重组纳豆激酶的研究进展
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摘要
纳豆激酶是一种碱性丝氨酸蛋白酶,具有较强的直接溶解血纤维蛋白的特性,与当前的溶栓药物相比具有诸多优点,已成为新型溶栓药物开发的焦点。本文首先简要介绍了纳豆激酶基因及纳豆激酶分子的理化特性和生物学功能,对当前国内外采用基因工程技术(密码子优化、点突变、高效表达系统构建等)重组表达纳豆激酶的研究进展进行了综述,分析了其中存在的主要问题和不足以及商业化生产重组纳豆激酶的可能性,对纳豆激酶在医药、食品等行业中的应用前景进行了展望。
Nattokinase is a kind of alkaline serine protease with a strong and direct fibrinolytic activity,has been the focus of development of new antithrombotic agent to prevent cardiovascular disease because of its advantages over conventional drugs in recent decade. The gene cloning,physicochemical properties and biological function of nattokinase were first introduced briefly,then the main studies and progress of recombinant expression of nattokinase by genetic engineering technology including codon optimization,point mutation,efficient expression system and so on were summarized,meanwhile some technical bottlenecks and obstacles to nattokinase industrial production were analyzed. Finally,the application prospect of nattokinase in medicine,food and other industries in the near future was discussed.
Nattokinase is a kind of alkaline serine protease with a strong and direct fibrinolytic activity,has been the focus of development of new antithrombotic agent to prevent cardiovascular disease because of its advantages over conventional drugs in recent decade. The gene cloning,physicochemical properties and biological function of nattokinase were first introduced briefly,then the main studies and progress of recombinant expression of nattokinase by genetic engineering technology including codon optimization,point mutation,efficient expression system and so on were summarized,meanwhile some technical bottlenecks and obstacles to nattokinase industrial production were analyzed. Finally,the application prospect of nattokinase in medicine,food and other industries in the near future was discussed.
引文
[1]Nakamura,T.,Yamagata,Y.,Ichishima,E.Nucleotide sequence of the subtilisin NAT gene,aprN,of Bacillus subtilis(natto)[J].Bioscience Biotechnology and Biochemistry,1992,56(11):1869-1871.
[2]Weng Y.Q.,Yao J.,Sparks S.,et al.Nattokinase:an oral antithrombotic agent for the prevention of cardiovascular disease[J].International Journal of Molecular Sciences,2017,18(3):523.
[3]Chen,H.J.,Mc Gowan,E.M.,Ren,N.N.,et al.Nattokinase:a promising alternative in prevention and treatment of cardiovascular diseases[J].Biomarker Insights,2017(13):1-8.
[4]李静,宋艳志,王梦静,等.纳豆激酶与尿激酶的对比性研究[J].中国药剂学杂志,2016,14(4):125-134.
[5]Jensen,G.S.,Lenninger,M.,Ero,M.P.,et al.Consumption of nattokinase is associated with reduced blood pressure and von Willebrand factor,a cardiovascular risk marker:results from a randomized,double-blind,placebocontrolled,multicenter North American clinical trial[J].Integrated Blood Pressure Control,2016(9):95-104.
[6]齐少卿,李晨,卢海强,等.保加利亚乳杆菌异源表达纳豆激酶的性质研究[J].中国食品学报,2017,17(4):51-57.
[7]Peng,Y.,Yang,X.,Zhang,Y.Microbial fibrinolytic enzymes:an overview of source,production,properties,and thrombolytic activity in vivo[J].Applied Microbiology and Biotechnology,2005(69):126-132.
[8]Urano,T.,Ihara,H.,Umemura,K.,et al.The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis cleaves and inactivates plasminogen activator inhibitor type 1[J].Journal of Biology Chemistry,2001(276):24690-24696.
[9]Jang,J.Y.,Kim,T.S.,Cai,J.M.,et al.Nattokinase improves blood flow by inhibiting platelet aggregation and thrombus formation[J].Laboratory Animal Research,2013,29(4):221-225.
[10]韩岚,王欢,王佳琪,等.密码子优化的纳豆激酶基因在番茄果实中的瞬时表达[J].内蒙古大学学报(自然科学版),2016,47(1):73-79.
[11]韩岚,鞠林芳,牛一丁,等.纳豆激酶基因的优化设计、合成及其在烟草叶片中的瞬时表达[J].中国生物工程杂志,2015,35(9):14-20.
[12]Han,L.,Zhang,L.Q.,Liu,J.L.,et al.Transient expression of optimized and synthesized nattokinase gene in melon(Cucumis melo L.)fruit by agroinfiltration[J].Plant Biotechnology,2015,32(2):175-180.
[13]刘靓蕾,张妍,孙晓蕾,等.人工合成的纳豆激酶基因转化烟草的研究[J].生物技术通报,2014(10):94-100.
[14]刘朔.纳豆激酶基因密码子优化设计与合成及在毕赤酵母中的高效表达[D].南京:南京农业大学,2007.
[15]葛春蕾,刘中美,崔文璟,等.通过串联启动子实现纳豆激酶在枯草芽孢杆菌中的高效表达[J].现代食品科技,2016,32(11):72-77、15.
[16]何孝天.Bacillus natto纳豆激酶的重组表达及热稳定性改造[D].江苏无锡:江南大学,2014.
[17]Liang,X.B.,et al.Secretory expression of nattokinase from Bacillus subtilis YF38 in Escherichia coli[J].Molecular Biotechnology,2007,37(3):187-194.
[18]Chiang,C.J.,et al.Efficient system of artificial oil bodies for functional expression and purification of recombinant nattokinase in Escherichia coli[J].Journal of Agricultural Food Chemistry,2005,53(12):4799-4804.
[19]Wu,S.M.,Feng,C.F.,Zhong,J.,et al.Enhanced production of recombinant nattokinase in Bacillus subtilis by promoter optimization[J].World Journal of Microbiology Biotechnology,2011,27(1):99-106.
[20]赵菡,周丽,周哲敏.通过定点突变提高纳豆激酶的酶活及热稳定性[J].食品与发酵工业,2018(9):36-40、47.
[21]刘中美,等.通过定点突变增强纳豆激酶的热稳定性[J].现代食品科技,2015,31(2):37-41.
[22]刘朔,姜梅,陈晓红,等.纳豆激酶第36位氨基酸突变对其活性及热稳定性的影响[J].南京农业大学学报,2008,31(3):130-136.
[23]Wu,S.,Feng,C.,Zhong,J.,et al.Roles of S3 site residues of nattokinase on its activity and substrate specificity[J].The Journal of Biochemistry,2007,142(3):357-364.
[24]Zheng,Z.L.,Ye,M.Q.,Zuo,Z.Y.,et al.Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation[J].Biochemical Journal,2006,395(3):509-515.
[25]周波.枯草芽孢杆菌Na-002纳豆激酶基因的克隆、表达及定点突变研究[D].山东泰安:山东农业大学,2011.
[26]Cai,Y.J.,et al.Directed evolution improves the fibrinolytic activity of nattokinase from Bacillus natto[J].FEMS Microbiology Letters,2011,325(2):155-161.
[27]艾海新,张力,张新刚,等.纳豆激酶的原核表达、纯化及其特性分析[J].微生物杂志,2017,37(1):20-27.
[28]李莹,陈斌,敬俊锋,等.纳豆激酶基因的克隆及其在大肠杆菌中的表达[J].重庆师范大学学报(自然科学版),2012,29(1):77-81.
[29]蒋凡.纳豆激酶的表达与复性研究[D].兰州:兰州大学,2008.
[30]张淑梅,李晶,王玉霞,等.纳豆激酶基因的表达及纯化[J].生物技术,2003,13(6):12-15.
[31]童煜,陈守春,张思仲.纳豆激酶原核表达载体的构建及其活性鉴定[J].应用与环境生物学报,2007,13(3):369-372.
[32]郭文秀.前纳豆激酶原基因的克隆与表达[D].呼和浩特:内蒙古大学,2006.
[33]Chen,P.T.,Chiang,C.J.,Chao,Y.P.Medium optimization for the production of recombinant nattokinase by Bacillus subtilis using response surface methodology[J].Biotechnology Progress,2007,23(6):1327-1332.
[34]Cui,W.J.,et al.Stepwise modifications of genetic parts reinforce the secretory production of nattokinase in Bacillus subtilis[J].Microbial Biotechnology,2018(11):930-942.
[35]Wei,X.T.,Zhou,Y.H.,Chen,J.B.,et al.Efficient expression of nattokinase in Bacillus licheniformis:host strain construction and signal peptide optimization[J].Journal of Industrial Microbiology Biotechnology,2015(42):287-295.
[36]赵新宇,等.高产纳豆激酶地衣芽孢杆菌工程菌全合成培养基优化[J].食品科学,2016,37(7):140-145.
[37]敬俊锋,等.纳豆激酶基因的克隆及其在毕赤酵母中的表达[J].生物学杂志,2011,28(5):55-60.
[38]闫少华.重组纳豆激酶在毕赤酵母中表达及大规模发酵研究[D].长春:吉林大学,2010.
[39]Liang,X.B.,et al.Secretory expression of a heterologous nattokinase in Lactococcus lactis[J].Applied Microbiology and Biotechnology,2007,75(1):95-101.
[40]Li,X.X.,et al.Expression and purification of recombinant nattokinase in Spodoptera frugiperda cells[J].Biotechnology Letters,2007,29(10):1459-1464.
[41]朱立成.纳豆激酶和h GM-CSF在家蚕杆状病毒表达系统中表达纯化及活性研究[D].杭州:浙江大学,2005.
[42]Ikemura,H.,Takagi,H.,Inouye,M.Requirement of prosequence for the production of active subtilisin E in Escherichia coli[J].The Journal of Biological Chemistry,1987,262(16):7859-7864.
[43]蔡立涛,徐祥,王婷婷,等.纳豆激酶基因在毕赤酵母中的表达纯化及抗体制备[J].中国生化药物杂志,2010,31(1):10-13.
[44]舒敏.纳豆激酶在毕赤酵母中的表达及在大鼠体内的溶栓作用的研究[D].武汉:湖北大学,2016.
[45]Nguyen,T.T.,Quyen,T.D.,Le,H.T.Cloning and enhancing production of a detergent-and organic-solventresistant nattokinase from Bacillus subtilis VTCC-DVN-12-01by using an eight-protease-gene-deficient Bacillus subtilis WB800[J].Microbial Cell Factories,2013(12):79.
[2]Weng Y.Q.,Yao J.,Sparks S.,et al.Nattokinase:an oral antithrombotic agent for the prevention of cardiovascular disease[J].International Journal of Molecular Sciences,2017,18(3):523.
[3]Chen,H.J.,Mc Gowan,E.M.,Ren,N.N.,et al.Nattokinase:a promising alternative in prevention and treatment of cardiovascular diseases[J].Biomarker Insights,2017(13):1-8.
[4]李静,宋艳志,王梦静,等.纳豆激酶与尿激酶的对比性研究[J].中国药剂学杂志,2016,14(4):125-134.
[5]Jensen,G.S.,Lenninger,M.,Ero,M.P.,et al.Consumption of nattokinase is associated with reduced blood pressure and von Willebrand factor,a cardiovascular risk marker:results from a randomized,double-blind,placebocontrolled,multicenter North American clinical trial[J].Integrated Blood Pressure Control,2016(9):95-104.
[6]齐少卿,李晨,卢海强,等.保加利亚乳杆菌异源表达纳豆激酶的性质研究[J].中国食品学报,2017,17(4):51-57.
[7]Peng,Y.,Yang,X.,Zhang,Y.Microbial fibrinolytic enzymes:an overview of source,production,properties,and thrombolytic activity in vivo[J].Applied Microbiology and Biotechnology,2005(69):126-132.
[8]Urano,T.,Ihara,H.,Umemura,K.,et al.The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis cleaves and inactivates plasminogen activator inhibitor type 1[J].Journal of Biology Chemistry,2001(276):24690-24696.
[9]Jang,J.Y.,Kim,T.S.,Cai,J.M.,et al.Nattokinase improves blood flow by inhibiting platelet aggregation and thrombus formation[J].Laboratory Animal Research,2013,29(4):221-225.
[10]韩岚,王欢,王佳琪,等.密码子优化的纳豆激酶基因在番茄果实中的瞬时表达[J].内蒙古大学学报(自然科学版),2016,47(1):73-79.
[11]韩岚,鞠林芳,牛一丁,等.纳豆激酶基因的优化设计、合成及其在烟草叶片中的瞬时表达[J].中国生物工程杂志,2015,35(9):14-20.
[12]Han,L.,Zhang,L.Q.,Liu,J.L.,et al.Transient expression of optimized and synthesized nattokinase gene in melon(Cucumis melo L.)fruit by agroinfiltration[J].Plant Biotechnology,2015,32(2):175-180.
[13]刘靓蕾,张妍,孙晓蕾,等.人工合成的纳豆激酶基因转化烟草的研究[J].生物技术通报,2014(10):94-100.
[14]刘朔.纳豆激酶基因密码子优化设计与合成及在毕赤酵母中的高效表达[D].南京:南京农业大学,2007.
[15]葛春蕾,刘中美,崔文璟,等.通过串联启动子实现纳豆激酶在枯草芽孢杆菌中的高效表达[J].现代食品科技,2016,32(11):72-77、15.
[16]何孝天.Bacillus natto纳豆激酶的重组表达及热稳定性改造[D].江苏无锡:江南大学,2014.
[17]Liang,X.B.,et al.Secretory expression of nattokinase from Bacillus subtilis YF38 in Escherichia coli[J].Molecular Biotechnology,2007,37(3):187-194.
[18]Chiang,C.J.,et al.Efficient system of artificial oil bodies for functional expression and purification of recombinant nattokinase in Escherichia coli[J].Journal of Agricultural Food Chemistry,2005,53(12):4799-4804.
[19]Wu,S.M.,Feng,C.F.,Zhong,J.,et al.Enhanced production of recombinant nattokinase in Bacillus subtilis by promoter optimization[J].World Journal of Microbiology Biotechnology,2011,27(1):99-106.
[20]赵菡,周丽,周哲敏.通过定点突变提高纳豆激酶的酶活及热稳定性[J].食品与发酵工业,2018(9):36-40、47.
[21]刘中美,等.通过定点突变增强纳豆激酶的热稳定性[J].现代食品科技,2015,31(2):37-41.
[22]刘朔,姜梅,陈晓红,等.纳豆激酶第36位氨基酸突变对其活性及热稳定性的影响[J].南京农业大学学报,2008,31(3):130-136.
[23]Wu,S.,Feng,C.,Zhong,J.,et al.Roles of S3 site residues of nattokinase on its activity and substrate specificity[J].The Journal of Biochemistry,2007,142(3):357-364.
[24]Zheng,Z.L.,Ye,M.Q.,Zuo,Z.Y.,et al.Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation[J].Biochemical Journal,2006,395(3):509-515.
[25]周波.枯草芽孢杆菌Na-002纳豆激酶基因的克隆、表达及定点突变研究[D].山东泰安:山东农业大学,2011.
[26]Cai,Y.J.,et al.Directed evolution improves the fibrinolytic activity of nattokinase from Bacillus natto[J].FEMS Microbiology Letters,2011,325(2):155-161.
[27]艾海新,张力,张新刚,等.纳豆激酶的原核表达、纯化及其特性分析[J].微生物杂志,2017,37(1):20-27.
[28]李莹,陈斌,敬俊锋,等.纳豆激酶基因的克隆及其在大肠杆菌中的表达[J].重庆师范大学学报(自然科学版),2012,29(1):77-81.
[29]蒋凡.纳豆激酶的表达与复性研究[D].兰州:兰州大学,2008.
[30]张淑梅,李晶,王玉霞,等.纳豆激酶基因的表达及纯化[J].生物技术,2003,13(6):12-15.
[31]童煜,陈守春,张思仲.纳豆激酶原核表达载体的构建及其活性鉴定[J].应用与环境生物学报,2007,13(3):369-372.
[32]郭文秀.前纳豆激酶原基因的克隆与表达[D].呼和浩特:内蒙古大学,2006.
[33]Chen,P.T.,Chiang,C.J.,Chao,Y.P.Medium optimization for the production of recombinant nattokinase by Bacillus subtilis using response surface methodology[J].Biotechnology Progress,2007,23(6):1327-1332.
[34]Cui,W.J.,et al.Stepwise modifications of genetic parts reinforce the secretory production of nattokinase in Bacillus subtilis[J].Microbial Biotechnology,2018(11):930-942.
[35]Wei,X.T.,Zhou,Y.H.,Chen,J.B.,et al.Efficient expression of nattokinase in Bacillus licheniformis:host strain construction and signal peptide optimization[J].Journal of Industrial Microbiology Biotechnology,2015(42):287-295.
[36]赵新宇,等.高产纳豆激酶地衣芽孢杆菌工程菌全合成培养基优化[J].食品科学,2016,37(7):140-145.
[37]敬俊锋,等.纳豆激酶基因的克隆及其在毕赤酵母中的表达[J].生物学杂志,2011,28(5):55-60.
[38]闫少华.重组纳豆激酶在毕赤酵母中表达及大规模发酵研究[D].长春:吉林大学,2010.
[39]Liang,X.B.,et al.Secretory expression of a heterologous nattokinase in Lactococcus lactis[J].Applied Microbiology and Biotechnology,2007,75(1):95-101.
[40]Li,X.X.,et al.Expression and purification of recombinant nattokinase in Spodoptera frugiperda cells[J].Biotechnology Letters,2007,29(10):1459-1464.
[41]朱立成.纳豆激酶和h GM-CSF在家蚕杆状病毒表达系统中表达纯化及活性研究[D].杭州:浙江大学,2005.
[42]Ikemura,H.,Takagi,H.,Inouye,M.Requirement of prosequence for the production of active subtilisin E in Escherichia coli[J].The Journal of Biological Chemistry,1987,262(16):7859-7864.
[43]蔡立涛,徐祥,王婷婷,等.纳豆激酶基因在毕赤酵母中的表达纯化及抗体制备[J].中国生化药物杂志,2010,31(1):10-13.
[44]舒敏.纳豆激酶在毕赤酵母中的表达及在大鼠体内的溶栓作用的研究[D].武汉:湖北大学,2016.
[45]Nguyen,T.T.,Quyen,T.D.,Le,H.T.Cloning and enhancing production of a detergent-and organic-solventresistant nattokinase from Bacillus subtilis VTCC-DVN-12-01by using an eight-protease-gene-deficient Bacillus subtilis WB800[J].Microbial Cell Factories,2013(12):79.