黑胸散白蚁体内产纤维素酶细菌的筛选及其纤维素酶基因的鉴定与原核表达
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摘要
白蚁(Isoptera)常以富含纤维素的木材为食,体内存在纤维素酶产生菌。从源于陕西佛坪的白蚁(经鉴定为黑胸散白蚁(Reticulitermes chinensis))体内筛选出了4株产纤维素酶菌株,采用单因素实验设计对酶活最高的菌株进行了产酶条件优化,并对其所产纤维素酶进行了酶学性质研究;同时,依据NCBI数据库设计的引物扩增出了该菌株的内切-β-1,4-葡聚糖酶基因Cbs和β-葡萄糖苷酶基因Ba G,之后在大肠杆菌(Escherichia coli)中进行了表达实验。结果表明:从黑胸散白蚁体内筛选出4株菌分别是阿氏肠杆菌(Enterobacter asburiae)、炭疽芽胞杆菌(Bacillus cereus)、枯草芽胞杆菌(Bacillus subtilis)、解淀粉芽胞杆菌(Bacillus amyloliquefaciens),其中枯草芽胞杆菌所产纤维素酶的活力最高,该菌发酵产酶的最适温度和pH分别是42℃、6.5,酶促反应的最适温度和pH分别是70℃、4.5,在此条件下该菌株酶活力为2.36 U/mL。该菌株所产的纤维素酶在50℃、pH 6.5的条件下热稳定性和pH稳定性最佳,使用该菌株所产的粗酶液发酵粗饲料,结果发现其对玉米(Zea mays)秸秆、小麦(Triticum aestivum)秸秆、苜蓿(Medicago polymorpha)干草和青贮饲料的粗纤维降解率分别为12.21%、1.3%、3.24%和17.42%。基因克隆结果表明该菌株具有Ba G、Cbs 2个纤维素酶基因,且大肠杆菌表达结果显示Ba G基因的粗酶液pro Ba G无纤维素酶活性,而Cbs基因的粗酶液pro Cbs在60℃、pH 5.0时酶活可达4.14 U/mL,将Ba G和Cbs基因进行原核融合表达,产生的蛋白约35 k D,其在最适条件70℃、pH 4.5时的酶活为4.57 U/mL,说明无纤维素酶活性的Ba G基因与Cbs基因融合后可能表达出了一个活性更高的纤维素酶蛋白。本研究对后期构建可降解木质纤维素的人工重组菌具有重要的理论价值。
Termites(Isoptera) often eat wood rich in cellulose,and there are much bacteria producing cellulase in vivo.The aim of this study was to screen a strain of producing highly active cellulase from termites to amplify the cellulase gene of this strain and express it in Escherichia coli,which had a potential basis for the the construction of cellulase gene recombinant bacteria.Collecting termites samples from Foping County Shaanxi province,this study screened 4 strains of producing cellulase from termites(Reticulitermes chinensin vivo by cellulase screening medium and measurements of cellulase activity;then optimized the fermentaticonditions were conducted by single factor experiment on the strain which producing the highest acticellulase,and studied the enzymatic properties of cellulase.Finally,according to the data of NCBI primewhich amplified the cellulase genes were designed and prokaryotic expression was completed.The resushowed that the 4 cellulase producing strains screening from termites were Enterobacter asburiae,Bacillcereus,Bacillus subtilis and Bacillus amyloliquefaciens,respectively.The cellulase activity of the Bacillsubtilis was the highest,and the optimum fermentation temperature and pH of the strain was 42 ℃ and 6.5.Toptimum temperature and pH of enzymatic reaction was 70 ℃ and 4.5,under this condition,the cellulaactivity reached 2.36 U/mL.Under the condition of 50 ℃ and pH 6.5,the thermal stability and pH stability the cellulase produced by the strain were optimum.Using crude enzyme solution to ferment crude fiber,it wfound that the degradation rate of crude enzyme solution to four kinds of feed,corn(Zea mays) stalk,whe(Triticum aestivum) straw,alfalfa(Medicago polymorpha) hay and silage corn stalk was 12.21%,1.30%,3.24 and 17.42%,respectively.Gene cloning results showed that the Bacillus cereus strain contained two cellulagenes Ba G and Cbs.Under prokaryotic expression of the cellulase gene,the pro Ba G didn't show any cellulaactivity,while the pro Cbs showed the optimum conditions of 60 ℃,pH 5.0,cellulase activity reached 4.14 mL.Under the condition of 50 ℃ and pH 5.0,the thermal stability and pH stability of the cellulase weoptimum.Finally,the fusion expression of Ba G and Cbs gene showed a new protein of about 35 k D while Band Cbs gene expressed the protein with a molecular weight of 27.4 k D and 55.2 k D,respectively.In toptimum conditions of 70 ℃ and pH 4.5,the cellulase activity of the fusion product expression reached 4.57 mL.Under the condition of 50 ℃ and pH 4.5,the thermal stability and pH stability of the cellulase weoptimum.This study indicated that the Ba G gene without cellulase activity could interact with the Cbs geand may produce a new protein with higher cellulase activity and smaller molecular weight.This study shouhave important theoretical value for the construction of recombinant bacteria which could degradalignocellulose.
Termites(Isoptera) often eat wood rich in cellulose,and there are much bacteria producing cellulase in vivo.The aim of this study was to screen a strain of producing highly active cellulase from termites to amplify the cellulase gene of this strain and express it in Escherichia coli,which had a potential basis for the the construction of cellulase gene recombinant bacteria.Collecting termites samples from Foping County Shaanxi province,this study screened 4 strains of producing cellulase from termites(Reticulitermes chinensin vivo by cellulase screening medium and measurements of cellulase activity;then optimized the fermentaticonditions were conducted by single factor experiment on the strain which producing the highest acticellulase,and studied the enzymatic properties of cellulase.Finally,according to the data of NCBI primewhich amplified the cellulase genes were designed and prokaryotic expression was completed.The resushowed that the 4 cellulase producing strains screening from termites were Enterobacter asburiae,Bacillcereus,Bacillus subtilis and Bacillus amyloliquefaciens,respectively.The cellulase activity of the Bacillsubtilis was the highest,and the optimum fermentation temperature and pH of the strain was 42 ℃ and 6.5.Toptimum temperature and pH of enzymatic reaction was 70 ℃ and 4.5,under this condition,the cellulaactivity reached 2.36 U/mL.Under the condition of 50 ℃ and pH 6.5,the thermal stability and pH stability the cellulase produced by the strain were optimum.Using crude enzyme solution to ferment crude fiber,it wfound that the degradation rate of crude enzyme solution to four kinds of feed,corn(Zea mays) stalk,whe(Triticum aestivum) straw,alfalfa(Medicago polymorpha) hay and silage corn stalk was 12.21%,1.30%,3.24 and 17.42%,respectively.Gene cloning results showed that the Bacillus cereus strain contained two cellulagenes Ba G and Cbs.Under prokaryotic expression of the cellulase gene,the pro Ba G didn't show any cellulaactivity,while the pro Cbs showed the optimum conditions of 60 ℃,pH 5.0,cellulase activity reached 4.14 mL.Under the condition of 50 ℃ and pH 5.0,the thermal stability and pH stability of the cellulase weoptimum.Finally,the fusion expression of Ba G and Cbs gene showed a new protein of about 35 k D while Band Cbs gene expressed the protein with a molecular weight of 27.4 k D and 55.2 k D,respectively.In toptimum conditions of 70 ℃ and pH 4.5,the cellulase activity of the fusion product expression reached 4.57 mL.Under the condition of 50 ℃ and pH 4.5,the thermal stability and pH stability of the cellulase weoptimum.This study indicated that the Ba G gene without cellulase activity could interact with the Cbs geand may produce a new protein with higher cellulase activity and smaller molecular weight.This study shouhave important theoretical value for the construction of recombinant bacteria which could degradalignocellulose.
引文
曹平华.2016.多元纤维素酶基因乳酸杆菌共表达载体的构建及无抗标记乳酸杆菌重组技术的研究[D].博士学位论文.西北农林科技大学.导师:陈玉林.pp.105-108(Cao P H.2016.Construction of lactobacillus coexpression vector of multiple cellulase genes and study on the non-antibiotic marker site-directed integration technology in Lactobacillus[D].Thesis for p H.D.,Northwest A&F University,Supervisor:Chen Y L,pp.105-108)
程东保,杨兆芬.2014.白蚁学[M].北京:科学出版社,pp.77-107.(Cheng D B,Yang Z F.2014.Termites to learn[M].Beijing:Science Press,pp.77-107.)
丁轲,邱静静,罗伟光,等.2017.2个枯草芽孢杆菌源纤维素酶基因的克隆、融合表达及其酶学性质分析[J].动物营养学报,29(08):2725-2733.(Ding K,Qiu J J,Luo W G,et al.2017.Cloning,Fusion expression of two cellulase genes from Bacillus subtilis and its enzymatic properties[J].Chinese Journal of Animal Nutrition,29(08):2725-2733.)
董丹,车振明,关统伟.2015.白酒酒糟中产纤维素酶菌株的筛选及其酶活力特性检测[J].中国酿造,34(08):44-48.(Dong D,Che Z M,Guan T W.2015.Screening of cellulose-producing strains and enzyme activity characteristic detection in distiller's grains[J].China Brewing,34(08):44-48.)
韩学易.2006.枯草芽孢杆菌纤维素酶产酶条件及酶学性质研究[D].硕士学位论文.四川农业大学.导师:陈惠.pp.14-30.(Han X Y.2006.Fermentation condition and properties of Bacillus Subtilis C-36 cellulase[D].Thesis for M.S.,Sichuan Agricultural University.Supervisor:Chen H,pp.14-30.)
何刚强,堵国成,刘立明,等.2009.从白蚁中分离筛选纤维素分解菌及其产酶性质[J].食品与生物技术学报,28(03):352-355.(He G Q,Zhu G C,Liu LM,et al.Screening and crude enzyme property of a cellulose-decoposing strain[J].Journal of Food Science and Biotechnology,28(03):352-355.)
胡艳平,王磊,曹平华,等.2013.纤维素酶产生菌的筛选、其酶学性质及对饲料粗纤维降解效果的研究[J].饲料工业,34(08):21-27.(Hu Y P,Wang L,Cao P H,et al.Study of screening of a strain producing cellulase,its enzymatic properties and the degradation rate of crude fiber in the feed[J].Feed Industry,34(08):21-27.)
胡艳平.2013.纤维素酶产生菌的筛选、其酶学性质与纤维素酶基因的克隆及表达[D].硕士学位论文.西北农林科技大学.导师:陈玉林.pp.1-17(Hu Y P.2013.Screening of a strain producing cellulase,its enzymatic properties and clone and expression of the cellulase gene[D].Thesis for M.S.,Northwest A&F University,Supervisor:Chen Y L,pp.1-17)
黄复生,张英俊,张志端.1986.陕西省白蚁的分布及其新种记述[J].昆虫分类学报,(03):215-219.(Huang F S,Zhang Y J,Zhang Z D.1986.The distribution of termites and their new species in Shaanxi[J].Entomotaxonomia,(03):215-219.)
黄复生,朱世模,李桂祥.1988.白蚁的外部形态和分类系统[J].动物学研究,(03):301-307.(Huang F S,Zhu S M,Li G X.1988.The external morphology and classification system of termites[J].Zoological Research,(03):301-307.)
李博.2017.白蚁肠道细菌的多样性和生物活性研究[D].硕士学位论文,郑州大学,导师:秦广雍.pp.1~8.(Li B.2017.Study on diversity and bioactivity of intestinal microorganisms in termite[D].Thesis for M.S.,Zhengzhou University,Supervisor:Qin G Y,pp.1~8.)
李春梅,何江波,陈惠等.2011.高产中性纤维素酶的巨大芽胞杆菌基因工程菌发酵工艺条件优化[J].农业生物技术学报,19(3):557-564.(Li C M,He J B,Chen H,et al.2011.Optimization of fermentation conditions for producing neutral cellulase from a high-yield Bacillus megaterium genetic engineering bacteria[J].Journal of Agricultural Biotechnology,19(3):557~564.)
李丹红,王誉,杨红.2017.高效降解木质纤维素的白蚁肠道微生物组[J].微生物学报,57(6):876-884.(Li D H,Wang Y,Yang H.2017.Gut microbiome of wood-feeding termites[J].Acta Microbiologica Sinica,57(6):876~884.)
李永博,暴金磊,万敏,等.2017.酒醅中高产纤维素酶菌株的筛选及其酶学性质研究[J].食品工业科技,38(24):109-113.(Li Y B,Bao J L,Wan M,et al.2017.Study on screening of high-yield cellulase strains in the fermented grains and its enzymatic properties[J].Science and Technology of Food Industry,38(24):109-113.)
龙海波,魏丽莎子,赵志祥等.2017.象耳豆根结线虫纤维素结合蛋白Me-cbp-1基因的克隆与功能分析[J].农业生物技术学报,25(2):196~204.(Long H B,Wei L S Z,Zhao Z X,et al.2017.Cloning and Functional Analysis of a Cellulose Binding Protein Gene Me-cbp-1 from the root-knot nematode meloidogyne enterolobii[J].Journal of Agricultural Biotechnology,25(2):196~204.)
王胤.2012.黑胸散白蚁肠道内几种共生细菌的定量、定位及分离菌株Lactococcus sp.的初步研究[D].硕士学位论文.华中师范大学,导师:杨红.pp.21-36.(Wang Y.Quantification and localization of several groups in the gut of Reticulitermes chinensis snyder and primary characterization of a Lactococcus sp.strain[D].Thesis for M.S.Central China Normal University.Supervisor:Yang H,pp.21-36)
尉吉乾,莫建初,徐文,等.2010.黑胸散白蚁的研究进展[J].中国媒介生物学及控制杂志,21(06):635-637.(Wei JQ,Mo J C,Xu W,et al.2010.Advances in research on Reticulitermes chinensis(Isoptera:Rhinotermitidae)in China[J].Chinese Journal of Vector Biology and Control,21(06):635-637.)
Bhat M K,Bhat S.1997.Cellulose degrading enzymes and their potential industrial applications[J].Biotechnology Advances,15(3-4):583-620.
James S W,Patricia J H K,Mukram M M,et al.2017.Cloning,production and characterization of a glycoside hydrolase family 7 enzyme from the gut microbiota of the termite coptotermes curvignathus[J].Springer,59(7):271~283.
Li W,Huan X,Zhou Y,et al.2009.Simultaneous cloning and expression of two cellulase genes from Bacillus subtilis newly isolated from Golden Takin(Budorcas taxicolor Bedfordi)[J].Biochemical and Biophysical Research Communications,383:397-400.
Miller G.L.1959.Use of dinitrosalicylic acid reagent for determination of reducing sugar[J].Analytical chemistry,31(3):426-428.
Ni J,Tokuda G.2013.Lignocellulose-degrading enzymes from termites and their symbiotic microbiota[J].Biotechnology Advances,31:838-850.
Perez F,Garcia O,Martinez C,et al.2015.Genome sequence of Citrobacter sp.Ct B7.12,isolated from the gut of the desert subterranean termite Heterotermes aureus[J].Genome Announcements,3(6):1-2.
Tarayre C,Brognaux A,Bauwens J,et al.2014.Isolation of amylolytic,xylanolytic,and cellulolytic microorganisms extracted from the gut of the termite Reticulitermes santonensis by means of a micro-aerobic atmosphere[J].Springer,30(5):1655-1660.
Watanabe H,Noda H.1998.A cellulase gene of termite origin[J].Nature.394:330-331.
Zhao K,Duan J,Ma X,et al.2016.Isolation,identification and expression of microbial cellulases from the gut of Odontotermes formosanus[J].Journal of Microbiology and Biotechnology,27(1):1-28.
程东保,杨兆芬.2014.白蚁学[M].北京:科学出版社,pp.77-107.(Cheng D B,Yang Z F.2014.Termites to learn[M].Beijing:Science Press,pp.77-107.)
丁轲,邱静静,罗伟光,等.2017.2个枯草芽孢杆菌源纤维素酶基因的克隆、融合表达及其酶学性质分析[J].动物营养学报,29(08):2725-2733.(Ding K,Qiu J J,Luo W G,et al.2017.Cloning,Fusion expression of two cellulase genes from Bacillus subtilis and its enzymatic properties[J].Chinese Journal of Animal Nutrition,29(08):2725-2733.)
董丹,车振明,关统伟.2015.白酒酒糟中产纤维素酶菌株的筛选及其酶活力特性检测[J].中国酿造,34(08):44-48.(Dong D,Che Z M,Guan T W.2015.Screening of cellulose-producing strains and enzyme activity characteristic detection in distiller's grains[J].China Brewing,34(08):44-48.)
韩学易.2006.枯草芽孢杆菌纤维素酶产酶条件及酶学性质研究[D].硕士学位论文.四川农业大学.导师:陈惠.pp.14-30.(Han X Y.2006.Fermentation condition and properties of Bacillus Subtilis C-36 cellulase[D].Thesis for M.S.,Sichuan Agricultural University.Supervisor:Chen H,pp.14-30.)
何刚强,堵国成,刘立明,等.2009.从白蚁中分离筛选纤维素分解菌及其产酶性质[J].食品与生物技术学报,28(03):352-355.(He G Q,Zhu G C,Liu LM,et al.Screening and crude enzyme property of a cellulose-decoposing strain[J].Journal of Food Science and Biotechnology,28(03):352-355.)
胡艳平,王磊,曹平华,等.2013.纤维素酶产生菌的筛选、其酶学性质及对饲料粗纤维降解效果的研究[J].饲料工业,34(08):21-27.(Hu Y P,Wang L,Cao P H,et al.Study of screening of a strain producing cellulase,its enzymatic properties and the degradation rate of crude fiber in the feed[J].Feed Industry,34(08):21-27.)
胡艳平.2013.纤维素酶产生菌的筛选、其酶学性质与纤维素酶基因的克隆及表达[D].硕士学位论文.西北农林科技大学.导师:陈玉林.pp.1-17(Hu Y P.2013.Screening of a strain producing cellulase,its enzymatic properties and clone and expression of the cellulase gene[D].Thesis for M.S.,Northwest A&F University,Supervisor:Chen Y L,pp.1-17)
黄复生,张英俊,张志端.1986.陕西省白蚁的分布及其新种记述[J].昆虫分类学报,(03):215-219.(Huang F S,Zhang Y J,Zhang Z D.1986.The distribution of termites and their new species in Shaanxi[J].Entomotaxonomia,(03):215-219.)
黄复生,朱世模,李桂祥.1988.白蚁的外部形态和分类系统[J].动物学研究,(03):301-307.(Huang F S,Zhu S M,Li G X.1988.The external morphology and classification system of termites[J].Zoological Research,(03):301-307.)
李博.2017.白蚁肠道细菌的多样性和生物活性研究[D].硕士学位论文,郑州大学,导师:秦广雍.pp.1~8.(Li B.2017.Study on diversity and bioactivity of intestinal microorganisms in termite[D].Thesis for M.S.,Zhengzhou University,Supervisor:Qin G Y,pp.1~8.)
李春梅,何江波,陈惠等.2011.高产中性纤维素酶的巨大芽胞杆菌基因工程菌发酵工艺条件优化[J].农业生物技术学报,19(3):557-564.(Li C M,He J B,Chen H,et al.2011.Optimization of fermentation conditions for producing neutral cellulase from a high-yield Bacillus megaterium genetic engineering bacteria[J].Journal of Agricultural Biotechnology,19(3):557~564.)
李丹红,王誉,杨红.2017.高效降解木质纤维素的白蚁肠道微生物组[J].微生物学报,57(6):876-884.(Li D H,Wang Y,Yang H.2017.Gut microbiome of wood-feeding termites[J].Acta Microbiologica Sinica,57(6):876~884.)
李永博,暴金磊,万敏,等.2017.酒醅中高产纤维素酶菌株的筛选及其酶学性质研究[J].食品工业科技,38(24):109-113.(Li Y B,Bao J L,Wan M,et al.2017.Study on screening of high-yield cellulase strains in the fermented grains and its enzymatic properties[J].Science and Technology of Food Industry,38(24):109-113.)
龙海波,魏丽莎子,赵志祥等.2017.象耳豆根结线虫纤维素结合蛋白Me-cbp-1基因的克隆与功能分析[J].农业生物技术学报,25(2):196~204.(Long H B,Wei L S Z,Zhao Z X,et al.2017.Cloning and Functional Analysis of a Cellulose Binding Protein Gene Me-cbp-1 from the root-knot nematode meloidogyne enterolobii[J].Journal of Agricultural Biotechnology,25(2):196~204.)
王胤.2012.黑胸散白蚁肠道内几种共生细菌的定量、定位及分离菌株Lactococcus sp.的初步研究[D].硕士学位论文.华中师范大学,导师:杨红.pp.21-36.(Wang Y.Quantification and localization of several groups in the gut of Reticulitermes chinensis snyder and primary characterization of a Lactococcus sp.strain[D].Thesis for M.S.Central China Normal University.Supervisor:Yang H,pp.21-36)
尉吉乾,莫建初,徐文,等.2010.黑胸散白蚁的研究进展[J].中国媒介生物学及控制杂志,21(06):635-637.(Wei JQ,Mo J C,Xu W,et al.2010.Advances in research on Reticulitermes chinensis(Isoptera:Rhinotermitidae)in China[J].Chinese Journal of Vector Biology and Control,21(06):635-637.)
Bhat M K,Bhat S.1997.Cellulose degrading enzymes and their potential industrial applications[J].Biotechnology Advances,15(3-4):583-620.
James S W,Patricia J H K,Mukram M M,et al.2017.Cloning,production and characterization of a glycoside hydrolase family 7 enzyme from the gut microbiota of the termite coptotermes curvignathus[J].Springer,59(7):271~283.
Li W,Huan X,Zhou Y,et al.2009.Simultaneous cloning and expression of two cellulase genes from Bacillus subtilis newly isolated from Golden Takin(Budorcas taxicolor Bedfordi)[J].Biochemical and Biophysical Research Communications,383:397-400.
Miller G.L.1959.Use of dinitrosalicylic acid reagent for determination of reducing sugar[J].Analytical chemistry,31(3):426-428.
Ni J,Tokuda G.2013.Lignocellulose-degrading enzymes from termites and their symbiotic microbiota[J].Biotechnology Advances,31:838-850.
Perez F,Garcia O,Martinez C,et al.2015.Genome sequence of Citrobacter sp.Ct B7.12,isolated from the gut of the desert subterranean termite Heterotermes aureus[J].Genome Announcements,3(6):1-2.
Tarayre C,Brognaux A,Bauwens J,et al.2014.Isolation of amylolytic,xylanolytic,and cellulolytic microorganisms extracted from the gut of the termite Reticulitermes santonensis by means of a micro-aerobic atmosphere[J].Springer,30(5):1655-1660.
Watanabe H,Noda H.1998.A cellulase gene of termite origin[J].Nature.394:330-331.
Zhao K,Duan J,Ma X,et al.2016.Isolation,identification and expression of microbial cellulases from the gut of Odontotermes formosanus[J].Journal of Microbiology and Biotechnology,27(1):1-28.