化突变体产生菌的高密度发酵80-81
    6.3 结果与讨论81-90
        6.3.1 链霉菌胰蛋白酶突变体 VD4Kmt 的构建和验证81-82
        6.3.2 杂交链霉菌胰蛋白酶 VD4Kmt 的体外激活82-83
        6.3.3 杂交链霉菌胰蛋白酶 VD4Kmt 的模拟结构分析83-84
        6.3.4 自活化融合突变体 TLmt(D4K)的构建和验证84-85
        6.3.5 自活化融合突变体 TLmt(D4K)的模拟结构分析85-86
        6.3.6 融合突变体 TLmt(D4K)自活化的 SDS-PAGE 验证86-87
        6.3.7 自活化融合突变体的设计及验证87-88
        6.3.8 自活化融合突变体 TLmt(D4K)的 3 L 罐高密度发酵88-90
    6.4 本章小结90-91
主要结论及展望91-93
    主要结论91-92
    展望92-93
论文主要创新点93-94
致谢94-95
参考文献95-102
附录: 作者在攻读博士学位期间发表论文102-103
附录: 英文缩略语注释103-104
附录: 氨基酸名称中英文对照104-105
附录: Britton-Robinson pH 缓冲液配制105


本文地址:

上一篇:普鲁兰酶的产生菌筛选及其表达与分泌调控
下一篇:染色质重塑相关基因对玉米杂种优势作用的研究

分享到: 分享Streptomyces griseus胰蛋白酶的分子改造到腾讯微博           收藏
发表网-Streptomyces griseus胰蛋白酶的分子改造-在线咨询