来源于豆豉的枯草芽孢杆菌MX-6所产纳豆激酶的稳定性研究
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摘要
以中国豆豉中筛选到的1株纤溶酶高产菌株———枯草芽孢杆菌MX-6为研究对象,应用聚合酶链式反应(PCR)成功扩增到1473bp的纤溶酶编码基因aprN,系统进化树结果显示该纤溶酶为纳豆激酶。研究了纳豆激酶对热、pH、金属离子、化学物质、抑制剂和变性剂的稳定性及传代稳定性。结果表明:纳豆激酶在-20,4,30～50℃的热稳定性较好,pH在3～11之间酶活性相对稳定,Mg~(2+)、Ca~(2+)对纳豆激酶具有显著的激活作用,K~+、Fe~(2+)对纳豆激酶的稳定性基本无影响,而Mn~(2+)、Zn~(2+)、Cu~(2+)、Al ~(3+)、Ba~(2+)有显著的抑制作用,Hg~(2+)导致纳豆激酶完全失活,牛血清蛋白、明胶、蛋白胨能够显著提高纳豆激酶的稳定性,丙二醇、海藻酸钠对纳豆激酶的稳定性基本无影响,而乙醇显著降低纳豆激酶的稳定性。PMSF导致纳豆激酶的活性完全丧失,EDAT和DTT对纳豆激酶的活性基本无影响,10mmol/L的SDS显著降低纳豆激酶的稳定性,说明此纳豆激酶是一种丝氨酸蛋白酶,不是金属酶,不含二硫键,且具有良好的传代稳定性。结果显示该酶的稳定性较好,具有开发为溶栓药物的应用价值,有利于纳豆激酶的工业化应用。
Bacillus subtilis MX-6,a high-yield strain of fibrinolytic enzyme,is screened from Chinese fermented soya beans.The fibrinolytic gene aprN is successfully amplified to 1473bp by polymerase chain reaction(PCR).The phylogenetic tree results show that the fibrinolytic enzyme is nattokinase.The stability and generation stability of nattokinase to heat,pH,metal ions,chemicals,inhibitors and modifiers are studied.The results show that the thermal stability of nattokinase is good at-20,4,30～50℃,and the activity of nattokinase is relatively stable at pH 3～11.Mg~(2+),Ca~(2+)have significant activation effect on nattokinase.K~+,Fe~(2+)have little effect on the stability of nattokinase,while Mn~(2+),Zn~(2+),Cu~(2+),Al ~(3+),Ba~(2+)have significant inhibition effect.Hg~(2+)leads to complete inactivation of nattokinase,bovine serum albumin,gelatin and peptone can significantly improve the stability of nattokinase,propylene glycol and sodium alginate have little effect on the stability of nattokinase,while ethanol significantly reduces the stability of nattokinase.PMSF results in the complete loss of nattokinase activity.EDAT and DTT have little effect on the activity of nattokinase.10mmol/L SDS significantly reduces the stability of nattokinase,which indicates that the nattokinase is a serine protease,not a metalloenzyme,does not contain disulfide bond,and has good generation stability.The results show that the enzyme has good stability and could be used as a thrombolytic agent,which is beneficial to the industrial application of nattokinase.
Bacillus subtilis MX-6,a high-yield strain of fibrinolytic enzyme,is screened from Chinese fermented soya beans.The fibrinolytic gene aprN is successfully amplified to 1473bp by polymerase chain reaction(PCR).The phylogenetic tree results show that the fibrinolytic enzyme is nattokinase.The stability and generation stability of nattokinase to heat,pH,metal ions,chemicals,inhibitors and modifiers are studied.The results show that the thermal stability of nattokinase is good at-20,4,30～50℃,and the activity of nattokinase is relatively stable at pH 3～11.Mg~(2+),Ca~(2+)have significant activation effect on nattokinase.K~+,Fe~(2+)have little effect on the stability of nattokinase,while Mn~(2+),Zn~(2+),Cu~(2+),Al ~(3+),Ba~(2+)have significant inhibition effect.Hg~(2+)leads to complete inactivation of nattokinase,bovine serum albumin,gelatin and peptone can significantly improve the stability of nattokinase,propylene glycol and sodium alginate have little effect on the stability of nattokinase,while ethanol significantly reduces the stability of nattokinase.PMSF results in the complete loss of nattokinase activity.EDAT and DTT have little effect on the activity of nattokinase.10mmol/L SDS significantly reduces the stability of nattokinase,which indicates that the nattokinase is a serine protease,not a metalloenzyme,does not contain disulfide bond,and has good generation stability.The results show that the enzyme has good stability and could be used as a thrombolytic agent,which is beneficial to the industrial application of nattokinase.
引文
[1]Mohanasrinivasan V,Mohanapriya A,Potdar S,et al.In vitro and in silico studies on fibrinolytic activity of nattokinase:a clot buster fromBacillus sp.[J].Frontiers in Biology,2017(3):219-225.
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[3]张超凤,严美婷,杜霞,等.响应面法优化纳豆激酶液态发酵条件[J].中国生物制品学,2017(6):658-662.
[4]Wei X,Zhou Y,Chen J,et al.Efficient expression of nattokinase in Bacillus licheniforms:host strain construction and signal peptide optimization[J].JournalofIndustrialMicrobiology&Biotechnology,2015(2):287-295.
[5]Wang H,Sun X,Wang L,et al.Coproduction of menaquinone-7and nattokinase by Bacillus subtilis using soybean curd residue as a renewable substrate combined with a dissolved oxygen control strategy[J].Annals of Microbiology,2018(10):655-665.
[6]Cai D,Zhu C,Chen S.Microbial production of nattokinase:current progress,challenge and prospect[J].World Journal of Microbiology and Biotechnology,2017(3):84.
[7]赵菡.纳豆激酶热稳定性及pH稳定性的改造研究[D].无锡:江南大学,2018.
[8]Vojcic L,Pitzler C,Krfer G,et al.Advances in protease engineering for laundry detergents[J].New Biotechnology,2015,32(6):629-634.
[9]Mahajan P M,Gokhale S V,Lele S S.Production of nattokinase using Bacillus natto NRRL 3666:media optimization,scale up,and kinetic modeling[J].Food Science and Biotechnology,2010(6):1593-1603.
[10]Zhang X,Yun L,Peng L,et al.Optimization of douchi fibrinolytic enzyme production by statistical experimental methods[J].Journal of Huazhong University of Science and Technology-Medical Sciences,2013(1):153-158.
[11]Zhang F,Zhang J,Linhardt R J.Interactions between nattokinase and heparin/GAGs[J].Glycoconjugate Journal,2015(9):695-702.
[12]刘诚.纳豆激酶液体发酵条件及其酶学稳定性研究[D].南京:南京农业大学,2000.
[13]Dabbagh F,Negahdaripour M,Berenjian A,et al.Nattokinase:production and application[J].Applied Microbiology and Biotechnology,2014(22):9199-9206.
[14]Yatagai C,Maruyama M,Kawahara T,et al.Nattokinase promoted tissue plasminogen activator release from human cells[J].Pathophysiology of Haemostasis and Thrombosis,2008(5):227-232.
[2]Subathra D C,Mohanasrinivasan V,Sharma P,et al.Production,purification and stability studies on nattokinase:a therapeutic protein extracted from mutant Pseudomonas aeruginosa CMSS isolated from bovine milk[J].International Journal of Peptide Research and Therapeutics,2016(2):263-269.
[3]张超凤,严美婷,杜霞,等.响应面法优化纳豆激酶液态发酵条件[J].中国生物制品学,2017(6):658-662.
[4]Wei X,Zhou Y,Chen J,et al.Efficient expression of nattokinase in Bacillus licheniforms:host strain construction and signal peptide optimization[J].JournalofIndustrialMicrobiology&Biotechnology,2015(2):287-295.
[5]Wang H,Sun X,Wang L,et al.Coproduction of menaquinone-7and nattokinase by Bacillus subtilis using soybean curd residue as a renewable substrate combined with a dissolved oxygen control strategy[J].Annals of Microbiology,2018(10):655-665.
[6]Cai D,Zhu C,Chen S.Microbial production of nattokinase:current progress,challenge and prospect[J].World Journal of Microbiology and Biotechnology,2017(3):84.
[7]赵菡.纳豆激酶热稳定性及pH稳定性的改造研究[D].无锡:江南大学,2018.
[8]Vojcic L,Pitzler C,Krfer G,et al.Advances in protease engineering for laundry detergents[J].New Biotechnology,2015,32(6):629-634.
[9]Mahajan P M,Gokhale S V,Lele S S.Production of nattokinase using Bacillus natto NRRL 3666:media optimization,scale up,and kinetic modeling[J].Food Science and Biotechnology,2010(6):1593-1603.
[10]Zhang X,Yun L,Peng L,et al.Optimization of douchi fibrinolytic enzyme production by statistical experimental methods[J].Journal of Huazhong University of Science and Technology-Medical Sciences,2013(1):153-158.
[11]Zhang F,Zhang J,Linhardt R J.Interactions between nattokinase and heparin/GAGs[J].Glycoconjugate Journal,2015(9):695-702.
[12]刘诚.纳豆激酶液体发酵条件及其酶学稳定性研究[D].南京:南京农业大学,2000.
[13]Dabbagh F,Negahdaripour M,Berenjian A,et al.Nattokinase:production and application[J].Applied Microbiology and Biotechnology,2014(22):9199-9206.
[14]Yatagai C,Maruyama M,Kawahara T,et al.Nattokinase promoted tissue plasminogen activator release from human cells[J].Pathophysiology of Haemostasis and Thrombosis,2008(5):227-232.