白蚁及其共生菌来源的4种木质纤维素酶的共表达
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摘要
天然的木质纤维素材料含有纤维素、半纤维素和木质素等成分。降解天然木质纤维素底物时,需要木质纤维素酶共同作用。近年在木质纤维素酶的相互协同作用方面的研究引起人们的关注,成为一个新的研究热点,文中使用两个不同的共表达载体pETDuet-1和pRSFDuet-1,在大肠杆菌中共表达了白蚁及其肠道微生物来源的β-葡萄糖苷酶、内切β-1,4-葡聚糖酶、漆酶和木聚糖酶这4种木质纤维素酶,经过SDS-PAGE分析得到了与理论值一致的蛋白条带,同时经过酶活验证,这4种蛋白都具有酶活性。以磷酸处理的微晶纤维素(PASC)为底物,测定了共表达酶粗酶液与单独表达酶混合液的协同作用因子,从还原糖的产量上经计算共表达的粗酶液比单独表达酶的混合液对PASC的降解协同作用提高44%;以滤纸和磷酸处理的玉米芯为底物,测定降解协同作用,分别提高了34%和20%。结果表明,共表达酶的降解效率要高于混合的单组分酶液降解效率的总和。
Natural lignocellulosic materials contain cellulose, hemicellulose, and lignin. Cellulose hydrolysis to glucose requires a series of lignocellulases. Recently, the research on the synergistic effect of lignocellulases has become a new research focus. Here, four lignocellulase genes encoding β-glucosidase, endo-1,4-β-glucanase, xylanase and laccase from termite and their endosymbionts were cloned into pETDuet-1 and pRSFDuet-1 and expressed in Escherichia coli. After SDS-PAGE analysis, the corresponding protein bands consistent with the theoretical values were observed and all the proteins showed enzyme activities. We used phosphoric acid swollen cellulose(PASC) as substrate to measure the synergistic effect of crude extracts of co-expressing enzymes and the mixture of single enzyme. The co-expressed enzymes increased the degradation efficiency of PASC by 44% compared with the single enzyme mixture; while the degradation rate increased by 34% and 20%, respectively when using filter paper and corn cob pretreated with phosphoric acid as substrates. The degradation efficiency of the co-expressed enzymes was higher than the total efficiency of the single enzyme mixture.
Natural lignocellulosic materials contain cellulose, hemicellulose, and lignin. Cellulose hydrolysis to glucose requires a series of lignocellulases. Recently, the research on the synergistic effect of lignocellulases has become a new research focus. Here, four lignocellulase genes encoding β-glucosidase, endo-1,4-β-glucanase, xylanase and laccase from termite and their endosymbionts were cloned into pETDuet-1 and pRSFDuet-1 and expressed in Escherichia coli. After SDS-PAGE analysis, the corresponding protein bands consistent with the theoretical values were observed and all the proteins showed enzyme activities. We used phosphoric acid swollen cellulose(PASC) as substrate to measure the synergistic effect of crude extracts of co-expressing enzymes and the mixture of single enzyme. The co-expressed enzymes increased the degradation efficiency of PASC by 44% compared with the single enzyme mixture; while the degradation rate increased by 34% and 20%, respectively when using filter paper and corn cob pretreated with phosphoric acid as substrates. The degradation efficiency of the co-expressed enzymes was higher than the total efficiency of the single enzyme mixture.
引文
[1]Ohkuma M.Termite symbiotic systems:efficientbio-recycling of lignocellulose.Appl Microbiol Biotechnol,2003,61(1):1-9.
[2]Li HJ,Yelle DJ,Li C,et al.Lignocellulose pretreatment in a fungus-cultivating termite.Proc Natl Acad Sci USA,2017,114(18):4709-4714.
[3]Gardner KH,Blackwell J.The structure of native cellulose.Biopolymers,1974,13(10):1975-2001.
[4]Woodward J,Wiseman A.Fungal and otherβ-D-glucosidases-their properties and applications.Enzyme Microbial Technol,1982,4(2):73-79.
[5]Angelov A,Pham VTT,übelacker M,et al.Ametagenome-derived thermostableβ-glucanase with an unusual module architecture which defines the new glycoside hydrolase family GH148.Sci Rep,2017,7(1):17306.
[6]Watanabe H,Tokuda G.Cellulolytic systems in insects.Ann Rev Entomol,2010,55(1):609-632.
[7]Polizeli MLTM,Rizzatti ACS,Monti R,et al.Xylanases from fungi:properties and industrial applications.Appl Microbiol Biotechnol,2005,67(5):577-591.
[8]Chávez R,Bull P,Eyzaguirre J.The xylanolytic enzyme system from the genus Penicillium.JBiotechnol,2006,123(4):413-433.
[9]Shang TT,Si DY,Zhang DY,et al.Enhancement of thermoalkaliphilic xylanase production by Pichia pastoris through novel fed-batch strategy in high cell-density fermentation.BMC Biotechnol,2017,17:55.
[10]Raud M,Tutt M,Olt J,et al.Effect of lignin content of lignocellulosic material on hydrolysis efficiency.Agron Res,2015,13(2):405-412.
[11]Donovan SE,Eggleton P,Bignell DE.Gut content analysis and a new feeding group classification of termites.Ecol Entomol,2001,26(4):356-366.
[12]Nakashima K,Watanabe H,Saitoh H,et al.Dual cellulose-digesting system of the wood-feeding termite,Coptotermes formosanus Shiraki.Insect Biochem Molec Biol,2002,32(7):777-784.
[13]Ni JF,Tokuda G,Takehara M,et al.Heterologous expression and enzymatic characterization ofβ-glucosidase from the drywood-eating termite,Neotermes koshunensis.Appl Entomol Zool,2007,42(3):457-463.
[14]Ni JF,Takehara M,Miyazawa M,et al.Random exchanges of non-conserved amino acid residues among four parental termite cellulases by family shuffling improved thermostability.Protein Eng Des Select,2007,20(11):535-542.
[15]Ning N.Cloning and expression of lignocellulase genes from termites and their endosymbionts[D].Ji’nan:Shandong University,2017(in Chinese).宁娜.白蚁及其肠道微生物来源木质纤维素酶基因的克隆与表达[D].济南:山东大学,2017.
[16]Shi XY.Purification and cloning the bacterial xylanase from the hindgut of Macrotermes barneyi and the microbial diversity in combs of fungus-growing termites[D].Ji’nan:Shandong University,2015(in Chinese).石小玉.黄翅大白蚁后肠细菌来源木聚糖酶的纯化与基因克隆以及培菌白蚁菌圃微生物多样性研究[D].济南:山东大学,2015.
[17]Martins LO,Soares CM,Pereira MM,et al.Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat.J Biol Chem,2002,277(21):18849-18859.
[18]Sawant SS,Salunke BK,Kim BS.Degradation of corn stover by fungal cellulase cocktail for production of polyhydroxyalkanoates by moderate halophile Paracoccus sp.LL1.Bioresource Technol,2015,194:247-255.
[19]Dong WL,Xue ML,Zhang Y,et al.Characterization of aβ-glucosidase from Paenibacillus species and its application for succinic acid production from sugarcane bagasse hydrolysate.Bioresource Technol,2017,241:309-316.
[20]Gao HY,Liu ZC,Duan SW,et al.Coexpression ofβ-mannanase and xylanase genes in Escherichia coli.Microbiology China,2012,39(3):344-352(in Chinese).高海有,刘正初,段盛文,等.β-甘露聚糖酶和木聚糖酶基因在大肠杆菌中共表达.微生物学通报,2012,39(3):344-352.
[21]Riou C,Salmon JM,Vallier MJ,et al.Purification,characterization,and substrate specificity of a novel highly glucose-tolerantβ-glucosidase from Aspergillus oryzae.Appl Environ Microbiol,1998,64(10):3607-3614.
[22]Ni JF,Wu Y,Yun C,et al.cDNA cloning and heterologous expression of an endo-β-1,4-glucanase from the fun-growing termite Macrotermes barneyi.Arch Insect Biochem Physiol,2014,86(3):151-164.
[23]Miller GL.Use of dinitrosalicylic acid reagent for determination of reducing sugar.Anal Chem,1959,31(3):426-428.
[24]Geng AL,Wu J,Xie RR,et al.Characterization of a laccase from a wood-feeding termite,Coptotermes formosanus.Insect Sci,2018,25(2):251-258.
[25]Yu XX,Liu Y,Cui YX,et al.Measurement of filter paper activities of cellulase with microplate-based assay.Saudi J Biol Sci,2016,23(S1):S93-S98.
[26]Zhang YHP,Cui JB,Lynd LR,et al.A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid:evidence from enzymatic hydrolysis and supramolecular structure.Biomacromolecules,2006,7(2):644-648.
[27]Banerjee G,Car S,Scott-Craig JS,et al.Alkaline peroxide pretreatment of corn stover:effects of biomass,peroxide,and enzyme loading and composition on yields of glucose and xylose.Biotechnol Biofuels,2011,4:16.
[28]Liu JM.Screening for cellobiohydrolase genes and the secretion of thermostable cellulase in Bacillus subtilis[D].Shanghai:East China University of Science and Technology,2011(in Chinese).刘建明.细菌纤维素外切酶基因的筛选及耐热纤维素酶基因在枯草杆菌系统里的表达[D].上海:华东理工大学,2011.
[29]Zhang YHP,Lynd LR.Toward an aggregated understanding of enzymatic hydrolysis of cellulose:noncomplexed cellulase systems.Biotechnol Bioeng,2004,88(7):797-824.
[30]Fonseca-Maldonado R,Ribeiro LF,Furtado GP,et al.Synergistic action of co-expressed xylanase/laccase mixtures against milled sugar cane bagasse.Process Biochem,2014,49(7):1152-1161.
[31]Kumar S,Jain KK,Bhardwaj KN,et al.Multiple genes in a single host:cost-effective production of bacterial laccase(cotA),pectate lyase(pel),and endoxylanase(xyl)by simultaneous expression and cloning in single vector in E.coli.PLoS ONE,2015,10(12):e0144379.
[2]Li HJ,Yelle DJ,Li C,et al.Lignocellulose pretreatment in a fungus-cultivating termite.Proc Natl Acad Sci USA,2017,114(18):4709-4714.
[3]Gardner KH,Blackwell J.The structure of native cellulose.Biopolymers,1974,13(10):1975-2001.
[4]Woodward J,Wiseman A.Fungal and otherβ-D-glucosidases-their properties and applications.Enzyme Microbial Technol,1982,4(2):73-79.
[5]Angelov A,Pham VTT,übelacker M,et al.Ametagenome-derived thermostableβ-glucanase with an unusual module architecture which defines the new glycoside hydrolase family GH148.Sci Rep,2017,7(1):17306.
[6]Watanabe H,Tokuda G.Cellulolytic systems in insects.Ann Rev Entomol,2010,55(1):609-632.
[7]Polizeli MLTM,Rizzatti ACS,Monti R,et al.Xylanases from fungi:properties and industrial applications.Appl Microbiol Biotechnol,2005,67(5):577-591.
[8]Chávez R,Bull P,Eyzaguirre J.The xylanolytic enzyme system from the genus Penicillium.JBiotechnol,2006,123(4):413-433.
[9]Shang TT,Si DY,Zhang DY,et al.Enhancement of thermoalkaliphilic xylanase production by Pichia pastoris through novel fed-batch strategy in high cell-density fermentation.BMC Biotechnol,2017,17:55.
[10]Raud M,Tutt M,Olt J,et al.Effect of lignin content of lignocellulosic material on hydrolysis efficiency.Agron Res,2015,13(2):405-412.
[11]Donovan SE,Eggleton P,Bignell DE.Gut content analysis and a new feeding group classification of termites.Ecol Entomol,2001,26(4):356-366.
[12]Nakashima K,Watanabe H,Saitoh H,et al.Dual cellulose-digesting system of the wood-feeding termite,Coptotermes formosanus Shiraki.Insect Biochem Molec Biol,2002,32(7):777-784.
[13]Ni JF,Tokuda G,Takehara M,et al.Heterologous expression and enzymatic characterization ofβ-glucosidase from the drywood-eating termite,Neotermes koshunensis.Appl Entomol Zool,2007,42(3):457-463.
[14]Ni JF,Takehara M,Miyazawa M,et al.Random exchanges of non-conserved amino acid residues among four parental termite cellulases by family shuffling improved thermostability.Protein Eng Des Select,2007,20(11):535-542.
[15]Ning N.Cloning and expression of lignocellulase genes from termites and their endosymbionts[D].Ji’nan:Shandong University,2017(in Chinese).宁娜.白蚁及其肠道微生物来源木质纤维素酶基因的克隆与表达[D].济南:山东大学,2017.
[16]Shi XY.Purification and cloning the bacterial xylanase from the hindgut of Macrotermes barneyi and the microbial diversity in combs of fungus-growing termites[D].Ji’nan:Shandong University,2015(in Chinese).石小玉.黄翅大白蚁后肠细菌来源木聚糖酶的纯化与基因克隆以及培菌白蚁菌圃微生物多样性研究[D].济南:山东大学,2015.
[17]Martins LO,Soares CM,Pereira MM,et al.Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat.J Biol Chem,2002,277(21):18849-18859.
[18]Sawant SS,Salunke BK,Kim BS.Degradation of corn stover by fungal cellulase cocktail for production of polyhydroxyalkanoates by moderate halophile Paracoccus sp.LL1.Bioresource Technol,2015,194:247-255.
[19]Dong WL,Xue ML,Zhang Y,et al.Characterization of aβ-glucosidase from Paenibacillus species and its application for succinic acid production from sugarcane bagasse hydrolysate.Bioresource Technol,2017,241:309-316.
[20]Gao HY,Liu ZC,Duan SW,et al.Coexpression ofβ-mannanase and xylanase genes in Escherichia coli.Microbiology China,2012,39(3):344-352(in Chinese).高海有,刘正初,段盛文,等.β-甘露聚糖酶和木聚糖酶基因在大肠杆菌中共表达.微生物学通报,2012,39(3):344-352.
[21]Riou C,Salmon JM,Vallier MJ,et al.Purification,characterization,and substrate specificity of a novel highly glucose-tolerantβ-glucosidase from Aspergillus oryzae.Appl Environ Microbiol,1998,64(10):3607-3614.
[22]Ni JF,Wu Y,Yun C,et al.cDNA cloning and heterologous expression of an endo-β-1,4-glucanase from the fun-growing termite Macrotermes barneyi.Arch Insect Biochem Physiol,2014,86(3):151-164.
[23]Miller GL.Use of dinitrosalicylic acid reagent for determination of reducing sugar.Anal Chem,1959,31(3):426-428.
[24]Geng AL,Wu J,Xie RR,et al.Characterization of a laccase from a wood-feeding termite,Coptotermes formosanus.Insect Sci,2018,25(2):251-258.
[25]Yu XX,Liu Y,Cui YX,et al.Measurement of filter paper activities of cellulase with microplate-based assay.Saudi J Biol Sci,2016,23(S1):S93-S98.
[26]Zhang YHP,Cui JB,Lynd LR,et al.A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid:evidence from enzymatic hydrolysis and supramolecular structure.Biomacromolecules,2006,7(2):644-648.
[27]Banerjee G,Car S,Scott-Craig JS,et al.Alkaline peroxide pretreatment of corn stover:effects of biomass,peroxide,and enzyme loading and composition on yields of glucose and xylose.Biotechnol Biofuels,2011,4:16.
[28]Liu JM.Screening for cellobiohydrolase genes and the secretion of thermostable cellulase in Bacillus subtilis[D].Shanghai:East China University of Science and Technology,2011(in Chinese).刘建明.细菌纤维素外切酶基因的筛选及耐热纤维素酶基因在枯草杆菌系统里的表达[D].上海:华东理工大学,2011.
[29]Zhang YHP,Lynd LR.Toward an aggregated understanding of enzymatic hydrolysis of cellulose:noncomplexed cellulase systems.Biotechnol Bioeng,2004,88(7):797-824.
[30]Fonseca-Maldonado R,Ribeiro LF,Furtado GP,et al.Synergistic action of co-expressed xylanase/laccase mixtures against milled sugar cane bagasse.Process Biochem,2014,49(7):1152-1161.
[31]Kumar S,Jain KK,Bhardwaj KN,et al.Multiple genes in a single host:cost-effective production of bacterial laccase(cotA),pectate lyase(pel),and endoxylanase(xyl)by simultaneous expression and cloning in single vector in E.coli.PLoS ONE,2015,10(12):e0144379.
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