一个新的内切葡聚糖酶基因umcel5K的克隆、表达及其表达产物的酶学特性
|
摘要
本工作从牛瘤胃的富集培养物中提取未培养细菌的总DNA，用柯斯质粒pWEB：：TNC为载体构建宏基因组文库，得到一个包含约8千个克隆的宏基因组文库。该文库外源片段最大为50.8kb，最小约20.5kb，平均大小为35.6kb，外源总DNA容量约为2.86×108bp。对文库进行活性筛选，获得1个表达既表达内切酶活性又表达4-MUC活性的克隆，对具有活性的克隆进行亚克隆、测序分析。鉴定分析表明：该克隆上潜在的内切酶基因umce／5K具1个1002bp的ORF(Open Reading Frame)，可编码含333个氨基酸的蛋白质。umce／5K蛋白与一个来源于未培养细菌的糖苷水解酶家族5的纤维素酶CelA(ABA02176)同源性最高，一致性为53％、相似性为68％；其次与来自混合纤维弧菌的糖苷水解酶家族5的纤维素酶CelA(AAB61461)一致性为51％、相似性为67％。将该ORF转入表达载体后用IPTG诱导表达Umcel 5K蛋白，经过Ni-NTA纯化后测定其酶学特性。Umcel 5K最适pH和最适温度分别为5.0和50℃。从Umcel 5K的pH耐受性和热稳定性实验中可知，Umcel 5K较适合偏酸酸性的环境，是个中温酶，不耐高温。其Km值为3.843mg／ml，Vmax为152.4U／mg。部分金属如Fe3+、Cr2+或Cu2+会抑制该酶的酶活，而另外一些金属离子如K~+、Li~+等对Umcel 5K的活性影响不大。
In this study, total DNA of uncultured microorganisms from the enrichment cultures of the contents of buffalo rumens was isolated . A metagenomic cosmid library that contained approximate 8×10~3 clones was constructed. By functional screening, a clone expressing both exoglucanase and endoglucanase activity was found from the library, and subcloning and sequencing analysis identified one gene (designated umcel5K) that contained an open reading frame (ORF) encoding a protein of 333 amino acid residues. The encoded products of the gene shared 53% identities and 68% similarities with a cellulase CelA(ABA02176) of glycosyl hydrolase family 5 from a uncultured bacterium. The ORF of umcel5K amplified by PCR was clonded into vecter pET30a(+), and over-expressed in Escherichia coli. The Ni-NTA purified expressed product was characterized. The optimum pH of the recombinant enzyme was 4.5～5.0, and optimum temperature was 50℃for endoglucanase activity. The enzyme kinetics experiment determined that the values for Km and Vmax of Umcel5K is 3.843mg/ml and 152.4 U/ mg. Certain ions such as Fe~(3+)、Cr~(2+) and Cu~(2+) had inhibitory effect on the activity of the enzyme, while K~+、Li~+ showed little influence.
In this study, total DNA of uncultured microorganisms from the enrichment cultures of the contents of buffalo rumens was isolated . A metagenomic cosmid library that contained approximate 8×10~3 clones was constructed. By functional screening, a clone expressing both exoglucanase and endoglucanase activity was found from the library, and subcloning and sequencing analysis identified one gene (designated umcel5K) that contained an open reading frame (ORF) encoding a protein of 333 amino acid residues. The encoded products of the gene shared 53% identities and 68% similarities with a cellulase CelA(ABA02176) of glycosyl hydrolase family 5 from a uncultured bacterium. The ORF of umcel5K amplified by PCR was clonded into vecter pET30a(+), and over-expressed in Escherichia coli. The Ni-NTA purified expressed product was characterized. The optimum pH of the recombinant enzyme was 4.5～5.0, and optimum temperature was 50℃for endoglucanase activity. The enzyme kinetics experiment determined that the values for Km and Vmax of Umcel5K is 3.843mg/ml and 152.4 U/ mg. Certain ions such as Fe~(3+)、Cr~(2+) and Cu~(2+) had inhibitory effect on the activity of the enzyme, while K~+、Li~+ showed little influence.
引文
[1] Lynd L R, Paul J W,Van Zyl W H,et al.Micribial cellulose utilization:Fundatnentals and biotechnology [J]. Microbiology and molecular biology reviews,2002,66(3):506—577.
[2] Christian P.The triehoderrna cdlulase regulatory puzzle [J].enzyme,Microb.Technol.,1993, 15(2): 90- 99.
[3] Sibtola, H., and Neimo, L1., In: Symp. on Enz. hyd. of Cellulase. Bailey, M., Enari, T. M., Linko, M.,(Eds), SITRA, Helsinki, p9(1975)
[4] Chanzy,H. (1991), Aspects of cellulose structure. In: Kennedy, J.F., Phillips, GO.Williams, P.A. (Eds), Cellulose Sources and Exploitation, Ellis Horwood, Chichester, pp3—12
[5] Hon, D., Cellulose:a random walk along its historical path. [J].Cellulose, 1994,1 (1):1-25
[6] Li Li,Jur Gen F,Peter P.et al.Termite gut symbiotic archaezoa are becoming living metabolic fossils[J]. Eukaryotic Cell,2003,2(5): 1091 — 1098.
[7] BeWch, J. P, C. Tardif, A. Belaich and C.Gaudin. (1997). The biological degradation of Cellulose[J].FEMS Microbiol.R ev. 13 ,219—248.
[8] Ahsan, M. M,T. Kimura, S. Karita, K. Sakka and K. Ohmiya.( 1996). Cloning, DNA sequencing, And expression of the gene encoding Clostridium thermocellum cellulose CelJ,the largest Catalytic component of the cellulosime [J]..Bacteriol. 178,5732—5740
[9] Berger,E .,W.A.Jo nes,D .T Jones and D .R .Woods.(1989).Clonning and sequencing of an Endoglucanase gene from Butyrivibrio fibrisolvens H17c.Mol.Gen.Genet 219,193—198
[10] Vercoe, P. E., J. L. Finks, and B. A. White.(1995). DNA sequence and transcriptional Characterization of a beta-glucanase gene from Ruminococcus flavefaciens. FD-1. Can. [J]. Microbiol.41,869—876.
[11] Karen M K L,Matrl S A,Teeri T T,et al,The celulases endoglucanase I and cellobiohydrolase II of Trichoderma reesei act synergistically to solubilize native cotton cellulose but not to decrease its molecular size[J].Applied and Environmental Microbiology, 1996,62(8):2883—2887.
[12] Alinda A Ester D,Marc Van Mil,et al. EglC a new endoglucanase from Aspergillus niger with major activity towards xylogulcan. [J]. Applied and Environmental Microbiology, 2002,68(4): 1556—1560.
[13] Singh A., Hayashi, K. Microbial cellulases: protein architecture, molecular properties and biosynthesis [J].Appl.Microbiol, 1995,40:1～44.
[14] Rachel.G.Larusa R.Sima Y, ed al.Cell,a nomelhlosomal fanfily 9 enzyme from Ciostridlum thermocellum,is a processive endoglucanase that degrades crystalline cellulose[J].J Bact,2003, 185(2):391～398.
[15] Te'o Vet al.Extrempophiles,1998, 32:24～32.
[16] Rouvinen J, Bergforst, Teeri T Three dimisional structure of cellobiohydrolase Ⅱ From T [J].reerei.Science, 1990,24 9(4967):380～386.
[17] Juy M, Amit A G, Alzari P M,et al.Crystal structure of a thermostable bacterial cellulose degrading enzyme [J].Nature, 1992,357～373.
[18] Cavaro Paulo A.et al.Enzyme Microb Techo. 1999,25:639～643.
[19] Linder Met al. protein Sci[M]. 1995,4:1056～1064.
[20] Reesi.E.T.;Stu.R.G.H.;Levinson,H.S.J.Becterid[M] 1960,69,485-497
[21] Humpheg A E.The hydlolysis of cellulosic matelialo to uletrl products[J]. AdvChem Se1,1979,181: 25-53.
[22] Wood T M,Maccpae S L.In bioconversion of cellulose substance into energy,chemicals and microbial protein [M]. Edited by T K Ghose Gal India,1978 111～141.
[23] Eunki Kim, et al.Factorial optimization of a six cellulose mixture [J] Biotechnolog and Bioengineering, 1998, 58 (5):496～501.
[24] 陈力宏．纤维素酶在食品发酵中的应用[J]．中国酿造，1990(5)：2～5．
[25] 谭宏等．长梗木霉纤维素酶的产生及提取，微生物学通报，1993，20(2)：90～93．
[26] 崔福绵，刘菡，韩辉，等．康宁木霉Cp8829纤维素酶生产条件的研究[J]．微生物学通报，1995，22(2)：72～75．
[27] Lee R.Lynd et al,Fuel Ethanol form Cellulosic Biomass[J], Science,1991, (1251): 1318～1323.
[28] 梁靖，须海荣．纤维素酶在速溶茶中的应用研究[J]．茶叶，2002，28(1)：25～26．
[29] 周正红，贾宗剑，高孔荣．纤维素酶在食品发酵工业中的应用及前景[J]．西南大学学报(自科与医学版)．
[30] 邱立友，朱德育．黑曲霉酶学特性及其在洒精生产中的应用[J]．河南农业大学学报，1993，27(3)：291～295．
[31] 明景熙．啤酒工业中可再生资源开发[J]．中国酿造，2000，4：28～29．
[32] 肖春玲，徐常新．微生物纤维素酶的应用研究[J]．微生物学杂志，2002，22(2)：33～35．
[33] 宋国安．纤维素酶的应用开发前景[J]．河南化工，1999,(6)：5～6．
[34] 仁大明．浅谈饲用纤维素酶生产与应用[J]．饲料工业，1995，(3)：1～3．
[35] Kamaya, Y, Role of endoglucanase in enzymatic modification of bleached kraft pulp.[J] Ferm Bioeng 1996,82:549～553.
[36] Oksanen,T, Pere, J. Buchert, J. and Viikari, L,The efect of T. reesei cellulases and hemicellulases on the paper technical properties of never-dried bleached kraft pulp[J]. Cellulose 1997,4:329～339.
[37] 乞永立，耿月霞，任章启．纤维索酶的生产及应用[J]．河北化工，2000，(1)：25～26．
[38] Torsvik V, φbvreas L. Curr Opin Microbio, 2002, 5: 240～245.
[39] 梁威，邱东茹，熊丽，等．生命科学[M]，2002，14(3)：142～144．
[40] Wang G Y, Graziani E,Waters B,et al.Novel natural products from soil DNA libraries in a streptomycete host.Org Lett,2000,2:2401-2404
[41] 阎冰，洪葵，许云，马超，宏基冈组克隆——微生物活性物质筛选的新途径[J]．微生物学通报，2005，32(1)：113-117
[42] Handelsman J,Rondon M R,Brady S F, Clardy J,Goodman R M.Molecular biological accesses to the chemistry of unknown soil microbes:A new frontier for natural p products[j].Chem Biol,1998,5(10):245～249.
[43] Streit Wr, Daniel R, Jaeger KE. Prospecting for biocatalysts and drugs in the genomes of non-cultured microorganisms[J].Curr Opin Biotechnol,2004,15 (4):285～290.
[44] Patrick Lorenz,Christa Schleper. Metagenome-a challenging source of enzyme discovery [J].J Mol Catal B:Enzymatic, 2002,19 (20):13～19.
[45] 徐平，李文均，姜成林，等．微波炉法快速提取放线菌基冈组DNA．微生物学通报，2003，30(4)：82-84．
[46] Courtois S,Cappellano Cm,Ball M,et al, Recombinant en2vironmental libraries provide access to microbial diversity for drug discovery from natural products[J]. Appl Environ Micro-biol,2003,69 (1):49～55.
[47] Henne A,Daniel R, Schmitz RA,et al.App EnvironMicrobiol[M], 1999,65:3901～39071.
[48] Knietsch A, Waschkowitz T, Bowien S,et al,Appl EnvironMicrobiol[M],2003, 69: 1408～14161.
[49] RondonM R, August P R, Bettermann A D,et al.Appl EnvironMicrobiol[M], 2000, 66:2541～2547.
[50] Schioss PD,Handelsman J.Biotechnological prospects from metagenomics[J]. Curr Opin Biotechnol,2003, 14 (3):303～310.
[51] Tyson GW, Chapman J,Hugenholtz P, et al,Community structure and metabolism through reconstruction of microbial genomes from the environment [J].Nature,2004,428 (2):37～43.
[52] Schmeisser C,Stockigt C,Raasch C,et al,Metagenome survey of biofiims in drinking2water networks[J].Appl Environ Microbiol,2003,69 (12):7298～7309.
[53] Hugenholtz P, Goebel BM, Pace NR. Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. [J] Bacteriol. 1998, 180(18): 4765～4774
[54] Rappe MS, Giovannoni SJ. The uncultured microbial majority[J]. Annu Rev Microbial. 2003, 57: 369～394
[55] 萨姆布鲁克 J．，弗里奇 E．F．，曼尼阿蒂斯 T，分子克隆实验指南(第二版)，北京：科学出版社，1999．19～22
[56] Zhou J, Brouns MA, Tiedje JM. DNA recovery from soil of diverse composition.[J]. Appl Environ Microbiol. 1996, 62(2): 316～322
[57] Richardson TH, Tan X, Frey Get al. A novel, high performance enzyme for starch liquefaction. Discovery and optimization of a low pH, thermostable a-amylase[J]. Biol Chem. 2002, 277: 26501～26507
[58] Teather RM, Wood PJ. Use of Congo red-polysaccharide interactions in enumeration and characterisation of cellulolytic bacteria from the bovine rumen.[J]. Appl Environ Microbiol. 1982, 43(4): 777-780
[59] Kwon Ki-sun, Lee Jaehoon, Kang Hyung-gyoo et al. Detection of β-Glucosidase activity in polyacrylamide gels with esculin as substrate.[J]. Appl Environ Microbiol. 1994, 60(12): 4584-4586
[60] Heptinstall J, Stewart JC, Seras M. Fluorimetric estimation ofexo-cellobiohydrolase and β-D-glucosidase activities in cellulase from Aspergillus fumigatus Fresenius.[J]. Enzyme Microb Technol. 1986, 8(2): 70-74
[61] Sebat JL, Colwell FS, Crawford RL. Metagenomic profiling: microarray analysis of an environmental genomic library[J]. Appl Environ Microbiol. 2003, 69(8): 4927-4934
[62] Laemmli. Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J]. Nature. 1970, 224(5259): 680-685
[63] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Anal Biochem. 1976, 72:248-254
[64] Miller GL. Use of dinitrosalicyclic acid reagent for determination of reducing sugar[J]. Biotechnol Bioeng Symp. 1959, 5:193-219
[65] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Anal Biochem. 1976, 72:248-254
[66] 胡莹，杨凌，杨胜利．糖苷酶序列分类法和作用机理的研究进展[J]．药物生物技术，2006，13(1)：066～070
[67] 王玢，汪天虹，张刚等．产低温纤维素酶海洋嗜冷菌的筛选及研究[J]．海洋科学，2003，27(5)：42～45
[68] 曾胤新，俞勇，陈波等．低温纤维素酶产生菌的筛选、鉴定、生长特性及酶学性质，高技术通讯，2005，15(4)：58～61
[69] 白玉，杨大群，王建辉等．天山冻土耐冷茵的分离与产低温酶菌株的筛选[J]，冰川冻土．2005，27(4)：615～618
[70] Ando S, et al. Appl Environ Microbiol, 2002, 68(1): 430～433.
[71] 强郁荣，李丽，封晔等．黄河中上游半干旱区典型盐渍土中细菌耐盐性及产酶特性研究[J]．西北农业学报2006，15(3)：65～68，82
[72] Susan Grant,Dimitry Y. Sorokin, William D. et al. A phylogenetic analysis of Wadi el Natrun soda lake cellulase enrichment cultures and identification ofcellulase genes from these cultures[J]. Extremophiles (2004) 8:421-429
[73] 黄永光，黄旭，黄平．茅台酒酿酒极端环境与极端酿酒微生物[J]，酿酒科技2006，12：47～50
[74] Daniel R. The soil metagenome-a rich resource for the discovery of novel natural products[J]. Curr Opin Biotechnol. 2004, 15(3): 199-204
[75] Schloss PD, Handelsman J. Biotechnological prospects from metagenomics[J]. Curr. Opin. Biotechnol. 2003, 14:303-310
[76] Handelsman J, Rondon MR, Brady SF et al. Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products[J]. Chem Biol. 1998, 5(10): R245-249
[77] Voget S, Leggewie C, Uesbeck A et al. Prospecting for novel biocatalysts in a soil metagenome[J]. Appl Environ Microbiol. 2003, 69(10): 6235-6242
[78] Streit WR, Daniel R, Jaeger KE. Prospecting for biocatalysts and drugs in the genomes of non-cultured microorganisms[J]. Curr Opin Biotechno, 2004, 15(4): 285-290.
[79] 杜连起，李香艳．酶制剂在啤酒生产中的应用[J]．酿酒科技，1996，75(3)：48～49．
[80] 许梓荣，王振来，王敏奇．高麦麸饲粮添加酶类物质对仔猪生长忡能和胴体组成的影响[J]．浙江大学学报(农业与生命科学版)，1998，6：7～10．
[81] 郭小权，胡国良，刘妹，β-葡聚糖酶的抗营养作用及β-葡聚糖酶在饲料中的应用[J]，江西饲料，2001，2：11～13
[2] Christian P.The triehoderrna cdlulase regulatory puzzle [J].enzyme,Microb.Technol.,1993, 15(2): 90- 99.
[3] Sibtola, H., and Neimo, L1., In: Symp. on Enz. hyd. of Cellulase. Bailey, M., Enari, T. M., Linko, M.,(Eds), SITRA, Helsinki, p9(1975)
[4] Chanzy,H. (1991), Aspects of cellulose structure. In: Kennedy, J.F., Phillips, GO.Williams, P.A. (Eds), Cellulose Sources and Exploitation, Ellis Horwood, Chichester, pp3—12
[5] Hon, D., Cellulose:a random walk along its historical path. [J].Cellulose, 1994,1 (1):1-25
[6] Li Li,Jur Gen F,Peter P.et al.Termite gut symbiotic archaezoa are becoming living metabolic fossils[J]. Eukaryotic Cell,2003,2(5): 1091 — 1098.
[7] BeWch, J. P, C. Tardif, A. Belaich and C.Gaudin. (1997). The biological degradation of Cellulose[J].FEMS Microbiol.R ev. 13 ,219—248.
[8] Ahsan, M. M,T. Kimura, S. Karita, K. Sakka and K. Ohmiya.( 1996). Cloning, DNA sequencing, And expression of the gene encoding Clostridium thermocellum cellulose CelJ,the largest Catalytic component of the cellulosime [J]..Bacteriol. 178,5732—5740
[9] Berger,E .,W.A.Jo nes,D .T Jones and D .R .Woods.(1989).Clonning and sequencing of an Endoglucanase gene from Butyrivibrio fibrisolvens H17c.Mol.Gen.Genet 219,193—198
[10] Vercoe, P. E., J. L. Finks, and B. A. White.(1995). DNA sequence and transcriptional Characterization of a beta-glucanase gene from Ruminococcus flavefaciens. FD-1. Can. [J]. Microbiol.41,869—876.
[11] Karen M K L,Matrl S A,Teeri T T,et al,The celulases endoglucanase I and cellobiohydrolase II of Trichoderma reesei act synergistically to solubilize native cotton cellulose but not to decrease its molecular size[J].Applied and Environmental Microbiology, 1996,62(8):2883—2887.
[12] Alinda A Ester D,Marc Van Mil,et al. EglC a new endoglucanase from Aspergillus niger with major activity towards xylogulcan. [J]. Applied and Environmental Microbiology, 2002,68(4): 1556—1560.
[13] Singh A., Hayashi, K. Microbial cellulases: protein architecture, molecular properties and biosynthesis [J].Appl.Microbiol, 1995,40:1～44.
[14] Rachel.G.Larusa R.Sima Y, ed al.Cell,a nomelhlosomal fanfily 9 enzyme from Ciostridlum thermocellum,is a processive endoglucanase that degrades crystalline cellulose[J].J Bact,2003, 185(2):391～398.
[15] Te'o Vet al.Extrempophiles,1998, 32:24～32.
[16] Rouvinen J, Bergforst, Teeri T Three dimisional structure of cellobiohydrolase Ⅱ From T [J].reerei.Science, 1990,24 9(4967):380～386.
[17] Juy M, Amit A G, Alzari P M,et al.Crystal structure of a thermostable bacterial cellulose degrading enzyme [J].Nature, 1992,357～373.
[18] Cavaro Paulo A.et al.Enzyme Microb Techo. 1999,25:639～643.
[19] Linder Met al. protein Sci[M]. 1995,4:1056～1064.
[20] Reesi.E.T.;Stu.R.G.H.;Levinson,H.S.J.Becterid[M] 1960,69,485-497
[21] Humpheg A E.The hydlolysis of cellulosic matelialo to uletrl products[J]. AdvChem Se1,1979,181: 25-53.
[22] Wood T M,Maccpae S L.In bioconversion of cellulose substance into energy,chemicals and microbial protein [M]. Edited by T K Ghose Gal India,1978 111～141.
[23] Eunki Kim, et al.Factorial optimization of a six cellulose mixture [J] Biotechnolog and Bioengineering, 1998, 58 (5):496～501.
[24] 陈力宏．纤维素酶在食品发酵中的应用[J]．中国酿造，1990(5)：2～5．
[25] 谭宏等．长梗木霉纤维素酶的产生及提取，微生物学通报，1993，20(2)：90～93．
[26] 崔福绵，刘菡，韩辉，等．康宁木霉Cp8829纤维素酶生产条件的研究[J]．微生物学通报，1995，22(2)：72～75．
[27] Lee R.Lynd et al,Fuel Ethanol form Cellulosic Biomass[J], Science,1991, (1251): 1318～1323.
[28] 梁靖，须海荣．纤维素酶在速溶茶中的应用研究[J]．茶叶，2002，28(1)：25～26．
[29] 周正红，贾宗剑，高孔荣．纤维素酶在食品发酵工业中的应用及前景[J]．西南大学学报(自科与医学版)．
[30] 邱立友，朱德育．黑曲霉酶学特性及其在洒精生产中的应用[J]．河南农业大学学报，1993，27(3)：291～295．
[31] 明景熙．啤酒工业中可再生资源开发[J]．中国酿造，2000，4：28～29．
[32] 肖春玲，徐常新．微生物纤维素酶的应用研究[J]．微生物学杂志，2002，22(2)：33～35．
[33] 宋国安．纤维素酶的应用开发前景[J]．河南化工，1999,(6)：5～6．
[34] 仁大明．浅谈饲用纤维素酶生产与应用[J]．饲料工业，1995，(3)：1～3．
[35] Kamaya, Y, Role of endoglucanase in enzymatic modification of bleached kraft pulp.[J] Ferm Bioeng 1996,82:549～553.
[36] Oksanen,T, Pere, J. Buchert, J. and Viikari, L,The efect of T. reesei cellulases and hemicellulases on the paper technical properties of never-dried bleached kraft pulp[J]. Cellulose 1997,4:329～339.
[37] 乞永立，耿月霞，任章启．纤维索酶的生产及应用[J]．河北化工，2000，(1)：25～26．
[38] Torsvik V, φbvreas L. Curr Opin Microbio, 2002, 5: 240～245.
[39] 梁威，邱东茹，熊丽，等．生命科学[M]，2002，14(3)：142～144．
[40] Wang G Y, Graziani E,Waters B,et al.Novel natural products from soil DNA libraries in a streptomycete host.Org Lett,2000,2:2401-2404
[41] 阎冰，洪葵，许云，马超，宏基冈组克隆——微生物活性物质筛选的新途径[J]．微生物学通报，2005，32(1)：113-117
[42] Handelsman J,Rondon M R,Brady S F, Clardy J,Goodman R M.Molecular biological accesses to the chemistry of unknown soil microbes:A new frontier for natural p products[j].Chem Biol,1998,5(10):245～249.
[43] Streit Wr, Daniel R, Jaeger KE. Prospecting for biocatalysts and drugs in the genomes of non-cultured microorganisms[J].Curr Opin Biotechnol,2004,15 (4):285～290.
[44] Patrick Lorenz,Christa Schleper. Metagenome-a challenging source of enzyme discovery [J].J Mol Catal B:Enzymatic, 2002,19 (20):13～19.
[45] 徐平，李文均，姜成林，等．微波炉法快速提取放线菌基冈组DNA．微生物学通报，2003，30(4)：82-84．
[46] Courtois S,Cappellano Cm,Ball M,et al, Recombinant en2vironmental libraries provide access to microbial diversity for drug discovery from natural products[J]. Appl Environ Micro-biol,2003,69 (1):49～55.
[47] Henne A,Daniel R, Schmitz RA,et al.App EnvironMicrobiol[M], 1999,65:3901～39071.
[48] Knietsch A, Waschkowitz T, Bowien S,et al,Appl EnvironMicrobiol[M],2003, 69: 1408～14161.
[49] RondonM R, August P R, Bettermann A D,et al.Appl EnvironMicrobiol[M], 2000, 66:2541～2547.
[50] Schioss PD,Handelsman J.Biotechnological prospects from metagenomics[J]. Curr Opin Biotechnol,2003, 14 (3):303～310.
[51] Tyson GW, Chapman J,Hugenholtz P, et al,Community structure and metabolism through reconstruction of microbial genomes from the environment [J].Nature,2004,428 (2):37～43.
[52] Schmeisser C,Stockigt C,Raasch C,et al,Metagenome survey of biofiims in drinking2water networks[J].Appl Environ Microbiol,2003,69 (12):7298～7309.
[53] Hugenholtz P, Goebel BM, Pace NR. Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. [J] Bacteriol. 1998, 180(18): 4765～4774
[54] Rappe MS, Giovannoni SJ. The uncultured microbial majority[J]. Annu Rev Microbial. 2003, 57: 369～394
[55] 萨姆布鲁克 J．，弗里奇 E．F．，曼尼阿蒂斯 T，分子克隆实验指南(第二版)，北京：科学出版社，1999．19～22
[56] Zhou J, Brouns MA, Tiedje JM. DNA recovery from soil of diverse composition.[J]. Appl Environ Microbiol. 1996, 62(2): 316～322
[57] Richardson TH, Tan X, Frey Get al. A novel, high performance enzyme for starch liquefaction. Discovery and optimization of a low pH, thermostable a-amylase[J]. Biol Chem. 2002, 277: 26501～26507
[58] Teather RM, Wood PJ. Use of Congo red-polysaccharide interactions in enumeration and characterisation of cellulolytic bacteria from the bovine rumen.[J]. Appl Environ Microbiol. 1982, 43(4): 777-780
[59] Kwon Ki-sun, Lee Jaehoon, Kang Hyung-gyoo et al. Detection of β-Glucosidase activity in polyacrylamide gels with esculin as substrate.[J]. Appl Environ Microbiol. 1994, 60(12): 4584-4586
[60] Heptinstall J, Stewart JC, Seras M. Fluorimetric estimation ofexo-cellobiohydrolase and β-D-glucosidase activities in cellulase from Aspergillus fumigatus Fresenius.[J]. Enzyme Microb Technol. 1986, 8(2): 70-74
[61] Sebat JL, Colwell FS, Crawford RL. Metagenomic profiling: microarray analysis of an environmental genomic library[J]. Appl Environ Microbiol. 2003, 69(8): 4927-4934
[62] Laemmli. Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J]. Nature. 1970, 224(5259): 680-685
[63] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Anal Biochem. 1976, 72:248-254
[64] Miller GL. Use of dinitrosalicyclic acid reagent for determination of reducing sugar[J]. Biotechnol Bioeng Symp. 1959, 5:193-219
[65] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Anal Biochem. 1976, 72:248-254
[66] 胡莹，杨凌，杨胜利．糖苷酶序列分类法和作用机理的研究进展[J]．药物生物技术，2006，13(1)：066～070
[67] 王玢，汪天虹，张刚等．产低温纤维素酶海洋嗜冷菌的筛选及研究[J]．海洋科学，2003，27(5)：42～45
[68] 曾胤新，俞勇，陈波等．低温纤维素酶产生菌的筛选、鉴定、生长特性及酶学性质，高技术通讯，2005，15(4)：58～61
[69] 白玉，杨大群，王建辉等．天山冻土耐冷茵的分离与产低温酶菌株的筛选[J]，冰川冻土．2005，27(4)：615～618
[70] Ando S, et al. Appl Environ Microbiol, 2002, 68(1): 430～433.
[71] 强郁荣，李丽，封晔等．黄河中上游半干旱区典型盐渍土中细菌耐盐性及产酶特性研究[J]．西北农业学报2006，15(3)：65～68，82
[72] Susan Grant,Dimitry Y. Sorokin, William D. et al. A phylogenetic analysis of Wadi el Natrun soda lake cellulase enrichment cultures and identification ofcellulase genes from these cultures[J]. Extremophiles (2004) 8:421-429
[73] 黄永光，黄旭，黄平．茅台酒酿酒极端环境与极端酿酒微生物[J]，酿酒科技2006，12：47～50
[74] Daniel R. The soil metagenome-a rich resource for the discovery of novel natural products[J]. Curr Opin Biotechnol. 2004, 15(3): 199-204
[75] Schloss PD, Handelsman J. Biotechnological prospects from metagenomics[J]. Curr. Opin. Biotechnol. 2003, 14:303-310
[76] Handelsman J, Rondon MR, Brady SF et al. Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products[J]. Chem Biol. 1998, 5(10): R245-249
[77] Voget S, Leggewie C, Uesbeck A et al. Prospecting for novel biocatalysts in a soil metagenome[J]. Appl Environ Microbiol. 2003, 69(10): 6235-6242
[78] Streit WR, Daniel R, Jaeger KE. Prospecting for biocatalysts and drugs in the genomes of non-cultured microorganisms[J]. Curr Opin Biotechno, 2004, 15(4): 285-290.
[79] 杜连起，李香艳．酶制剂在啤酒生产中的应用[J]．酿酒科技，1996，75(3)：48～49．
[80] 许梓荣，王振来，王敏奇．高麦麸饲粮添加酶类物质对仔猪生长忡能和胴体组成的影响[J]．浙江大学学报(农业与生命科学版)，1998，6：7～10．
[81] 郭小权，胡国良，刘妹，β-葡聚糖酶的抗营养作用及β-葡聚糖酶在饲料中的应用[J]，江西饲料，2001，2：11～13