Streptomyces griseus胰蛋白酶的分子改造
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摘要
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~(–1)wasinduced with20g·L~(–1)methanol for5days at30°C co-feeded with4g·L~(–1)glycerol in shakeflask fermentation. The recombinant P. pastoris GS115/pAOX1/mt had the highest trypsinexpression level of15.6U·mL~(–1)under 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~(–1)towards BPANA.Compared with Exmt and wSGT, it increased1.46-fold and3.61-fold. Moreover, And it hasthe specific activity101491.58±1225.56U·mg~(–1)towards 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~(–1)under 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~(–1)in 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~(–1)with the optimized condition by the high cell density fermentation method in3L fermentor.
（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~(–1)wasinduced with20g·L~(–1)methanol for5days at30°C co-feeded with4g·L~(–1)glycerol in shakeflask fermentation. The recombinant P. pastoris GS115/pAOX1/mt had the highest trypsinexpression level of15.6U·mL~(–1)under 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~(–1)towards BPANA.Compared with Exmt and wSGT, it increased1.46-fold and3.61-fold. Moreover, And it hasthe specific activity101491.58±1225.56U·mg~(–1)towards 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~(–1)under 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~(–1)in 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~(–1)with the optimized condition by the high cell density fermentation method in3L fermentor.
引文
[1] Walsh KA. Trypsinogens and trypsins of various species [J], Methods Enzymol,1970,19:41-63.
[2] Muhlia-Almazan A, Sanchez-Paz A&Garcia-Carreno FL. Invertebrate trypsins: a review [J], JComp Physiol B,2008,178(6):655-672.
[3] Stroud RM, Kossiakoff AA&Chambers JL. Mechanisms of zymogen activation [J], Annu RevBiophys Bioeng,1977,6:177-193.
[4] Lopes AR, Juliano MA, Juliano L, et al. Coevolution of insect trypsins and inhibitors [J], ArchInsect Biochem Physiol,2004,55(3):140-152.
[5] Kato JY, Chi WJ, Ohnishi Y, et al. Transcriptional control by A-factor of two trypsin genes inStreptomyces griseus [J], J Bacteriol,2005,187(1):286-295.
[6] Olsen JV, Ong SE&Mann M. Trypsin cleaves exclusively C-terminal to arginine and lysineresidues [J], Mol Cel Proteomics,2004,3(6):608-614.
[7] Erlanger BF, Kokowsky N&Cohen W. The preparation and properties of two new chromogenicsubstrates of trypsin [J], Arch Biochem Biophys,1961,95:271-278.
[8] Zugno LA. The Effect of Trypsin on Soaking of Salt Cured Hides [J], J Am Leather Chem As,1992,87(6):207-220.
[9] Gudmundsdottir A, Hilmarsson H&Stefansson B. Potential Use of Atlantic Cod Trypsin inBiomedicine [J], Biomed Res Int,2013.
[10] Torrissen KR&Shearer KD. protein Digestion, Growth and Food Conversion in Atlantic Salmonand Arctic Charr with Different Trypsin-Like Isozyme Patterns [J], J Fish Biology,1992,41(3):409-415.
[11] Trop M&Birk Y. The trypsin-like enzyme from Streptomyces griseus (pronase)[J], Biochem J,1968,109(3):475-476.
[12] Trop M&Birk Y. The specificity of proteinases from Streptomyces griseus (pronase)[J], BiochemJ,1970,116(1):19-25.
[13] Hohenblum H, Vorauer-Uhl K, Katinger H, et al. Bacterial expression and refolding of humantrypsinogen [J], J Biotechnol,2004,109(1-2):3-11.
[14] Yao LY, Man CX, Zhao F, et al. Expression of bovine trypsin in Lactococcus lactis [J], Int Dairy J,2010,20(11):806-809.
[15] Hanquier J, Sorlet Y, Desplancq D, et al. A single mutation in the activation site of bovinetrypsinogen enhances its accumulation in the fermentation broth of the yeast Pichia pastoris [J],Appl Environ Microbiol,2003,69(2):1108-1113.
[16] Kim J-H&Hong S-K. Overproduction of Bacterial Trypsin in Streptomyces-Optimization forStreptomyces griseus Trypsin production by Recombinant Streptomyces [J], Kor J MicrobiolBiotechnol,2008,36(1):28-33.
[17] Page MJ, Wong SL, Hewitt J, et al. Engineering the primary substrate specificity of Streptomycesgriseus trypsin [J], Biochemistry,2003,42(30):9060-9066.
[18] Kim JC, Cha SH, Jeong ST, et al. Molecular cloning and nucleotide sequence of Streptomycesgriseus trypsin gene [J], Biochem Biophys Res Commun,1991,181(2):707-713.
[19] Olafson RW, Jurasek L, Carpenter MR, et al. Amino acid sequence of Streptomyces griseus trypsin.Cyanogen bromide fragments and complete sequence [J], Biochemistry,1975,14(6):1168-1177.
[20] Read RJ&James MN. Refined crystal structure of Streptomyces griseus trypsin at1.7A resolution[J], J Mol Biol,1988,200(3):523-551.
[21] Kraut J. Serine proteases: structure and mechanism of catalysis [J], Annu Rev Biochem,1977,46:331-358.
[22] Weiner SJ, Seibel GL&Kollman PA. The nature of enzyme catalysis in trypsin [J], Proc Natl AcadSci U S A,1986,83(3):649-653.
[23] Bode W. The transition of bovine trypsinogen to a trypsin-like state upon strong ligand binding. II.The binding of the pancreatic trypsin inhibitor and of isoleucine-valine and of sequentially relatedpeptides to trypsinogen and to p-guanidinobenzoate-trypsinogen [J], J Mol Biol,1979,127(4):357-374.
[24] Bode W, Schwager P&Huber R. The transition of bovine trypsinogen to a trypsin-like state uponstrong ligand binding. The refined crystal structures of the bovine trypsinogen-pancreatic trypsininhibitor complex and of its ternary complex with Ile-Val at1.9resolution [J], J Mol Biol,1978,118(1):99-112.
[25] Higaki JN, Gibson BW&Craik CS. Evolution of catalysis in the serine proteases [J], Cold SpringHarb Symp Quant Biol,1987,52:615-621.
[26] Craik CS, Largman C, Fletcher T, et al. Redesigning trypsin: alteration of substrate specificity [J],Science,1985,228(4697):291-297.
[27] Stratowa C&Rutter WJ. Selective regulation of trypsin gene expression by calcium and byglucose starvation in a rat exocrine pancreas cell line [J], Proc Natl Acad Sci U S A,1986,83(12):4292-4296.
[28] Vasquez JR, Evnin LB, Higaki JN, et al. An expression system for trypsin [J], J Cell Biochem,1989,39(3):265-276.
[29] Higaki JN, Evnin LB&Craik CS. Introduction of a cysteine protease active site into trypsin [J],Biochemistry,1989,28(24):9256-9263.
[30] Yee L&Blanch HW. Recombinant trypsin production in high cell density fed-batch cultures inEscherichia coli [J], Biotechnol Bioeng,1993,41(8):781-790.
[31]干路.胰蛋白酶非保守性二硫键C22-C157与C129-C232的重要性研究[D]:[硕士学位论文].北京大学,1998
[32]王军.鼠胰蛋白酶二硫键Cys129-Cys232的定位改造及性质研究[D]:[硕士学位论文].北京大学,1995
[33] Li XF, Tang JG&Zhang LX. Autolytic site mutant R105C of rat trypsin [J], Biochem Mol Biol Int,1997,42(5):991-996.
[34] Sahin-Toth M, Graf L&Toth M. Trypsinogen stabilization by mutation Arg117->His: a unifyingpathomechanism for hereditary pancreatitis?[J], Biochem Biophys Res Commun,1999,264(2):505-508.
[35] Szilagyi L, Kenesi E, Katona G, et al. Comparative in vitro studies on native and recombinanthuman cationic trypsins. Cathepsin B is a possible pathological activator of trypsinogen inpancreatitis [J], J Biol Chem,2001,276(27):24574-24580.
[36] Chen JM&Ferec C. Genes, cloned cDNAs, and proteins of human trypsinogens andpancreatitis-associated cationic trypsinogen mutations [J], Pancreas,2000,21(1):57-62.
[37] Feng W&Zhang S. A trypsin homolog in amphioxus: expression, enzymatic activity andevolution [J], Mol Biol Rep,2012,39(2):1745-1753.
[38] Jonsdottir G, Bjarnason JB&Gudmundsdottir A. Recombinant cold-adapted trypsin I fromAtlantic cod-expression, purification, and identification [J], Protein Expr Purif,2004,33(1):110-122.
[39] Palsdottir HM&Gudmundsdottir A. Expression and purification of a cold-adapted group IIItrypsin in Escherichia coli [J], Protein Expr Purif,2007,51(2):243-252.
[40] Caspeta L, Flores N, Perez NO, et al. The effect of heating rate on Escherichia coli metabolism,physiological stress, transcriptional response, and production of temperature-induced recombinantprotein: a scale-down study [J], Biotechnol Bioeng,2009,102(2):468-482.
[41] Hoffmann F&Rinas U. Stress induced by recombinant protein production in Escherichia coli [J],Adv Biochem Eng Biotechnol,2004,89:73-92.
[42] Wang ECW, Hung SH, Cahoon M, et al. The role of the Cys191-Cys220disulfide bond in trypsin:New targets for engineering substrate specificity [J], Protein Eng,1997,10(4):405-411.
[43] Niles AL, Maffitt M, Haak-Frendscho M, et al. Recombinant human mast cell tryptase beta: stableexpression in Pichia pastoris and purification of fully active enzyme [J], Biotechnol Appl Biochem,1998,28(Pt2):125-131.
[44] Macouzet M, Simpson BK&Lee BH. Expression of a cold-adapted fish trypsin in Pichia pastoris[J], FEMS Yeast Res,2005,5(9):851-857.
[45] Guerrero-Olazaran M, Escamilla-Trevino LL, Castillo-Galvan M, et al. Recombinant Shrimp(Litopenaeus vannamei) Trypsinogen production in Pichia pastoris [J], Biotechnol Progr,2009,25(5):1310-1316.
[46] Viader-Salvado JM, Fuentes-Garibay JA, Galan-Wong LJ, et al. Recombinant shrimp trypsinogenproduction in Pichia pastoris [J], J Biotechnol,2005,118:S40-S40.
[47] Kim NS, Yu HY, Chung ND, et al. production of functional recombinant bovine trypsin intransgenic rice cell suspension cultures [J], Protein Expr Purif,2011,76(1):121-126.
[48] Rypniewski WR, Perrakis A, Vorgias CE, et al. Evolutionary divergence and conservation oftrypsin [J], protein Eng,1994,7(1):57-64.
[49] Barata RA, Andrade MH, Rodrigues RD, et al. Purification and characterization of an extracellulartrypsin-like protease of Fusarium oxysporum var. lini [J], J Biosci Bioeng,2002,94(4):304-308.
[50] Nagaminenatsuka Y, Norioka S&Sakiyama F. Molecular-Cloning, Nucleotide-Sequence, andExpression of the Gene Encoding a Trypsin-Like protease from Streptomyces Erythraeus [J], JBiochem,1995,118(2):338-346.
[51] Meng K, Li J, Cao Y, et al. Gene cloning and heterologous expression of a serine protease fromStreptomyces fradiae var.k11[J], Can J Microbiol,2007,53(2):186-195.
[52] Li J, Shi PJ, Zhang WZ, et al. Gene cloning and expression of serine protease SFP2fromStreptomyces fradiae var. k11[J], Sheng Wu Gong Cheng Xue Bao,2005,21(5):782-788.
[53] Olafson RW&Smillie LB. Enzymic and physicochemical properties of Streptomyces griseustrypsin [J], Biochemistry,1975,14(6):1161-1167.
[54] Ohnishi Y, Ishikawa J, Hara H, et al. Genome sequence of the streptomycin-producingmicroorganism Streptomyces griseus IFO13350[J], J Bacteriol,2008,190(11):4050-4060.
[55] Li J, Shi PJ, Han XY, et al. Functional expression of the keratinolytic serine protease gene sfp2from Streptomyces fradiae var. k11in Pichia pastoris [J], protein Expr Purif,2007,54(1):79-86.
[56] Koo BJ, Bae KH, Byun SM, et al. Purification and characterization of Streptomyces griseus trypsinoverexpressed in Streptomyces lividans [J], J Microbiol Biotechnol,1998,8(4):333-340.
[57] Chi WJ, Song JH, Oh EA, et al. Medium optimization and application of an affinity columnchromatography for streptomyces griseus trypsin production from the recombinant Streptomycesgriseus [J], J Microbiol Biotechnol,2009,19(10):1191-1196.
[58] Oh EA, Kim MS, Chi WJ, et al. Characterization of the sgtR1and sgtR2genes and their role inregulating expression of the sprT gene encoding Streptomyces griseus trypsin [J], FEMS MicrobiolLett,2007,276(1):75-82.
[59] Nohara D, Sugiura H, Sakakibara H, et al. High performance in refolding of Streptomyces griseustrypsin by the aid of a mutant of Streptomyces subtilisin inhibitor designed as trypsin inhibitor [J],J Biochem,1999,125(2):343-347.
[60] Choi JH&Lee SY. Secretory and extracellular production of recombinant proteins usingEscherichia coli [J], Appl Microbiol Biotechnol,2004,64(5):625-635.
[61] Baneyx F&Mujacic M. Recombinant protein folding and misfolding in Escherichia coli [J], NatBiotechnol,2004,22(11):1399-1408.
[62] Salamin K, Sriranganadane D, Lechenne B, et al. Secretion of an Endogenous Subtilisin by Pichiapastoris Strains GS115and KM71[J], Appl Environ Microbiol,2010,76(13):4269-4276.
[63] Jiang X, Zhang M, Ding Y, et al. Escherichia coli prlC gene encodes a trypsin-like proteinaseregulating the cell cycle [J], J Biochem,1998,124(5):980-985.
[64] Abita JP, Delaage M&Lazdunski M. The mechanism of activation of trypsinogen. The role of thefour N-terminal aspartyl residues [J], Eur J Biochem,1969,8(3):314-324.
[65] Davie E&Neurath H. Identification of a peptide released during autocatalytic activation oftrypsinogen [J], J Biol Chem,1955,212:515-530.
[66] Yamashina I. The action of enterokinase on trypsinogen [J], Biochim Biophys Acta,1956,20(2):739-743.
[67] Kato JY, Chi WJ, Ohnishi Y, et al. Transcriptional control by A-factor of two trypsin genes inStreptomyces griseus [J], J Bacteriol,2005,187(1):286-295.
[68] Shinde U, Fu X&Inouye M. A pathway for conformational diversity in proteins mediated byintramolecular chaperones [J], J Biol Chem,1999,274(22):15615-11521.
[69] Graycar TP, Bott RR, Caldwell RM, et al. Altering the proteolytic Activity of Subtilisin throughprotein Engineering [J], Ann N Y Acad Sci,1992,672:71-79.
[70] Takagi H, Matsuzawa H, Ohta T, et al. Studies on the Structure and Function of Subtilisin E byprotein Engineering [J], Ann N Y Acad Sci,1992,672:52-59.
[71] Bryan PN. protein engineering of subtilisin [J], Biochim Biophys Acta-protein Structure andMolecular Enzymology,2000,1543(2):203-222.
[72] Briand L, Chobert JM, Gantier R, et al. Impact of the lysine-188and aspartic acid-189inversionon activity of trypsin [J], FEBS Lett,1999,442(1):43-47.
[73] Uesugi Y, Usuki H, Iwabuchi M, et al. The role of Tyr71in Streptomyces trypsin on therecognition mechanism of structural protein substrates [J], FEBS J,2009,276(19):5634-5646.
[74] Page MJ, Bleackley MR, Wong S, et al. Conversion of trypsin into a Na+-activated enzyme [J],Biochemistry,2006,45(9):2987-2993.
[75] Lee WS, Park CH&Byun SM. Streptomyces griseus trypsin is stabilized against autolysis by thecooperation of a salt bridge and cation-pi interaction [J], J Biochem,2004,135(1):93-99.
[76] Li XF, Nie X&Tang JG. Anti-autolysis of trypsin by modification of autolytic site Arg117[J],Biochem Biophys Res Commun,1998,250(2):235-239.
[77] Cregg JM, Cereghino JL, Shi J, et al. Recombinant protein expression in Pichia pastoris [J], MolBiotechnol,2000,16(1):23-52.
[78] Cereghino GP, Cereghino JL, Ilgen C, et al. production of recombinant proteins in fermentercultures of the yeast Pichia pastoris [J], Curr Opin Biotechnol,2002,13(4):329-332.
[79] Kim SJ, Lee JA, Kim YH, et al. Optimization of the functional expression of Coprinus cinereusperoxidase in Pichia pastoris by varying the host and promoter [J], J Microbiol Biotechnol,2009,19(9):966-971.
[80] Daly R&Hearn MT. Expression of heterologous proteins in Pichia pastoris: a useful experimentaltool in protein engineering and production [J], J Mol Recognit,2005,18(2):119-138.
[81] Macauley-Patrick S, Fazenda ML, McNeil B, et al. Heterologous protein production using thePichia pastoris expression system [J], Yeast,2005,22(4):249-270.
[82] Arnau C, Ramon R, Casas C, et al. Optimization of the heterologous production of a Rhizopusoryzae lipase in Pichia pastoris system using mixed substrates on controlled fed-batch bioprocess[J], Enzyme and Microb Technol,2010,46(6):494-500.
[83] Jungo C, Marison I&von Stockar U. Mixed feeds of glycerol and methanol can improve theperformance of Pichia pastoris cultures: A quantitative study based on concentration gradients intransient continuous cultures [J], J Biotechnol,2007,128(4):824-837.
[84] Ramchuran SO, Mateus B, Holst O, et al. The methylotrophic yeast Pichia pastoris as a host forthe expression and production of thermostable xylanase from the bacterium Rhodothermusmarinus [J], FEMS Yeast Res,2005,5(9):839-850.
[85] Oka C, Tanaka M, Muraki M, et al. Human lysozyme secretion increased by alpha-factorpro-sequence in Pichia pastoris [J], Biosci Biotechnol Biochem,1999,63(11):1977-1983.
[86] Vadhana AK, Samuel P, Berin RM, et al. Improved secretion of Candida antarctica lipase B withits native signal peptide in Pichia pastoris [J], Enzyme Microb Technol,2013,52(3):177-183.
[87] Hartner FS&Glieder A. Regulation of methanol utilisation pathway genes in yeasts [J], MicrobCell Fact,2006,5:39.
[88] Wang Z, Wang Y, Zhang D, et al. Enhancement of cell viability and alkaline polygalacturonatelyase production by sorbitol co-feeding with methanol in Pichia pastoris fermentation [J],Bioresour Technol,2010,101(4):1318-1323.
[89] Wang Y, Wang Z, Du G, et al. Enhancement of alkaline polygalacturonate lyase production inrecombinant Pichia pastoris according to the ratio of methanol to cell concentration [J], BioresourTechnol,2009,100(3):1343-1349.
[90] Inan M&Meagher MM. Non-repressing carbon sources for alcohol oxidase (AOX1) promoter ofPichia pastoris [J], J Biosci Bioeng,2001,92(6):585-589.
[91] Ohashi R, Mochizuki E&Suzuki T. A mini-scale mass production and separation system forsecretory heterologous proteins by perfusion culture of recombinant Pichia pastoris using a shakenceramic membrane flask [J], J Biosci Bioeng,1999,87(5):655-660.
[92] Werten MW&de Wolf FA. Reduced proteolysis of secreted gelatin and Yps1-mediatedalpha-factor leader processing in a Pichia pastoris kex2disruptant [J], Appl Environ Microbiol,2005,71(5):2310-2317.
[93] Baumann K, Carnicer M, Dragosits M, et al. A multi-level study of recombinant Pichia pastoris indifferent oxygen conditions [J], BMC Syst Biol,2010,4:141.
[94] Chen JM, Kukor Z, Le Marechal U, et al. Evolution of trypsinogen activation peptides [J], MolecBiol Evol,2003,20(11):1767-1777.
[95] Chen JM, Kukor Z, Le Marechal C, et al. Understanding the evolution of trypsinogen activationpeptides through integration of functional characterization of disease-associated mutations withcomparative genomic analysis.[J], Am J Hum Genet,2003,73(5):430-430.
[96] Phillips JC, Braun R, Wang W, et al. Scalable molecular dynamics with NAMD [J], J ComputChem,2005,26(16):1781-1802.
[97] Brake AJ, Merryweather JP, Coit DG, et al. Alpha-factor-directed synthesis and secretion of matureforeign proteins in Saccharomyces cerevisiae [J], Proc Natl Acad Sci U S A,1984,81(15):4642-4646.
[98] Vadhana AKP, Samuel P, Berin RM, et al. Improved secretion of Candida antarctica lipase B withits native signal peptide in Pichia pastoris [J], Enzyme Microb Technol,2013,52(3):177-183.
[99] Zhang Z, Norris J, Schwartz C, et al. In silico and in vitro investigations of the mutability ofdisease-causing missense mutation sites in spermine synthase [J], PLoS One,2011,6(5):e20373.
[100] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of proteinutilizing the principle of protein-dye binding [J], Anal Biochem,1976,72:248-254.
[101] Jiang W, Hardy DJ, Phillips JC, et al. High-performance scalable molecular dynamics simulationsof a polarizable force field based on classical Drude oscillators in NAMD [J], J Phys Chem Lett,2011,2(2):87-92.
[102] Gallivan JP&Dougherty DA. Cation-π interactions in structural biology [J], Proc Natl Acad Sci US A,1999,96(17):9459-9464.
[103] Brandsdal BO, Aqvist J&Smalas AO. Computational analysis of binding of P1variants to trypsin[J], Protein Sci,2001,10(8):1584-1595.
[104] Liener IE. The essentiality of the disulfide linkages in trypsin [J], J Biol Chem,1957,225(2):1061-1069.
[105] Huber R&Bode W. Structural Basis of the Activation and Action of Trypsin [J], Accounts ChemRes1978,11:114-122.
[106] Shinde U&Inouye M. Intramolecular chaperones: polypeptide extensions that modulate proteinfolding [J], Semin Cell Dev Biol,2000,11(1):35-44.
[107] Zhao X, Nagai Y, Reeves PJ, et al. Designer short peptide surfactants stabilize G protein-coupledreceptor bovine rhodopsin [J], Proc Natl Acad Sci U S A,2006,103(47):17707-17712.
[108] Fehlhammer H, Bode W&Huber R. Crystal structure of bovine trypsinogen at1-8A resolution. II.Crystallographic refinement, refined crystal structure and comparison with bovine trypsin [J], JMol Biol,1977,111(4):415-438.
[109] Ge B, Yang F, Yu D, et al. Designer amphiphilic short peptides enhance thermal stability ofisolated photosystem-I [J], PLoS One,2010,5(4):e10233.
[110] Qi X, Guo Q, Wei Y, et al. Enhancement of pH stability and activity of glycerol dehydratase fromKlebsiella pneumoniae by rational design [J], Biotechnol Lett,2012,34(2):339-346.
[111] Park HS, Nam SH, Lee JK, et al. Design and evolution of new catalytic activity with an existingprotein scaffold [J], Science,2006,311(5760):535-538.
[112] Hu HY, Yang JC, Chen JH, et al. Enzymatic characterization of Bacillus licheniformisgamma-glutamyltranspeptidase fused with N-terminally truncated forms of Bacillus sp. TS-23alpha-amylase [J], Enzyme Microb Technol,2012,51(2):86-94.
[113] Sekizaki H, Itoh K, Murakami M, et al. Anionic trypsin from chum salmon: activity withp-amidinophenyl ester and comparison with bovine and Streptomyces griseus trypsins [J], CompBiochem Physiol B-Biochem Mol Biol,2000,127(3):337-346.
[114] Mosbah H, Horchani H, Sayari A, et al. The insertion of (LK) residues at the N-terminus ofStaphylococcus xylosus lipase affects its catalytic properties and its enantioselectivity [J], ProcessBiochem,2010,45(5):777-785.
[115] Charbonneau DM, Meddeb-Mouelhi F&Beauregard M. N-terminal purification tag alters thermalstability of the carboxylesterase EstGtA2from G. thermodenitrificans by impairing reversibility ofthermal unfolding [J], Protein Pept Lett,2012,19(3):264-269.
[116] Green NM&Neurath H. The effects of divalent cations on trypsin [J], J Biol Chem,1953,204(1):379-390.
[117] Tanizawa K, Nakano M&Kanaoka Y. Comparison of Streptomyces griseus and bovine trypsin byactive site analysis using fluorescent acyl groups [J], Biochim Biophys Acta,1987,913(3):292-299.
[118] Yokoyama T, Mizuguchi M, Nabeshima Y, et al. Hydrogen-bond network and pH sensitivity intransthyretin: Neutron crystal structure of human transthyretin [J], J Struct Biol,2012,177(2):283-290.
[119] Hedstrom L, Szilagyi L&Rutter WJ. Converting trypsin to chymotrypsin: the role of surface loops[J], Science,1992,255(5049):1249-1253.
[120] Vogt G&Argos P. protein thermal stability: hydrogen bonds or internal packing?[J], Fold Des,1997,2(4):S40-S46.
[121] Huang SY, Zhang YH&Zhong JJ. A thermostable recombinant transaldolase with high activityover a broad pH range [J], Appl Microbiol Biotechnol,2012,93(6):2403-2410.
[122] Dougherty DA. Cation-π interactions in chemistry and biology: a new view of benzene, Phe, Tyr,and Trp [J], Science,1996,271(5246):163-168.
[123] Kumar S, Tsai CJ&Nussinov R. Factors enhancing protein thermostability [J], Protein Eng,2000,13(3):179-191.
[124] Korkegian A, Black ME, Baker D, et al. Computational thermostabilization of an enzyme [J],Science,2005,308(5723):857-860.
[125] Gilis D&Rooman M. PoPMuSiC, an algorithm for predicting protein mutant stability changes:application to prion proteins [J], Protein Eng,2000,13(12):849-856.
[126] Kwasigroch JM, Gilis D, Dehouck Y, et al. PoPMuSiC, rationally designing point mutations inprotein structures [J], Bioinformatics,2002,18(12):1701-1702.
[127] Parthiban V, Gromiha MM&Schomburg D. CUPSAT: prediction of protein stability upon pointmutations [J], Nucleic Acids Res,2006,34:W239-W242.
[128] Dehouck Y, Kwasigroch JM, Gilis D, et al. PoPMuSiC2.1: a web server for the estimation ofprotein stability changes upon mutation and sequence optimality [J], BMC Bioinformatics,2011,12:151.
[129] Sinha J, Plantz BA, Inan M, et al. Causes of proteolytic degradation of secreted recombinantproteins produced in methylotrophic yeast Pichia pastoris: case study with recombinant ovineinterferon-tau [J], Biotechnol Bioeng,2005,89(1):102-112.
[130] Kay J&Kassell B. The autoactivation of trypsinogen [J], J Biol Chem,1971,246(21):6661-6665.
[2] Muhlia-Almazan A, Sanchez-Paz A&Garcia-Carreno FL. Invertebrate trypsins: a review [J], JComp Physiol B,2008,178(6):655-672.
[3] Stroud RM, Kossiakoff AA&Chambers JL. Mechanisms of zymogen activation [J], Annu RevBiophys Bioeng,1977,6:177-193.
[4] Lopes AR, Juliano MA, Juliano L, et al. Coevolution of insect trypsins and inhibitors [J], ArchInsect Biochem Physiol,2004,55(3):140-152.
[5] Kato JY, Chi WJ, Ohnishi Y, et al. Transcriptional control by A-factor of two trypsin genes inStreptomyces griseus [J], J Bacteriol,2005,187(1):286-295.
[6] Olsen JV, Ong SE&Mann M. Trypsin cleaves exclusively C-terminal to arginine and lysineresidues [J], Mol Cel Proteomics,2004,3(6):608-614.
[7] Erlanger BF, Kokowsky N&Cohen W. The preparation and properties of two new chromogenicsubstrates of trypsin [J], Arch Biochem Biophys,1961,95:271-278.
[8] Zugno LA. The Effect of Trypsin on Soaking of Salt Cured Hides [J], J Am Leather Chem As,1992,87(6):207-220.
[9] Gudmundsdottir A, Hilmarsson H&Stefansson B. Potential Use of Atlantic Cod Trypsin inBiomedicine [J], Biomed Res Int,2013.
[10] Torrissen KR&Shearer KD. protein Digestion, Growth and Food Conversion in Atlantic Salmonand Arctic Charr with Different Trypsin-Like Isozyme Patterns [J], J Fish Biology,1992,41(3):409-415.
[11] Trop M&Birk Y. The trypsin-like enzyme from Streptomyces griseus (pronase)[J], Biochem J,1968,109(3):475-476.
[12] Trop M&Birk Y. The specificity of proteinases from Streptomyces griseus (pronase)[J], BiochemJ,1970,116(1):19-25.
[13] Hohenblum H, Vorauer-Uhl K, Katinger H, et al. Bacterial expression and refolding of humantrypsinogen [J], J Biotechnol,2004,109(1-2):3-11.
[14] Yao LY, Man CX, Zhao F, et al. Expression of bovine trypsin in Lactococcus lactis [J], Int Dairy J,2010,20(11):806-809.
[15] Hanquier J, Sorlet Y, Desplancq D, et al. A single mutation in the activation site of bovinetrypsinogen enhances its accumulation in the fermentation broth of the yeast Pichia pastoris [J],Appl Environ Microbiol,2003,69(2):1108-1113.
[16] Kim J-H&Hong S-K. Overproduction of Bacterial Trypsin in Streptomyces-Optimization forStreptomyces griseus Trypsin production by Recombinant Streptomyces [J], Kor J MicrobiolBiotechnol,2008,36(1):28-33.
[17] Page MJ, Wong SL, Hewitt J, et al. Engineering the primary substrate specificity of Streptomycesgriseus trypsin [J], Biochemistry,2003,42(30):9060-9066.
[18] Kim JC, Cha SH, Jeong ST, et al. Molecular cloning and nucleotide sequence of Streptomycesgriseus trypsin gene [J], Biochem Biophys Res Commun,1991,181(2):707-713.
[19] Olafson RW, Jurasek L, Carpenter MR, et al. Amino acid sequence of Streptomyces griseus trypsin.Cyanogen bromide fragments and complete sequence [J], Biochemistry,1975,14(6):1168-1177.
[20] Read RJ&James MN. Refined crystal structure of Streptomyces griseus trypsin at1.7A resolution[J], J Mol Biol,1988,200(3):523-551.
[21] Kraut J. Serine proteases: structure and mechanism of catalysis [J], Annu Rev Biochem,1977,46:331-358.
[22] Weiner SJ, Seibel GL&Kollman PA. The nature of enzyme catalysis in trypsin [J], Proc Natl AcadSci U S A,1986,83(3):649-653.
[23] Bode W. The transition of bovine trypsinogen to a trypsin-like state upon strong ligand binding. II.The binding of the pancreatic trypsin inhibitor and of isoleucine-valine and of sequentially relatedpeptides to trypsinogen and to p-guanidinobenzoate-trypsinogen [J], J Mol Biol,1979,127(4):357-374.
[24] Bode W, Schwager P&Huber R. The transition of bovine trypsinogen to a trypsin-like state uponstrong ligand binding. The refined crystal structures of the bovine trypsinogen-pancreatic trypsininhibitor complex and of its ternary complex with Ile-Val at1.9resolution [J], J Mol Biol,1978,118(1):99-112.
[25] Higaki JN, Gibson BW&Craik CS. Evolution of catalysis in the serine proteases [J], Cold SpringHarb Symp Quant Biol,1987,52:615-621.
[26] Craik CS, Largman C, Fletcher T, et al. Redesigning trypsin: alteration of substrate specificity [J],Science,1985,228(4697):291-297.
[27] Stratowa C&Rutter WJ. Selective regulation of trypsin gene expression by calcium and byglucose starvation in a rat exocrine pancreas cell line [J], Proc Natl Acad Sci U S A,1986,83(12):4292-4296.
[28] Vasquez JR, Evnin LB, Higaki JN, et al. An expression system for trypsin [J], J Cell Biochem,1989,39(3):265-276.
[29] Higaki JN, Evnin LB&Craik CS. Introduction of a cysteine protease active site into trypsin [J],Biochemistry,1989,28(24):9256-9263.
[30] Yee L&Blanch HW. Recombinant trypsin production in high cell density fed-batch cultures inEscherichia coli [J], Biotechnol Bioeng,1993,41(8):781-790.
[31]干路.胰蛋白酶非保守性二硫键C22-C157与C129-C232的重要性研究[D]:[硕士学位论文].北京大学,1998
[32]王军.鼠胰蛋白酶二硫键Cys129-Cys232的定位改造及性质研究[D]:[硕士学位论文].北京大学,1995
[33] Li XF, Tang JG&Zhang LX. Autolytic site mutant R105C of rat trypsin [J], Biochem Mol Biol Int,1997,42(5):991-996.
[34] Sahin-Toth M, Graf L&Toth M. Trypsinogen stabilization by mutation Arg117->His: a unifyingpathomechanism for hereditary pancreatitis?[J], Biochem Biophys Res Commun,1999,264(2):505-508.
[35] Szilagyi L, Kenesi E, Katona G, et al. Comparative in vitro studies on native and recombinanthuman cationic trypsins. Cathepsin B is a possible pathological activator of trypsinogen inpancreatitis [J], J Biol Chem,2001,276(27):24574-24580.
[36] Chen JM&Ferec C. Genes, cloned cDNAs, and proteins of human trypsinogens andpancreatitis-associated cationic trypsinogen mutations [J], Pancreas,2000,21(1):57-62.
[37] Feng W&Zhang S. A trypsin homolog in amphioxus: expression, enzymatic activity andevolution [J], Mol Biol Rep,2012,39(2):1745-1753.
[38] Jonsdottir G, Bjarnason JB&Gudmundsdottir A. Recombinant cold-adapted trypsin I fromAtlantic cod-expression, purification, and identification [J], Protein Expr Purif,2004,33(1):110-122.
[39] Palsdottir HM&Gudmundsdottir A. Expression and purification of a cold-adapted group IIItrypsin in Escherichia coli [J], Protein Expr Purif,2007,51(2):243-252.
[40] Caspeta L, Flores N, Perez NO, et al. The effect of heating rate on Escherichia coli metabolism,physiological stress, transcriptional response, and production of temperature-induced recombinantprotein: a scale-down study [J], Biotechnol Bioeng,2009,102(2):468-482.
[41] Hoffmann F&Rinas U. Stress induced by recombinant protein production in Escherichia coli [J],Adv Biochem Eng Biotechnol,2004,89:73-92.
[42] Wang ECW, Hung SH, Cahoon M, et al. The role of the Cys191-Cys220disulfide bond in trypsin:New targets for engineering substrate specificity [J], Protein Eng,1997,10(4):405-411.
[43] Niles AL, Maffitt M, Haak-Frendscho M, et al. Recombinant human mast cell tryptase beta: stableexpression in Pichia pastoris and purification of fully active enzyme [J], Biotechnol Appl Biochem,1998,28(Pt2):125-131.
[44] Macouzet M, Simpson BK&Lee BH. Expression of a cold-adapted fish trypsin in Pichia pastoris[J], FEMS Yeast Res,2005,5(9):851-857.
[45] Guerrero-Olazaran M, Escamilla-Trevino LL, Castillo-Galvan M, et al. Recombinant Shrimp(Litopenaeus vannamei) Trypsinogen production in Pichia pastoris [J], Biotechnol Progr,2009,25(5):1310-1316.
[46] Viader-Salvado JM, Fuentes-Garibay JA, Galan-Wong LJ, et al. Recombinant shrimp trypsinogenproduction in Pichia pastoris [J], J Biotechnol,2005,118:S40-S40.
[47] Kim NS, Yu HY, Chung ND, et al. production of functional recombinant bovine trypsin intransgenic rice cell suspension cultures [J], Protein Expr Purif,2011,76(1):121-126.
[48] Rypniewski WR, Perrakis A, Vorgias CE, et al. Evolutionary divergence and conservation oftrypsin [J], protein Eng,1994,7(1):57-64.
[49] Barata RA, Andrade MH, Rodrigues RD, et al. Purification and characterization of an extracellulartrypsin-like protease of Fusarium oxysporum var. lini [J], J Biosci Bioeng,2002,94(4):304-308.
[50] Nagaminenatsuka Y, Norioka S&Sakiyama F. Molecular-Cloning, Nucleotide-Sequence, andExpression of the Gene Encoding a Trypsin-Like protease from Streptomyces Erythraeus [J], JBiochem,1995,118(2):338-346.
[51] Meng K, Li J, Cao Y, et al. Gene cloning and heterologous expression of a serine protease fromStreptomyces fradiae var.k11[J], Can J Microbiol,2007,53(2):186-195.
[52] Li J, Shi PJ, Zhang WZ, et al. Gene cloning and expression of serine protease SFP2fromStreptomyces fradiae var. k11[J], Sheng Wu Gong Cheng Xue Bao,2005,21(5):782-788.
[53] Olafson RW&Smillie LB. Enzymic and physicochemical properties of Streptomyces griseustrypsin [J], Biochemistry,1975,14(6):1161-1167.
[54] Ohnishi Y, Ishikawa J, Hara H, et al. Genome sequence of the streptomycin-producingmicroorganism Streptomyces griseus IFO13350[J], J Bacteriol,2008,190(11):4050-4060.
[55] Li J, Shi PJ, Han XY, et al. Functional expression of the keratinolytic serine protease gene sfp2from Streptomyces fradiae var. k11in Pichia pastoris [J], protein Expr Purif,2007,54(1):79-86.
[56] Koo BJ, Bae KH, Byun SM, et al. Purification and characterization of Streptomyces griseus trypsinoverexpressed in Streptomyces lividans [J], J Microbiol Biotechnol,1998,8(4):333-340.
[57] Chi WJ, Song JH, Oh EA, et al. Medium optimization and application of an affinity columnchromatography for streptomyces griseus trypsin production from the recombinant Streptomycesgriseus [J], J Microbiol Biotechnol,2009,19(10):1191-1196.
[58] Oh EA, Kim MS, Chi WJ, et al. Characterization of the sgtR1and sgtR2genes and their role inregulating expression of the sprT gene encoding Streptomyces griseus trypsin [J], FEMS MicrobiolLett,2007,276(1):75-82.
[59] Nohara D, Sugiura H, Sakakibara H, et al. High performance in refolding of Streptomyces griseustrypsin by the aid of a mutant of Streptomyces subtilisin inhibitor designed as trypsin inhibitor [J],J Biochem,1999,125(2):343-347.
[60] Choi JH&Lee SY. Secretory and extracellular production of recombinant proteins usingEscherichia coli [J], Appl Microbiol Biotechnol,2004,64(5):625-635.
[61] Baneyx F&Mujacic M. Recombinant protein folding and misfolding in Escherichia coli [J], NatBiotechnol,2004,22(11):1399-1408.
[62] Salamin K, Sriranganadane D, Lechenne B, et al. Secretion of an Endogenous Subtilisin by Pichiapastoris Strains GS115and KM71[J], Appl Environ Microbiol,2010,76(13):4269-4276.
[63] Jiang X, Zhang M, Ding Y, et al. Escherichia coli prlC gene encodes a trypsin-like proteinaseregulating the cell cycle [J], J Biochem,1998,124(5):980-985.
[64] Abita JP, Delaage M&Lazdunski M. The mechanism of activation of trypsinogen. The role of thefour N-terminal aspartyl residues [J], Eur J Biochem,1969,8(3):314-324.
[65] Davie E&Neurath H. Identification of a peptide released during autocatalytic activation oftrypsinogen [J], J Biol Chem,1955,212:515-530.
[66] Yamashina I. The action of enterokinase on trypsinogen [J], Biochim Biophys Acta,1956,20(2):739-743.
[67] Kato JY, Chi WJ, Ohnishi Y, et al. Transcriptional control by A-factor of two trypsin genes inStreptomyces griseus [J], J Bacteriol,2005,187(1):286-295.
[68] Shinde U, Fu X&Inouye M. A pathway for conformational diversity in proteins mediated byintramolecular chaperones [J], J Biol Chem,1999,274(22):15615-11521.
[69] Graycar TP, Bott RR, Caldwell RM, et al. Altering the proteolytic Activity of Subtilisin throughprotein Engineering [J], Ann N Y Acad Sci,1992,672:71-79.
[70] Takagi H, Matsuzawa H, Ohta T, et al. Studies on the Structure and Function of Subtilisin E byprotein Engineering [J], Ann N Y Acad Sci,1992,672:52-59.
[71] Bryan PN. protein engineering of subtilisin [J], Biochim Biophys Acta-protein Structure andMolecular Enzymology,2000,1543(2):203-222.
[72] Briand L, Chobert JM, Gantier R, et al. Impact of the lysine-188and aspartic acid-189inversionon activity of trypsin [J], FEBS Lett,1999,442(1):43-47.
[73] Uesugi Y, Usuki H, Iwabuchi M, et al. The role of Tyr71in Streptomyces trypsin on therecognition mechanism of structural protein substrates [J], FEBS J,2009,276(19):5634-5646.
[74] Page MJ, Bleackley MR, Wong S, et al. Conversion of trypsin into a Na+-activated enzyme [J],Biochemistry,2006,45(9):2987-2993.
[75] Lee WS, Park CH&Byun SM. Streptomyces griseus trypsin is stabilized against autolysis by thecooperation of a salt bridge and cation-pi interaction [J], J Biochem,2004,135(1):93-99.
[76] Li XF, Nie X&Tang JG. Anti-autolysis of trypsin by modification of autolytic site Arg117[J],Biochem Biophys Res Commun,1998,250(2):235-239.
[77] Cregg JM, Cereghino JL, Shi J, et al. Recombinant protein expression in Pichia pastoris [J], MolBiotechnol,2000,16(1):23-52.
[78] Cereghino GP, Cereghino JL, Ilgen C, et al. production of recombinant proteins in fermentercultures of the yeast Pichia pastoris [J], Curr Opin Biotechnol,2002,13(4):329-332.
[79] Kim SJ, Lee JA, Kim YH, et al. Optimization of the functional expression of Coprinus cinereusperoxidase in Pichia pastoris by varying the host and promoter [J], J Microbiol Biotechnol,2009,19(9):966-971.
[80] Daly R&Hearn MT. Expression of heterologous proteins in Pichia pastoris: a useful experimentaltool in protein engineering and production [J], J Mol Recognit,2005,18(2):119-138.
[81] Macauley-Patrick S, Fazenda ML, McNeil B, et al. Heterologous protein production using thePichia pastoris expression system [J], Yeast,2005,22(4):249-270.
[82] Arnau C, Ramon R, Casas C, et al. Optimization of the heterologous production of a Rhizopusoryzae lipase in Pichia pastoris system using mixed substrates on controlled fed-batch bioprocess[J], Enzyme and Microb Technol,2010,46(6):494-500.
[83] Jungo C, Marison I&von Stockar U. Mixed feeds of glycerol and methanol can improve theperformance of Pichia pastoris cultures: A quantitative study based on concentration gradients intransient continuous cultures [J], J Biotechnol,2007,128(4):824-837.
[84] Ramchuran SO, Mateus B, Holst O, et al. The methylotrophic yeast Pichia pastoris as a host forthe expression and production of thermostable xylanase from the bacterium Rhodothermusmarinus [J], FEMS Yeast Res,2005,5(9):839-850.
[85] Oka C, Tanaka M, Muraki M, et al. Human lysozyme secretion increased by alpha-factorpro-sequence in Pichia pastoris [J], Biosci Biotechnol Biochem,1999,63(11):1977-1983.
[86] Vadhana AK, Samuel P, Berin RM, et al. Improved secretion of Candida antarctica lipase B withits native signal peptide in Pichia pastoris [J], Enzyme Microb Technol,2013,52(3):177-183.
[87] Hartner FS&Glieder A. Regulation of methanol utilisation pathway genes in yeasts [J], MicrobCell Fact,2006,5:39.
[88] Wang Z, Wang Y, Zhang D, et al. Enhancement of cell viability and alkaline polygalacturonatelyase production by sorbitol co-feeding with methanol in Pichia pastoris fermentation [J],Bioresour Technol,2010,101(4):1318-1323.
[89] Wang Y, Wang Z, Du G, et al. Enhancement of alkaline polygalacturonate lyase production inrecombinant Pichia pastoris according to the ratio of methanol to cell concentration [J], BioresourTechnol,2009,100(3):1343-1349.
[90] Inan M&Meagher MM. Non-repressing carbon sources for alcohol oxidase (AOX1) promoter ofPichia pastoris [J], J Biosci Bioeng,2001,92(6):585-589.
[91] Ohashi R, Mochizuki E&Suzuki T. A mini-scale mass production and separation system forsecretory heterologous proteins by perfusion culture of recombinant Pichia pastoris using a shakenceramic membrane flask [J], J Biosci Bioeng,1999,87(5):655-660.
[92] Werten MW&de Wolf FA. Reduced proteolysis of secreted gelatin and Yps1-mediatedalpha-factor leader processing in a Pichia pastoris kex2disruptant [J], Appl Environ Microbiol,2005,71(5):2310-2317.
[93] Baumann K, Carnicer M, Dragosits M, et al. A multi-level study of recombinant Pichia pastoris indifferent oxygen conditions [J], BMC Syst Biol,2010,4:141.
[94] Chen JM, Kukor Z, Le Marechal U, et al. Evolution of trypsinogen activation peptides [J], MolecBiol Evol,2003,20(11):1767-1777.
[95] Chen JM, Kukor Z, Le Marechal C, et al. Understanding the evolution of trypsinogen activationpeptides through integration of functional characterization of disease-associated mutations withcomparative genomic analysis.[J], Am J Hum Genet,2003,73(5):430-430.
[96] Phillips JC, Braun R, Wang W, et al. Scalable molecular dynamics with NAMD [J], J ComputChem,2005,26(16):1781-1802.
[97] Brake AJ, Merryweather JP, Coit DG, et al. Alpha-factor-directed synthesis and secretion of matureforeign proteins in Saccharomyces cerevisiae [J], Proc Natl Acad Sci U S A,1984,81(15):4642-4646.
[98] Vadhana AKP, Samuel P, Berin RM, et al. Improved secretion of Candida antarctica lipase B withits native signal peptide in Pichia pastoris [J], Enzyme Microb Technol,2013,52(3):177-183.
[99] Zhang Z, Norris J, Schwartz C, et al. In silico and in vitro investigations of the mutability ofdisease-causing missense mutation sites in spermine synthase [J], PLoS One,2011,6(5):e20373.
[100] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of proteinutilizing the principle of protein-dye binding [J], Anal Biochem,1976,72:248-254.
[101] Jiang W, Hardy DJ, Phillips JC, et al. High-performance scalable molecular dynamics simulationsof a polarizable force field based on classical Drude oscillators in NAMD [J], J Phys Chem Lett,2011,2(2):87-92.
[102] Gallivan JP&Dougherty DA. Cation-π interactions in structural biology [J], Proc Natl Acad Sci US A,1999,96(17):9459-9464.
[103] Brandsdal BO, Aqvist J&Smalas AO. Computational analysis of binding of P1variants to trypsin[J], Protein Sci,2001,10(8):1584-1595.
[104] Liener IE. The essentiality of the disulfide linkages in trypsin [J], J Biol Chem,1957,225(2):1061-1069.
[105] Huber R&Bode W. Structural Basis of the Activation and Action of Trypsin [J], Accounts ChemRes1978,11:114-122.
[106] Shinde U&Inouye M. Intramolecular chaperones: polypeptide extensions that modulate proteinfolding [J], Semin Cell Dev Biol,2000,11(1):35-44.
[107] Zhao X, Nagai Y, Reeves PJ, et al. Designer short peptide surfactants stabilize G protein-coupledreceptor bovine rhodopsin [J], Proc Natl Acad Sci U S A,2006,103(47):17707-17712.
[108] Fehlhammer H, Bode W&Huber R. Crystal structure of bovine trypsinogen at1-8A resolution. II.Crystallographic refinement, refined crystal structure and comparison with bovine trypsin [J], JMol Biol,1977,111(4):415-438.
[109] Ge B, Yang F, Yu D, et al. Designer amphiphilic short peptides enhance thermal stability ofisolated photosystem-I [J], PLoS One,2010,5(4):e10233.
[110] Qi X, Guo Q, Wei Y, et al. Enhancement of pH stability and activity of glycerol dehydratase fromKlebsiella pneumoniae by rational design [J], Biotechnol Lett,2012,34(2):339-346.
[111] Park HS, Nam SH, Lee JK, et al. Design and evolution of new catalytic activity with an existingprotein scaffold [J], Science,2006,311(5760):535-538.
[112] Hu HY, Yang JC, Chen JH, et al. Enzymatic characterization of Bacillus licheniformisgamma-glutamyltranspeptidase fused with N-terminally truncated forms of Bacillus sp. TS-23alpha-amylase [J], Enzyme Microb Technol,2012,51(2):86-94.
[113] Sekizaki H, Itoh K, Murakami M, et al. Anionic trypsin from chum salmon: activity withp-amidinophenyl ester and comparison with bovine and Streptomyces griseus trypsins [J], CompBiochem Physiol B-Biochem Mol Biol,2000,127(3):337-346.
[114] Mosbah H, Horchani H, Sayari A, et al. The insertion of (LK) residues at the N-terminus ofStaphylococcus xylosus lipase affects its catalytic properties and its enantioselectivity [J], ProcessBiochem,2010,45(5):777-785.
[115] Charbonneau DM, Meddeb-Mouelhi F&Beauregard M. N-terminal purification tag alters thermalstability of the carboxylesterase EstGtA2from G. thermodenitrificans by impairing reversibility ofthermal unfolding [J], Protein Pept Lett,2012,19(3):264-269.
[116] Green NM&Neurath H. The effects of divalent cations on trypsin [J], J Biol Chem,1953,204(1):379-390.
[117] Tanizawa K, Nakano M&Kanaoka Y. Comparison of Streptomyces griseus and bovine trypsin byactive site analysis using fluorescent acyl groups [J], Biochim Biophys Acta,1987,913(3):292-299.
[118] Yokoyama T, Mizuguchi M, Nabeshima Y, et al. Hydrogen-bond network and pH sensitivity intransthyretin: Neutron crystal structure of human transthyretin [J], J Struct Biol,2012,177(2):283-290.
[119] Hedstrom L, Szilagyi L&Rutter WJ. Converting trypsin to chymotrypsin: the role of surface loops[J], Science,1992,255(5049):1249-1253.
[120] Vogt G&Argos P. protein thermal stability: hydrogen bonds or internal packing?[J], Fold Des,1997,2(4):S40-S46.
[121] Huang SY, Zhang YH&Zhong JJ. A thermostable recombinant transaldolase with high activityover a broad pH range [J], Appl Microbiol Biotechnol,2012,93(6):2403-2410.
[122] Dougherty DA. Cation-π interactions in chemistry and biology: a new view of benzene, Phe, Tyr,and Trp [J], Science,1996,271(5246):163-168.
[123] Kumar S, Tsai CJ&Nussinov R. Factors enhancing protein thermostability [J], Protein Eng,2000,13(3):179-191.
[124] Korkegian A, Black ME, Baker D, et al. Computational thermostabilization of an enzyme [J],Science,2005,308(5723):857-860.
[125] Gilis D&Rooman M. PoPMuSiC, an algorithm for predicting protein mutant stability changes:application to prion proteins [J], Protein Eng,2000,13(12):849-856.
[126] Kwasigroch JM, Gilis D, Dehouck Y, et al. PoPMuSiC, rationally designing point mutations inprotein structures [J], Bioinformatics,2002,18(12):1701-1702.
[127] Parthiban V, Gromiha MM&Schomburg D. CUPSAT: prediction of protein stability upon pointmutations [J], Nucleic Acids Res,2006,34:W239-W242.
[128] Dehouck Y, Kwasigroch JM, Gilis D, et al. PoPMuSiC2.1: a web server for the estimation ofprotein stability changes upon mutation and sequence optimality [J], BMC Bioinformatics,2011,12:151.
[129] Sinha J, Plantz BA, Inan M, et al. Causes of proteolytic degradation of secreted recombinantproteins produced in methylotrophic yeast Pichia pastoris: case study with recombinant ovineinterferon-tau [J], Biotechnol Bioeng,2005,89(1):102-112.
[130] Kay J&Kassell B. The autoactivation of trypsinogen [J], J Biol Chem,1971,246(21):6661-6665.