启动子及信号肽筛选提高普鲁兰酶在地衣芽孢杆菌中的表达
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摘要
普鲁兰酶(pullulanase)是一种淀粉脱支酶,在淀粉糖化加工等工业生产中具有重要的应用价值。选用地衣芽孢杆菌(Bacillus licheniformis) BL10作为表达宿主,为了提高其中的普鲁兰酶产量,对来自枯草芽孢杆菌(Bacillus subtilis) 168和地衣芽孢杆菌BL10中的15种不同信号肽以及4种不同启动子进行筛选,构建了不同的重组工程菌株,最终发现在以P43为启动子、apr为信号肽的条件下,上清液中普鲁兰酶的表达量最高,酶活可达12 U/m L,且测得其最适反应温度为60℃,最适p H为4.5。结果表明通过对启动子及信号肽进行筛选是提高重组工程菌株中目的蛋白表达量的一种有效手段。
Pullulanase is a well-known starch debranching enzyme with high application value in industrial production such as starch saccharification. In this study, in order to improve the production of pullulanase in Bacillus licheniformis BL10,fifteen signal peptides and four promoters from B. licheniformis BL10 and Bacillus subtilis 168 were screened,and the recombinant strains were constructed. The results showed that the recombinant strain with P43 as the promoter and apr as the signal peptide displayed the highest expression level of pullulanase in supernatant and its enzyme activity reached 12 U/m L. The optimal reaction temperature and p H of pullulanase were 60℃ and 4.5,respectively. The results indicated that it was an effective method to enhance the expression of a target protein by screening promoters and signal peptides in recombinant strain.
Pullulanase is a well-known starch debranching enzyme with high application value in industrial production such as starch saccharification. In this study, in order to improve the production of pullulanase in Bacillus licheniformis BL10,fifteen signal peptides and four promoters from B. licheniformis BL10 and Bacillus subtilis 168 were screened,and the recombinant strains were constructed. The results showed that the recombinant strain with P43 as the promoter and apr as the signal peptide displayed the highest expression level of pullulanase in supernatant and its enzyme activity reached 12 U/m L. The optimal reaction temperature and p H of pullulanase were 60℃ and 4.5,respectively. The results indicated that it was an effective method to enhance the expression of a target protein by screening promoters and signal peptides in recombinant strain.
引文
[1]段绪果.淀粉脱支酶的重组表达及分子改造[D].江苏无锡:江南大学,博士学位论文,2013.Duan X G.Overexpression and molecular modification of starch debranching enzymes[D].Jiangsu Wuxi:Jiangnan University,Doctoral Dissertation,2013.
[2]常美会.酸性普鲁兰酶的高效表达及其热稳定性改良研究[D].北京:中国农业科学院,硕士学位论文,2017.Chang M H.Efficient expression and improvement of the thermal stability of the aciduric pullulanase[D].Beijing:Chinese Academy of Agricultural Sciences,Master Dissertation,2017.
[3]Qoura F,Elleuche S,Brueck T,et al..Purification and characterization of a cold-adapted pullulanase from a psychrophilic bacterial isolate[J].Extremophiles,2014,18(6):1095-1102.
[4]Qiao Y,Peng Q,Yan J,et al..Gene cloning and enzymatic characterization of alkali-tolerant type I pullulanase from Exiguobacterium acetylicum[J].Lett.Appl.Microbiol.,2015,60(1):52-59.
[5]Qiu Y M,Zhang J Y,Li L,et al..Engineering Bacillus licheniformis for the production of meso-2,3-butanediol[J].Biotechnol.Biofuels,2016,9:117.
[6]俞玲.地衣芽孢杆菌pel B,amy X,yvdF基因的功能鉴定与表达[D].江苏无锡:江南大学,硕士学位论文,2009.Yu L.Indentification and expression of the gene pel B,amy Xand yvd F from Bacillus licheniformis[D].Jiangsu Wuxi:Jiangnan University,Master Dissertation,2009.
[7]方安然.地衣芽胞杆菌遗传改良及在淀粉脱支酶表达中的应用[D].天津:天津科技大学,硕士学位论文,2016.Fang A R.Genetic improvement of Bacillus licheniformis and application in the expression of starch debranching enzyme[D].Tianjin:Tianjin University of Science and Technology,Master Dissertation,2016.
[8]Yu X X,Xu J T,Liu X Q,et al..Identification of a highly efficient stationary phase promoter in Bacillus subtilis[J].Sci.Rep.,2015,5:18405.
[9]Cai D B,Wei X T,Qiu Y M,et al..High-level expression of nattokinase in Bacillus licheniformis by manipulating signal peptide and signal peptidase[J].J.Appl.Microbiol.,2016,121(3):704-712.
[10]Wang Y P,Liu Y H,Wang Z X,et al..Influence of promoter and signal peptide on the expression of pullulanase in Bacillus subtilis[J].Biotechnol.Lett.,2014,36(9):1783-1789.
[11]Cui Z L,Zhang X Z,Zhang Z H,et al..Construction and application of a promoter-trapping vector with methyl parathion hydrolase gene mpd as the reporter[J].Biotechnol.Lett.,2004,26(14):1115-1118.
[12]Song Y F,Nikoloff J M,Fu G,et al..Promoter screening from Bacillus subtilis in various conditions hunting for synthetic biology and industrial applications[J].PLoS ONE,2016,11(7):e0158447.
[13]余小霞,田健,刘晓青,等.枯草芽孢杆菌表达系统及其启动子研究进展[J].生物技术通报,2015,31(02):35-44.Yu X X,Tian J,Liu X Q,et al..Research progress of Bacillus subtilis expression system and its promoter regulatory elements[J].Biotechnol.Bull.,2015,31(02):35-44.
[14]Li X L,Pei J Y,Fei T,et al..Production of slowly digestible corn starch using hyperthermophilic Staphylothermus marinus amylopullulanase in Bacillus subtilis[J].Food Chem.,2019,277:1-5.
[15]Trung N T,Hung N M,Thuan N H,et al..An auto-inducible phosphate-controlled expression system of Bacillus licheniformis[J].BMC Biotechnol.,2019,19(1):3.
[16]Cai D B,Wang H,He P H,et al..A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis[J].Microb.Cell Fact.,2017,16(1):70.
[17]Freudl R.Signal peptides for recombinant protein secretion in bacterial expression systems[J].Microb.Cell Fact.,2018,17(1):52.
[18]Kang Z,Yang S,Du G C,et al..Molecular engineering of secretory machinery components for high-level secretion of proteins in Bacillus species[J].J.Ind.Microbiol.Biotechnol.,2014,41(11):1599-1607.
[19]Aw R,Mc Kay P F,Shattock R J,et al..A systematic analysis of the expression of the anti-HIV VRC01 antibody in Pichia pastoris through signal peptide optimization[J].Protein Expr.Purif.,2018,149:43-50.
[20]Han S,Machhi S,Berge M,et al..Novel signal peptides improve the secretion of recombinant Staphylococcus aureus Alpha toxinH35Lin Escherichia coli[J].AMB Express,2017,7(1):93.
[21]Degering C,Eggert T,Puls M,et al..Optimization of protease secretion in Bacillus subtilis and Bacillus licheniformis by screening of homologous and heterologous signal peptides[J].Appl.Environ.Microbiol.,2010,76(19):6370-6376.
[2]常美会.酸性普鲁兰酶的高效表达及其热稳定性改良研究[D].北京:中国农业科学院,硕士学位论文,2017.Chang M H.Efficient expression and improvement of the thermal stability of the aciduric pullulanase[D].Beijing:Chinese Academy of Agricultural Sciences,Master Dissertation,2017.
[3]Qoura F,Elleuche S,Brueck T,et al..Purification and characterization of a cold-adapted pullulanase from a psychrophilic bacterial isolate[J].Extremophiles,2014,18(6):1095-1102.
[4]Qiao Y,Peng Q,Yan J,et al..Gene cloning and enzymatic characterization of alkali-tolerant type I pullulanase from Exiguobacterium acetylicum[J].Lett.Appl.Microbiol.,2015,60(1):52-59.
[5]Qiu Y M,Zhang J Y,Li L,et al..Engineering Bacillus licheniformis for the production of meso-2,3-butanediol[J].Biotechnol.Biofuels,2016,9:117.
[6]俞玲.地衣芽孢杆菌pel B,amy X,yvdF基因的功能鉴定与表达[D].江苏无锡:江南大学,硕士学位论文,2009.Yu L.Indentification and expression of the gene pel B,amy Xand yvd F from Bacillus licheniformis[D].Jiangsu Wuxi:Jiangnan University,Master Dissertation,2009.
[7]方安然.地衣芽胞杆菌遗传改良及在淀粉脱支酶表达中的应用[D].天津:天津科技大学,硕士学位论文,2016.Fang A R.Genetic improvement of Bacillus licheniformis and application in the expression of starch debranching enzyme[D].Tianjin:Tianjin University of Science and Technology,Master Dissertation,2016.
[8]Yu X X,Xu J T,Liu X Q,et al..Identification of a highly efficient stationary phase promoter in Bacillus subtilis[J].Sci.Rep.,2015,5:18405.
[9]Cai D B,Wei X T,Qiu Y M,et al..High-level expression of nattokinase in Bacillus licheniformis by manipulating signal peptide and signal peptidase[J].J.Appl.Microbiol.,2016,121(3):704-712.
[10]Wang Y P,Liu Y H,Wang Z X,et al..Influence of promoter and signal peptide on the expression of pullulanase in Bacillus subtilis[J].Biotechnol.Lett.,2014,36(9):1783-1789.
[11]Cui Z L,Zhang X Z,Zhang Z H,et al..Construction and application of a promoter-trapping vector with methyl parathion hydrolase gene mpd as the reporter[J].Biotechnol.Lett.,2004,26(14):1115-1118.
[12]Song Y F,Nikoloff J M,Fu G,et al..Promoter screening from Bacillus subtilis in various conditions hunting for synthetic biology and industrial applications[J].PLoS ONE,2016,11(7):e0158447.
[13]余小霞,田健,刘晓青,等.枯草芽孢杆菌表达系统及其启动子研究进展[J].生物技术通报,2015,31(02):35-44.Yu X X,Tian J,Liu X Q,et al..Research progress of Bacillus subtilis expression system and its promoter regulatory elements[J].Biotechnol.Bull.,2015,31(02):35-44.
[14]Li X L,Pei J Y,Fei T,et al..Production of slowly digestible corn starch using hyperthermophilic Staphylothermus marinus amylopullulanase in Bacillus subtilis[J].Food Chem.,2019,277:1-5.
[15]Trung N T,Hung N M,Thuan N H,et al..An auto-inducible phosphate-controlled expression system of Bacillus licheniformis[J].BMC Biotechnol.,2019,19(1):3.
[16]Cai D B,Wang H,He P H,et al..A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis[J].Microb.Cell Fact.,2017,16(1):70.
[17]Freudl R.Signal peptides for recombinant protein secretion in bacterial expression systems[J].Microb.Cell Fact.,2018,17(1):52.
[18]Kang Z,Yang S,Du G C,et al..Molecular engineering of secretory machinery components for high-level secretion of proteins in Bacillus species[J].J.Ind.Microbiol.Biotechnol.,2014,41(11):1599-1607.
[19]Aw R,Mc Kay P F,Shattock R J,et al..A systematic analysis of the expression of the anti-HIV VRC01 antibody in Pichia pastoris through signal peptide optimization[J].Protein Expr.Purif.,2018,149:43-50.
[20]Han S,Machhi S,Berge M,et al..Novel signal peptides improve the secretion of recombinant Staphylococcus aureus Alpha toxinH35Lin Escherichia coli[J].AMB Express,2017,7(1):93.
[21]Degering C,Eggert T,Puls M,et al..Optimization of protease secretion in Bacillus subtilis and Bacillus licheniformis by screening of homologous and heterologous signal peptides[J].Appl.Environ.Microbiol.,2010,76(19):6370-6376.