基于foldon介导的寡聚化以提高阿魏酸酯酶催化效率
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摘要
将foldon结构域与阿魏酸酯酶C-末端进行融合表达并用组氨酸标签对融合蛋白进行纯化。实现基于foldon的寡聚化阿魏酸酯酶及单体阿魏酸酯酶在毕赤酵母GS115中表达,并应用目标蛋白与foldon结构域融合可自发形成三聚体结构的特性对阿魏酸酯酶进行改造,以提高阿魏酸酯酶的催化性能。经纯化获得寡聚化及单体阿魏酸酯酶,寡聚化阿魏酸酯酶表观分子量约为110kDa,单体阿魏酸酯酶表观分子量为40 kDa;寡聚化阿魏酸酯酶的最适反应温度和pH分别为50℃和5.0,而单体阿魏酸酯酶则分别为50℃和6.0。寡聚化阿魏酸酯酶的底物亲和力(K_m)及催化效率(k_(cat)/K_m)较单体阿魏酸酯酶分别提高3.42倍和7.57倍。结果表明,寡聚化及单体阿魏酸酯酶均成功表达,且寡聚化阿魏酸酯酶在底物亲和力和催化效率上具有明显优势,该提高阿魏酸酯酶催化效率的方法简单、高效,有很好的应用前景。
A new method to express oligomerized feruloyl esterase(FAE) in Pichia pastoris GS115 to improve the catalytic efficiency was developed. It was realized by fusing the foldon domain at the C-terminus of FAE, and the fusion protein was purified by histidine tag. Fusion of the feruloyl esterase with the foldon domain resulted spontaneously forming a trimer FAE to improve the catalytic performance. The oligomerized FAE and monomeric FAE were obtained by purification. The apparent molecular weight of the oligomerized FAE was about 110 kDa, while the monomeric FAE about 40 kDa, and the optimum temperature of the oligomerized FAE was 50 °C, which is the same as the monomeric one. The optimal pH of the oligomerized FAE is 5.0, while the optimal pH of the monomer FAE is 6.0. When compared with the monomeric ones, the catalytic efficiency(k_(cat)/K_m) of the oligomerized FAE increased 7.57-folds. The catalytic constant(k_(cat)) of the oligomerized FAE increased 3.42-folds. The oligomerized FAE induced by foldon have advantages in the catalytic performances, which represents a simple and effective enzyme-engineering tool. The method proposed here for improving the catalytic efficiency of FAE would have great potentials for improving the catalytic efficiency of other enzymes.
A new method to express oligomerized feruloyl esterase(FAE) in Pichia pastoris GS115 to improve the catalytic efficiency was developed. It was realized by fusing the foldon domain at the C-terminus of FAE, and the fusion protein was purified by histidine tag. Fusion of the feruloyl esterase with the foldon domain resulted spontaneously forming a trimer FAE to improve the catalytic performance. The oligomerized FAE and monomeric FAE were obtained by purification. The apparent molecular weight of the oligomerized FAE was about 110 kDa, while the monomeric FAE about 40 kDa, and the optimum temperature of the oligomerized FAE was 50 °C, which is the same as the monomeric one. The optimal pH of the oligomerized FAE is 5.0, while the optimal pH of the monomer FAE is 6.0. When compared with the monomeric ones, the catalytic efficiency(k_(cat)/K_m) of the oligomerized FAE increased 7.57-folds. The catalytic constant(k_(cat)) of the oligomerized FAE increased 3.42-folds. The oligomerized FAE induced by foldon have advantages in the catalytic performances, which represents a simple and effective enzyme-engineering tool. The method proposed here for improving the catalytic efficiency of FAE would have great potentials for improving the catalytic efficiency of other enzymes.
引文
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[2]de Vries R,Visser J.Aspergillus enzymes involved in degradation of plant cell wall polysaccharides.Microbiol Mol Biol Rev,2001,65(4):497-522.
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[7]Zhang SB,Zhai HC,Wang L,et al.Expression,purification and characterization of a feruloyl esterase A from Aspergillus flavus.Protein Expr Purif,2013,92(1):36-40.
[8]Sun JT.Construction of Thermostableβ-1,3-1,4-glucanase and its Enzymatic Properties[D].Wuxi:Jiangnan University,2012(in Chinese).孙军涛.耐热β-1,3-1,4-葡聚糖酶的构建及其酶学性质研究[D].无锡:江南大学,2012.
[9]Liu T.Improving stability of enzyme through the use of extremophiles.J Chem Ind Eng,2009,30(6):26-29(in Chinese).刘涛,利用嗜极微生物提高酶的稳定性.能源化工,2009,30(6):26-29.
[10]Lutz S.Beyond directed evolution-semi-rational protein engineering and design.Curr Opin Biotechnol,2010,21(6):734-743.
[11]Papanikolopoulou K,Forge V,Goeltz P,et al.Formation of highly stable chimeric trimers by fusion of an adenovirus fiber shaft fragment with the foldon domain of bacteriophage t4 fibritin.J Biol Chem,2004,279(10):8991-8998.
[12]Yang HQ,Li JH,Shin HD,et al.Molecular engineering of industrial enzymes:recent advances and future prospects.Appl Microbiol Biotechnol,2014,98(1):23-29.
[13]Can?,Holland NB.Utilizing avidity to improve antifreeze protein activity:a type III antifreeze protein trimer exhibits increased thermal hysteresis activity.Biochemistry,2013,52(48):8745-8752.
[14]Engel J,Kammerer RA.What are oligomerization domains good for?Matrix Biol,2000,19(4):283-288.
[15]Tao YZ,Strelkov SV,Mesyanzhinov VV,et al.Structure of bacteriophage T4 fibritin:a segmented coiled coil and the role of the C-terminal domain.Structure,1997,5(6):789-798.
[16]Stetefeld J,Frank S,Jenny M,et al.Collagen stabilization at atomic level:crystal structure of designed(GlyProPro)10 foldon.Structure,2003,11(3):339-346.
[17]Sisso?ff L,Mousli M,England P,et al.Stable trimerization of recombinant rabies virus glycoprotein ectodomain is required for interaction with the p75NTR receptor.J General Virol,2005,86:2543-2552.
[18]Malashkevich VN,Kammerer RA,Efimov VP,et al.The crystal structure of a five-stranded coiled coil in COMP:a prototype ion channel.Science,1996,274(5288):761-765.
[19]Li XL,Fan YM,Fang BS.Purification and characterization of ferulic acid esterase from Penicillium citrinum.Microbiology China,2010,50(8):1058-1064(in Chinese).李夏兰,范韵敏,方柏山.来自桔青霉的阿魏酸酯酶的分离纯化、理化性质.微生物学报,2010,50(8):1058-1064.
[20]Li CC,Zhang GY.The fusions of elastin-like polypeptides and xylanase self-assembled into insoluble active xylanase particles.J Biotechnol,2014,177:60-66.
[21]Juge N,Williamson G,Puigserver A,et al.High-level production of recombinant Aspergillus niger cinnamoyl esterase(FAEA)in the methylotrophic yeast Pichia pastoris.FEMS Yeast Res,2002,1(2):127-132.
[22]Bhardwaj A,Walker-Kopp N,Wilkens S,et al.Foldon-guided self-assembly of ultra-stable protein fibers.Protein Sci,2010,17(9):1475-1485.
[23]Fang Y,Ou SY,Zhang N.Preparation and enzymatic properties of ferulic acid esterase.J Jinan Univ:Nat Scie Med Ed,2012,33(5):481-485(in Chinese).方园,欧仕益,张宁.阿魏酸酯酶的制备及其酶学性质.暨南大学学报:自然科学与医学版,2012,33(5):481-485.
[24]Yang HQ,Lu XY,Liu L,et al.Fusion of an oligopeptide to the N terminus of an alkalineα-Amylase from Alkalimonas amylolytica simultaneously improves the enzyme’s catalytic efficiency,thermal stability,and resistance to oxidation.Appl Environ Microbiol,2013,79(9):3049-3058.
[25]Li JJ,Pei XQ,Zhang SB,et al.Improving the thermostability of feruloyl esterase by DNA shuffling and site-directed mutagenesis.Process Biochem,2015,50(11):1783-1787.
[26]Zhang SB,Wu ZL.Identification of amino acid residues responsible for increased thermostability of feruloyl esterase A from Aspergillus niger using the PoPMuSiC algorithm.Bioresour Technol,2011,102(2):2093-2096.
[27]Cao LC,Chen R,Xie W,et al.Enhancing the thermostability of feruloyl esterase EstF27 by directed evolution and the underlying structural basis.J Agric Food Chem,2015,63(37):8225-8233.
[28]Zhang SB,Pei XQ,Wu ZL.Multiple amino acid substitutions significantly improve the thermostability of feruloyl esterase A from Aspergillus niger.Bioresource Technology,2012,117:140-147.
[29]Yin X,Hu D,Li JF,et al.Contribution of disulfide bridges to the thermostability of a Type A feruloyl esterase from Aspergillus usamii.PLoS ONE,2015,10(5):e0126864.
[30]Bonzom C,Schild L,Gustafsson H,et al.Feruloyl esterase immobilization in mesoporous silica particles and characterization in hydrolysis and transesterification.BMC Biochem,2018,19(1):1.
[31]He FM,Zhang S,Liu XL.Immobilization of feruloyl esterases on magnetic nanoparticles and its potential in production of ferulic acid.J Biosci Bioeng,2015,120(3):330-334.
[32]Illanes A,Cauerhff A,Wilson L,et al.Recent trends in biocatalysis engineering.Bioresour Technol,2012,115:48-57.
[33]Dalby PA.Strategy and success for the directed evolution of enzymes.Curr Opin Struct Biol,2011,21(4):473-480.
[34]Pe?i?M,Bo?i?N,López C,et al.Chemical modification of chloroperoxidase for enhanced stability and activity.Process Biochem,2014,49(9):1472-1479.
[35]Luo TZ,Kiick K.Noncovalent modulation of the inverse temperature transition and self-assembly of elastin-b-collagen-like peptide bioconjugates.J Am Chem Soc,2015,137(49):15362-15365.
[36]Wang XZ,Ge HH,Zhang DD,et al.Oligomerization triggered by foldon:a simple method to enhance the catalytic efficiency of lichenase and xylanase.BMCBiotechnol,2017,17(1):57.
[2]de Vries R,Visser J.Aspergillus enzymes involved in degradation of plant cell wall polysaccharides.Microbiol Mol Biol Rev,2001,65(4):497-522.
[3]Faulds CB,Mandalari G,LoCurto R,et al.Arabinoxylan and mono-and dimeric ferulic acid release from brewer’s grain and wheat bran by feruloyl esterases and glycosyl hydrolases from Humicola insolens.Appl Microbiol Biotechnol,2004,64(5):644-650.
[4]Kroon PA,Faulds CB,Williamson G.Purification and characterization of a novel esterase induced by growth of Aspergillus niger on sugar-beet pulp.Biotechnol Appl Biochem,2011,23(3):255-262.
[5]Chen YH,Li H,Zhang GY,et al.Expression of feruloyl esterase O42807 in Pichia pastoris GS115.JHuaqiao Univ:Nat Sci,2016,37(2):224-229(in Chinese).陈云华,李慧,张光亚,等.阿魏酸酯酶O42807在毕赤酵母GS115中的表达.华侨大学学报:自然科学版,2016,37(2):224-229.
[6]Li B,Cai GL,Zhu DW,et al.Codon optimization of feruloyl esterase gene of Aspergillus niger and its high expression in Pichia pastoris.Microbiology China,2017,44(5):1065-1073(in Chinese).李兵,蔡国林,朱德伟,等.黑曲霉阿魏酸酯酶基因密码子优化及在毕赤酵母中的高效表达.微生物学通报,2017,44(5):1065-1073.
[7]Zhang SB,Zhai HC,Wang L,et al.Expression,purification and characterization of a feruloyl esterase A from Aspergillus flavus.Protein Expr Purif,2013,92(1):36-40.
[8]Sun JT.Construction of Thermostableβ-1,3-1,4-glucanase and its Enzymatic Properties[D].Wuxi:Jiangnan University,2012(in Chinese).孙军涛.耐热β-1,3-1,4-葡聚糖酶的构建及其酶学性质研究[D].无锡:江南大学,2012.
[9]Liu T.Improving stability of enzyme through the use of extremophiles.J Chem Ind Eng,2009,30(6):26-29(in Chinese).刘涛,利用嗜极微生物提高酶的稳定性.能源化工,2009,30(6):26-29.
[10]Lutz S.Beyond directed evolution-semi-rational protein engineering and design.Curr Opin Biotechnol,2010,21(6):734-743.
[11]Papanikolopoulou K,Forge V,Goeltz P,et al.Formation of highly stable chimeric trimers by fusion of an adenovirus fiber shaft fragment with the foldon domain of bacteriophage t4 fibritin.J Biol Chem,2004,279(10):8991-8998.
[12]Yang HQ,Li JH,Shin HD,et al.Molecular engineering of industrial enzymes:recent advances and future prospects.Appl Microbiol Biotechnol,2014,98(1):23-29.
[13]Can?,Holland NB.Utilizing avidity to improve antifreeze protein activity:a type III antifreeze protein trimer exhibits increased thermal hysteresis activity.Biochemistry,2013,52(48):8745-8752.
[14]Engel J,Kammerer RA.What are oligomerization domains good for?Matrix Biol,2000,19(4):283-288.
[15]Tao YZ,Strelkov SV,Mesyanzhinov VV,et al.Structure of bacteriophage T4 fibritin:a segmented coiled coil and the role of the C-terminal domain.Structure,1997,5(6):789-798.
[16]Stetefeld J,Frank S,Jenny M,et al.Collagen stabilization at atomic level:crystal structure of designed(GlyProPro)10 foldon.Structure,2003,11(3):339-346.
[17]Sisso?ff L,Mousli M,England P,et al.Stable trimerization of recombinant rabies virus glycoprotein ectodomain is required for interaction with the p75NTR receptor.J General Virol,2005,86:2543-2552.
[18]Malashkevich VN,Kammerer RA,Efimov VP,et al.The crystal structure of a five-stranded coiled coil in COMP:a prototype ion channel.Science,1996,274(5288):761-765.
[19]Li XL,Fan YM,Fang BS.Purification and characterization of ferulic acid esterase from Penicillium citrinum.Microbiology China,2010,50(8):1058-1064(in Chinese).李夏兰,范韵敏,方柏山.来自桔青霉的阿魏酸酯酶的分离纯化、理化性质.微生物学报,2010,50(8):1058-1064.
[20]Li CC,Zhang GY.The fusions of elastin-like polypeptides and xylanase self-assembled into insoluble active xylanase particles.J Biotechnol,2014,177:60-66.
[21]Juge N,Williamson G,Puigserver A,et al.High-level production of recombinant Aspergillus niger cinnamoyl esterase(FAEA)in the methylotrophic yeast Pichia pastoris.FEMS Yeast Res,2002,1(2):127-132.
[22]Bhardwaj A,Walker-Kopp N,Wilkens S,et al.Foldon-guided self-assembly of ultra-stable protein fibers.Protein Sci,2010,17(9):1475-1485.
[23]Fang Y,Ou SY,Zhang N.Preparation and enzymatic properties of ferulic acid esterase.J Jinan Univ:Nat Scie Med Ed,2012,33(5):481-485(in Chinese).方园,欧仕益,张宁.阿魏酸酯酶的制备及其酶学性质.暨南大学学报:自然科学与医学版,2012,33(5):481-485.
[24]Yang HQ,Lu XY,Liu L,et al.Fusion of an oligopeptide to the N terminus of an alkalineα-Amylase from Alkalimonas amylolytica simultaneously improves the enzyme’s catalytic efficiency,thermal stability,and resistance to oxidation.Appl Environ Microbiol,2013,79(9):3049-3058.
[25]Li JJ,Pei XQ,Zhang SB,et al.Improving the thermostability of feruloyl esterase by DNA shuffling and site-directed mutagenesis.Process Biochem,2015,50(11):1783-1787.
[26]Zhang SB,Wu ZL.Identification of amino acid residues responsible for increased thermostability of feruloyl esterase A from Aspergillus niger using the PoPMuSiC algorithm.Bioresour Technol,2011,102(2):2093-2096.
[27]Cao LC,Chen R,Xie W,et al.Enhancing the thermostability of feruloyl esterase EstF27 by directed evolution and the underlying structural basis.J Agric Food Chem,2015,63(37):8225-8233.
[28]Zhang SB,Pei XQ,Wu ZL.Multiple amino acid substitutions significantly improve the thermostability of feruloyl esterase A from Aspergillus niger.Bioresource Technology,2012,117:140-147.
[29]Yin X,Hu D,Li JF,et al.Contribution of disulfide bridges to the thermostability of a Type A feruloyl esterase from Aspergillus usamii.PLoS ONE,2015,10(5):e0126864.
[30]Bonzom C,Schild L,Gustafsson H,et al.Feruloyl esterase immobilization in mesoporous silica particles and characterization in hydrolysis and transesterification.BMC Biochem,2018,19(1):1.
[31]He FM,Zhang S,Liu XL.Immobilization of feruloyl esterases on magnetic nanoparticles and its potential in production of ferulic acid.J Biosci Bioeng,2015,120(3):330-334.
[32]Illanes A,Cauerhff A,Wilson L,et al.Recent trends in biocatalysis engineering.Bioresour Technol,2012,115:48-57.
[33]Dalby PA.Strategy and success for the directed evolution of enzymes.Curr Opin Struct Biol,2011,21(4):473-480.
[34]Pe?i?M,Bo?i?N,López C,et al.Chemical modification of chloroperoxidase for enhanced stability and activity.Process Biochem,2014,49(9):1472-1479.
[35]Luo TZ,Kiick K.Noncovalent modulation of the inverse temperature transition and self-assembly of elastin-b-collagen-like peptide bioconjugates.J Am Chem Soc,2015,137(49):15362-15365.
[36]Wang XZ,Ge HH,Zhang DD,et al.Oligomerization triggered by foldon:a simple method to enhance the catalytic efficiency of lichenase and xylanase.BMCBiotechnol,2017,17(1):57.
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