特异腐质霉来源漆酶基因的克隆及其在毕赤酵母中的表达
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摘要
漆酶是一种含铜的多酚氧化酶,在有机农药残留和木质素的降解方面具有潜在的工业价值。从特异腐质霉Y1(Humicola insolens Y1)中分离到了漆酶基因Lac1,其全长1 803 bp,编码600个氨基酸,与NCBI蛋白数据库比对结果表明,与来源于Chaetomium globosum CBS 148.51(XP_001228806)的多酚氧化酶序列相似性最高为71%,表明是一个新的漆酶基因。将其克隆入毕赤酵母表达载体pPIC9r中,通过PCR和SDS-PAGE检测证实基因已经整合入酵母基因组中且能分泌表达。在3 L发酵罐中,重组Lac1蛋白表达量达到3.79mg/mL发酵液,酶活性达到1.3 U/mL发酵液。酶学性质分析结果显示,漆酶的最适作用温度为65℃,最适反应pH为4.5,且在pH 6～11的范围内酶的相对活力均在70%以上。
Laccase is a copper-containing polyphenol oxidase,which has potential industrial value in the degradation of organic pesticide residues and lignin. In this study,the laccase gene Lac1 was isolated from a Humicola insolens Y1. The 1 803 bp DNA of Lac1 encoded a mature protein with 600 amino acids. The deduced amino acid sequence was aligned with available protein sequences held in the Gen Bank,the results showed that the highest similarity was71% which was the polyphenol oxidase sequence from Chaetomium globosum CBS 148. 51( XP _001228806). The recombinant vector pPIC9 r-Lac1 was constructed and transferred into Pichia pastris to integrate into the yeast genome and the recombinant laccase was secreted expression. It was confirmed by the PCR and SDS-PAGE. In 3 L fermentation,the expression of Lac1 protein reached 3.79 mg/mL,and the enzyme activity reached 1.3 U/mL. The analysis of enzymatic properties showed that the optimum temperature was 65℃,the optimum pH was 4.5,and the relative activity of enzyme was above 70% in pH 6 ～ 11.
Laccase is a copper-containing polyphenol oxidase,which has potential industrial value in the degradation of organic pesticide residues and lignin. In this study,the laccase gene Lac1 was isolated from a Humicola insolens Y1. The 1 803 bp DNA of Lac1 encoded a mature protein with 600 amino acids. The deduced amino acid sequence was aligned with available protein sequences held in the Gen Bank,the results showed that the highest similarity was71% which was the polyphenol oxidase sequence from Chaetomium globosum CBS 148. 51( XP _001228806). The recombinant vector pPIC9 r-Lac1 was constructed and transferred into Pichia pastris to integrate into the yeast genome and the recombinant laccase was secreted expression. It was confirmed by the PCR and SDS-PAGE. In 3 L fermentation,the expression of Lac1 protein reached 3.79 mg/mL,and the enzyme activity reached 1.3 U/mL. The analysis of enzymatic properties showed that the optimum temperature was 65℃,the optimum pH was 4.5,and the relative activity of enzyme was above 70% in pH 6 ～ 11.
引文
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[2]Aktas N,Cicek H,Taspinar A U,et al..Reaction kinetics for laccase-catalyzed polymerization of 1-naphthol[J].Bioresour.Technol.,2001.80(1):29-36.
[3]Leontievsky A A,Myasoedova N M,Baskunov B P,et al..Transformation of 2,4,6-trichlorophenol by the white rot fungi Panus tigrinus and Coriolus versicolor[J].Biodegradation,2000,11(5):331-340.
[4]陈辉,张剑波,刘小鹏,等.漆酶催化降解氯酚类有机污染物[J].北京大学自然期刊,2005,41(4):605-611.Chen H,Zhang J B,Liu X P,et al..Degradation of chlorophenols catalyzed by laccase[J].Acta Sci.Nat.Univ.Pekinensis,2005,41(4):605-611.
[5]张鑫璐,裴继诚,张方东,等.漆酶和APMP废液中酚类物质共同改善OCC纸浆纤维性能的研究[J].中国造纸学报,2011,26(2):16-20.Zhang X L,Pei J C,Zhang F D,et al..Modification of OCCpulp by laccase with the phenols contained in APMP waste liquor[J].Trans.China Pulp.Paper,2011,26(2):16-20.
[6]赵敏,魏兴东,汪春蕾,等.细菌漆酶研究方法进展[J].中国造纸学报,2008,23(3):107-114.Zhao M,Wei X D,Wang C L,et al..Research progress of bacterial laccase[J].Trans.China Pulp.Paper,2008,23(3):107-114.
[7]Givaudan A,Effosse A,Faure D,et al..Polyphenoloxidase in Azospirillum lipoferum isolated from rice rhizosphere:Evidence for laccase activity innonmotile strains of Azospi-rillumlipoferum[J].FEMS Microb.Lett.,1993,108:205-210.
[8]Aoife M M,Evelyn M D,Sarah B,et al..Biochemical characterization of the coexisting tyrosinase and laccase in the soil bacterium Pseudomonas putida F6[J].Enzyme Microb.Technol.,2007,40(5):1435-1441.
[9]Niladevi K N,Sukumaran R K,Jacob N,et al..Optimization of laccase production from a novel strain---Streptomyces psammoticus using response surface methodology[J].Microbiol.Res.,2009,164(1):105-113.
[10]Solano F,Garcia E,Egea E,et al..Isolation and characterization of strain MMB1 a novel melanogenic marine bacterium[J].Appl.Environ.Microbiol.,1997,63:3499-3506.
[11]Susana C S,Gloria M D,Yaacov O.Laccase activity in melanin-producing strains of Sinorhizobium meliloti[J].FEMSMicrob.Lett.,2002,209(1):119-125.
[12]Brown N L,Barrett S R,Camakaris J,et al..Molecular gene and transport analysis of the copper resistance determinant from Escherichia coli plasmid pdR J1004[J].Mol.Microbiol.,1995,17:1153-1166.
[13]Hisashi H,Mitsuhide N,Hidenobu K,et al..Isolation of five laccase gene sequences from the white-rot fungus Trametes sanguinea by PCR,and cloning,characterization and expression of the laccase c DNA in yeasts[J].J.Biosci.Bioengin.,2001,92(4):372-380.
[14]郭梅,蒲军,杜连祥,等.杂色云芝漆酶基因(Lcc1)的克隆及在甲醇毕赤酵母中的表达[J].菌物学报,2005,23(2):221-226.Guo M,Pu J,Du L X,et al..Cloning of Variegated luciferase gene(Lcc1)and expression in methanol pichia pastoris[J].Mycosystema,2005(2):221-226.
[15]张银波,姜琼,江木兰,等.金针菇漆酶基因的克隆及其在毕赤酵母中的表达研究[J].微生物学报,2004(6):775-778.Zhang Y B,Jiang Q,Jiang M L,et al..Cloning of a laccase gene from Flammulina velutipes and study on its expression in Pichia pastoris[J].Acta Microbiol.Sin.,2004,44(6):775-778.
[16]Liu W,Chao Y,Liu S,et al..Molecular cloning and characterization of a laccase gene from the basidiomycete Fome lignosus and expression in Pichia pastoris[J].Appl.Microbiol.Biotechnol.,2003,63(2):174-181.
[17]张银波,江木兰,胡小加,等.平菇漆酶基因在毕赤酵母中的分泌表达及酶学性质研究[J].微生物学报,2005,4(4):625-628.Zhang Y B,Jiang M L,Hu X J,et al..Study on the secretion expression and enzymatic properties of Pleurotus ostreatus laccase in Pichia pastoris[J].Acta Microb.Sin.,2005:4(4):625-628.
[18]杨建强,刘刚,汤国营,等.改构的野生革耳漆酶基因在毕赤酵母中的表达和活性鉴定[J].生物技术通讯,2005,6(3):225-258.Yang J Q,Liu G,Tang G Y,et al..Expression and activity identification of modified wild-ear lactase gene in Pichia coli[J].Lett.Biotechnol.,2005,6(3):225-58.
[19]Soden D M,Callaghan J,Dobson A D W.Molecular cloning of a laccase isozyme gene from Pleurotus sajorcaju and expression in the heterologous Pichia pastoris host[J].Microbiol.SGM,2002,14(8):4003-4014.
[20]Kiiskinen L L,Saloheimo M.Molecular cloning and expression in Saccharomyces cerevisiae of a laccase gene from the ascomycete Melan℃arpus albomyces[J].Appl.Environ.Microbiol.,2004,70(1):137-144.
[21]谌斌,唐雪明,沈微,等.粗糙脉孢菌漆酶基因的克隆及在毕赤酵母中的初步表达[J].食品与生物技术学报,2006,8(4):43-46.Chen B,Tang X M,Shen W,et al..Cloning and preliminary expression of Coarse neurospora laccae gene in Pichia pastoris[J].J.Food Sci.Biotechnol.,2006,8(4):43-46.
[22]O'Callaghan J,O'Brien M M,Mc Clean K,et al..Optimisation of the expression of a Trametes versicolor laccase gene in Pichia pastoris[J].J.Ind.Microbiol.Biotechnol.,2002,29(2):55-59.
[23]Guan Z B,Zhang N,Song C M,et al..Molecular cloning,characterization and dye-decolorizing ability of a temperature and p H-stable laccase from Bacillus subtilis X1[J].Appl.Biochem.Biotechnol.,2014,172(3):1147-1157.
[24]Pezzella C,Autore F,Giardina P,et al..The Pleurotus ostreatus laccase multi-gene family:Isolation and heterologous expression of new family members[J].Curr.Genet.,2009,55(1):45-57.
[25]Yang J,Ng T B,Lin J,et al..A novel laccase from basidiomycete Cerrena sp.cloning,heterologous expression,and characterization[J].Int.J.Biol.Macromol.,2015,7(7):344-349.
[26]Feng B Z,Li P Q.Cloning,characterization and expression of a novel laccase gene Pclac2 from Phytophthora capsici[J].Braz J.Microbiol.,2014,45(1):351-358.
[27]Garg N,Bieler N,Kenzom T,et al..Cloning,sequence analysis,expression of Cyathusbulleri laccase in Pichia pastoris and characterization of recombinant laccase[J].BMCBiotechnol.,2012,12(03)75-77.
[28]Kittl R,Gonaus C,Pillei C,et al..Constitutive expression of Botrytisnaclada laccase in Pichia pastoris[J].Bioengineered,2012,3(4):232-235.
[29]Hong Y Z,Zhou H M,Tu X M,et al..Cloning of a laccase gene from a novel basidiomycete Trametes sp.420 and its heterologous expression in Pichia pastori[J].Curr.Microbiol.,2007,54(4):260-265.
[30]李剑凤,洪宇植,肖亚中.栓菌420漆酶C基因的克隆、高效表达及重组酶的染料脱色潜能[J].微生物学报,2007,47(1):54-58.Li J F,Hong Y Z,Xiao Y Z.Cloning and heterologous expression of the gene of laccase from Trametes sp.420 and potential of recombinant laccase in dye decolorization[J].Acta Microbiol.Sin.,2007,47(1):54-58.
[31]Xu X,Li J,Zhang W,et al..A neutral thermostableβ-1,4-Glucanase from Humicola insolens Y1 with potential for applications in various industries[J].PLoS ONE,2015,10(4):e0124925.
[32]Sambrook J,Fritsch E F,Maniatis T,et al..分子克隆实验指南[M].(第二版).金冬雁,黎孟枫,等译.北京:科学出版社,2002.
[33]Ribeiro L F,Furtado G P,Lourenzoni M R,et al..Engineering bifunctional laccase-xylanase chimeras for improved catalytic performance[J].JBIC J.Biol.Inorganic Chem.,2011,286(50):43026-43038.
[34]李泰仑.枯草芽孢杆菌WN02芽孢漆酶的性质及染料脱色研究[D].哈尔滨:东北林业大学,硕士学位论文,2010.Li T L.Properties of spore laccase from Bacillus subtilis WN02and its use in dye decolorization[D].Harbin:Northeast Forestry University,Master Dissertation,2010.
[35]Gu C,Zheng F,Long L,et al..Engineering the expression and characterization of two novel laccase isoenzymes from Coprinus comatus in Pichia pastoris by fusing an additional ten amino acids tag at N-terminus[J].PLoS ONE,2014,9(4):86-92.
[36]华欣春,陈丽丽,毕云枫,等.Coprinopsis cinerea漆酶基因的克隆及其在毕赤酵母中的表达[J].纺织学报,2012,33(10):79-83.Hua X C,Chen L L,Bi Y F,et al..Cloning of laccase gene form Coprinopsis cinerea and study on its expression in Pichia pastoris[J].J.Textile Res.,2012,33(10):79-83.
[2]Aktas N,Cicek H,Taspinar A U,et al..Reaction kinetics for laccase-catalyzed polymerization of 1-naphthol[J].Bioresour.Technol.,2001.80(1):29-36.
[3]Leontievsky A A,Myasoedova N M,Baskunov B P,et al..Transformation of 2,4,6-trichlorophenol by the white rot fungi Panus tigrinus and Coriolus versicolor[J].Biodegradation,2000,11(5):331-340.
[4]陈辉,张剑波,刘小鹏,等.漆酶催化降解氯酚类有机污染物[J].北京大学自然期刊,2005,41(4):605-611.Chen H,Zhang J B,Liu X P,et al..Degradation of chlorophenols catalyzed by laccase[J].Acta Sci.Nat.Univ.Pekinensis,2005,41(4):605-611.
[5]张鑫璐,裴继诚,张方东,等.漆酶和APMP废液中酚类物质共同改善OCC纸浆纤维性能的研究[J].中国造纸学报,2011,26(2):16-20.Zhang X L,Pei J C,Zhang F D,et al..Modification of OCCpulp by laccase with the phenols contained in APMP waste liquor[J].Trans.China Pulp.Paper,2011,26(2):16-20.
[6]赵敏,魏兴东,汪春蕾,等.细菌漆酶研究方法进展[J].中国造纸学报,2008,23(3):107-114.Zhao M,Wei X D,Wang C L,et al..Research progress of bacterial laccase[J].Trans.China Pulp.Paper,2008,23(3):107-114.
[7]Givaudan A,Effosse A,Faure D,et al..Polyphenoloxidase in Azospirillum lipoferum isolated from rice rhizosphere:Evidence for laccase activity innonmotile strains of Azospi-rillumlipoferum[J].FEMS Microb.Lett.,1993,108:205-210.
[8]Aoife M M,Evelyn M D,Sarah B,et al..Biochemical characterization of the coexisting tyrosinase and laccase in the soil bacterium Pseudomonas putida F6[J].Enzyme Microb.Technol.,2007,40(5):1435-1441.
[9]Niladevi K N,Sukumaran R K,Jacob N,et al..Optimization of laccase production from a novel strain---Streptomyces psammoticus using response surface methodology[J].Microbiol.Res.,2009,164(1):105-113.
[10]Solano F,Garcia E,Egea E,et al..Isolation and characterization of strain MMB1 a novel melanogenic marine bacterium[J].Appl.Environ.Microbiol.,1997,63:3499-3506.
[11]Susana C S,Gloria M D,Yaacov O.Laccase activity in melanin-producing strains of Sinorhizobium meliloti[J].FEMSMicrob.Lett.,2002,209(1):119-125.
[12]Brown N L,Barrett S R,Camakaris J,et al..Molecular gene and transport analysis of the copper resistance determinant from Escherichia coli plasmid pdR J1004[J].Mol.Microbiol.,1995,17:1153-1166.
[13]Hisashi H,Mitsuhide N,Hidenobu K,et al..Isolation of five laccase gene sequences from the white-rot fungus Trametes sanguinea by PCR,and cloning,characterization and expression of the laccase c DNA in yeasts[J].J.Biosci.Bioengin.,2001,92(4):372-380.
[14]郭梅,蒲军,杜连祥,等.杂色云芝漆酶基因(Lcc1)的克隆及在甲醇毕赤酵母中的表达[J].菌物学报,2005,23(2):221-226.Guo M,Pu J,Du L X,et al..Cloning of Variegated luciferase gene(Lcc1)and expression in methanol pichia pastoris[J].Mycosystema,2005(2):221-226.
[15]张银波,姜琼,江木兰,等.金针菇漆酶基因的克隆及其在毕赤酵母中的表达研究[J].微生物学报,2004(6):775-778.Zhang Y B,Jiang Q,Jiang M L,et al..Cloning of a laccase gene from Flammulina velutipes and study on its expression in Pichia pastoris[J].Acta Microbiol.Sin.,2004,44(6):775-778.
[16]Liu W,Chao Y,Liu S,et al..Molecular cloning and characterization of a laccase gene from the basidiomycete Fome lignosus and expression in Pichia pastoris[J].Appl.Microbiol.Biotechnol.,2003,63(2):174-181.
[17]张银波,江木兰,胡小加,等.平菇漆酶基因在毕赤酵母中的分泌表达及酶学性质研究[J].微生物学报,2005,4(4):625-628.Zhang Y B,Jiang M L,Hu X J,et al..Study on the secretion expression and enzymatic properties of Pleurotus ostreatus laccase in Pichia pastoris[J].Acta Microb.Sin.,2005:4(4):625-628.
[18]杨建强,刘刚,汤国营,等.改构的野生革耳漆酶基因在毕赤酵母中的表达和活性鉴定[J].生物技术通讯,2005,6(3):225-258.Yang J Q,Liu G,Tang G Y,et al..Expression and activity identification of modified wild-ear lactase gene in Pichia coli[J].Lett.Biotechnol.,2005,6(3):225-58.
[19]Soden D M,Callaghan J,Dobson A D W.Molecular cloning of a laccase isozyme gene from Pleurotus sajorcaju and expression in the heterologous Pichia pastoris host[J].Microbiol.SGM,2002,14(8):4003-4014.
[20]Kiiskinen L L,Saloheimo M.Molecular cloning and expression in Saccharomyces cerevisiae of a laccase gene from the ascomycete Melan℃arpus albomyces[J].Appl.Environ.Microbiol.,2004,70(1):137-144.
[21]谌斌,唐雪明,沈微,等.粗糙脉孢菌漆酶基因的克隆及在毕赤酵母中的初步表达[J].食品与生物技术学报,2006,8(4):43-46.Chen B,Tang X M,Shen W,et al..Cloning and preliminary expression of Coarse neurospora laccae gene in Pichia pastoris[J].J.Food Sci.Biotechnol.,2006,8(4):43-46.
[22]O'Callaghan J,O'Brien M M,Mc Clean K,et al..Optimisation of the expression of a Trametes versicolor laccase gene in Pichia pastoris[J].J.Ind.Microbiol.Biotechnol.,2002,29(2):55-59.
[23]Guan Z B,Zhang N,Song C M,et al..Molecular cloning,characterization and dye-decolorizing ability of a temperature and p H-stable laccase from Bacillus subtilis X1[J].Appl.Biochem.Biotechnol.,2014,172(3):1147-1157.
[24]Pezzella C,Autore F,Giardina P,et al..The Pleurotus ostreatus laccase multi-gene family:Isolation and heterologous expression of new family members[J].Curr.Genet.,2009,55(1):45-57.
[25]Yang J,Ng T B,Lin J,et al..A novel laccase from basidiomycete Cerrena sp.cloning,heterologous expression,and characterization[J].Int.J.Biol.Macromol.,2015,7(7):344-349.
[26]Feng B Z,Li P Q.Cloning,characterization and expression of a novel laccase gene Pclac2 from Phytophthora capsici[J].Braz J.Microbiol.,2014,45(1):351-358.
[27]Garg N,Bieler N,Kenzom T,et al..Cloning,sequence analysis,expression of Cyathusbulleri laccase in Pichia pastoris and characterization of recombinant laccase[J].BMCBiotechnol.,2012,12(03)75-77.
[28]Kittl R,Gonaus C,Pillei C,et al..Constitutive expression of Botrytisnaclada laccase in Pichia pastoris[J].Bioengineered,2012,3(4):232-235.
[29]Hong Y Z,Zhou H M,Tu X M,et al..Cloning of a laccase gene from a novel basidiomycete Trametes sp.420 and its heterologous expression in Pichia pastori[J].Curr.Microbiol.,2007,54(4):260-265.
[30]李剑凤,洪宇植,肖亚中.栓菌420漆酶C基因的克隆、高效表达及重组酶的染料脱色潜能[J].微生物学报,2007,47(1):54-58.Li J F,Hong Y Z,Xiao Y Z.Cloning and heterologous expression of the gene of laccase from Trametes sp.420 and potential of recombinant laccase in dye decolorization[J].Acta Microbiol.Sin.,2007,47(1):54-58.
[31]Xu X,Li J,Zhang W,et al..A neutral thermostableβ-1,4-Glucanase from Humicola insolens Y1 with potential for applications in various industries[J].PLoS ONE,2015,10(4):e0124925.
[32]Sambrook J,Fritsch E F,Maniatis T,et al..分子克隆实验指南[M].(第二版).金冬雁,黎孟枫,等译.北京:科学出版社,2002.
[33]Ribeiro L F,Furtado G P,Lourenzoni M R,et al..Engineering bifunctional laccase-xylanase chimeras for improved catalytic performance[J].JBIC J.Biol.Inorganic Chem.,2011,286(50):43026-43038.
[34]李泰仑.枯草芽孢杆菌WN02芽孢漆酶的性质及染料脱色研究[D].哈尔滨:东北林业大学,硕士学位论文,2010.Li T L.Properties of spore laccase from Bacillus subtilis WN02and its use in dye decolorization[D].Harbin:Northeast Forestry University,Master Dissertation,2010.
[35]Gu C,Zheng F,Long L,et al..Engineering the expression and characterization of two novel laccase isoenzymes from Coprinus comatus in Pichia pastoris by fusing an additional ten amino acids tag at N-terminus[J].PLoS ONE,2014,9(4):86-92.
[36]华欣春,陈丽丽,毕云枫,等.Coprinopsis cinerea漆酶基因的克隆及其在毕赤酵母中的表达[J].纺织学报,2012,33(10):79-83.Hua X C,Chen L L,Bi Y F,et al..Cloning of laccase gene form Coprinopsis cinerea and study on its expression in Pichia pastoris[J].J.Textile Res.,2012,33(10):79-83.