粘细菌纤维堆囊菌降解纤维素多酶复合体的确证和性质分析
|
摘要
粘细菌是一类特殊的革兰氏阴性杆菌，能够进行滑行运动，具有复杂的多细胞行为和形态发生过程，是一类“生活在真核生物边缘”的高等的原核生物类群；产生众多结构新颖、作用机制独特的生物活性次级代谢产物是粘细菌的另一重要特征。根据“食性"的差异可将粘细菌简单的划分为溶细菌和溶纤维素两个生理类群。溶纤维素群中只有堆囊菌属一个属，纤维堆囊菌是其中的模式种。作为重要的微生物药物(例如著名的埃博霉素)产生菌，纤维堆囊菌逐渐引起全球的广泛关注。但是，对其最为基本的微生物学特性——粘细菌中唯一能够降解纤维素、半纤维素等生物大分子的类群，相关研究则涉及很少。本文利用实验室所建立的国内最大的粘细菌资源库的优势，对纤维堆囊菌降解纤维素的基本性质及相关酶类进行了研究。
纤维素是地球上最丰富的可再生有机资源，对纤维素的降解基本上是由微生物完成的。不同微生物降解纤维素时所采取的策略也不尽相同，主要表现在各种降解酶类的作用机制和组织形式上。纤维堆囊菌能够在以纤维素为唯一碳源的简单无机盐培养基(如CNST培养基)上生长，可产生多种纤维素和半纤维素酶类。所以，我们从表征其纤维素酶类的基本性质出发，开展了相关研究。广泛选取了13株纤维堆囊菌作为研究对象，对其在不同纤维素底物上的生长形态、降解纤维素特征、分化发育能力以及多种纤维素和半纤维素酶类的产生时间进行了测定和比较。通过对各菌株在滤纸纤维素底物上生长和子实体形成情况的分析，我们将其总结为两种不同的类型：一种类型是以接种点为中心，先向四周快速扩展，在扩展过程中伴随着滤纸纤维素的降解，达到一定程度以后，整个菌落的细胞同时开始聚集，并形成子实体结构，其子实体基本在生长同一时期形成，并布满整个菌落，这种类型的特点就是几乎所有的菌体细胞都处于相同的生长时期，细胞生长和分化发育具有一定的同步性；另一种类型是在菌落外围菌体细胞仍在增殖扩展时，靠近中心部位的菌体则已开始聚集和进入分化发育阶段，首先形成子实体结构，随着培养时间的延长，外围细胞也逐渐形成子实体结构，其子实体最终布满菌落中心部位或整个菌落，这种类型的特点就是菌落中不同部位的细胞所处
The myxobacteria are a special kind of Gram-negative unicellular bacteria with rod-shaped vegetative cells. Because of their gliding movement, complicated multicellular behavior and morphogenesis process, the myxobacteria are generally considered as advanced prokaryotes, which may live on the edge of eukaryotes. Another outstanding trait of the myxobacteria is that they can produce a large number of bioactive secondary metabolites with some novel structures and unique mechanism of action. Based on their specialization in degradation of biomacromolecules, they are divided into two groups, one is bacteriolytic, and the other is cellulolytic. Within cellulolytic group, there is only one genus named Sorangium, in which Sorangium cellulosum is the type species in this genus. As an important medicine microbial producer (e.g. the famous antitumor drug epothilone), S. cellulosum draws worldwide attention. However, very few studies have been focused on the activity of degrading biomacromolecular materials, such as cellulose and hemicellulose, which is the unique and basic property of S. cellulosum among the myxobacteria. In virtue of the biggest domestic myxobacteria strain bank established by our laboratory, this paper tried to explore the characteristics and mechanism of Sorangium's cellulolytic activity.
Cellulose is the most abundant and renewable organic biopolymer on earth and the degradation of cellulose is mainly carried out by microorganisms. Different microorganisms select different strategies to hydrolyze cellulose, represented by the organization and mechanism of action of their cellulolytic enzymes. Sorangium strains can grow on simple inorganic salt medium (e.g. CNST medium) with cellulose as the only carbon source by producing cellulases and hemicellulases. Accordingly, to elucidate basic property of cellulases produced by S. cellulosum was set as our research starting point. The morphologies, cellulolytic characteristics and
纤维素是地球上最丰富的可再生有机资源，对纤维素的降解基本上是由微生物完成的。不同微生物降解纤维素时所采取的策略也不尽相同，主要表现在各种降解酶类的作用机制和组织形式上。纤维堆囊菌能够在以纤维素为唯一碳源的简单无机盐培养基(如CNST培养基)上生长，可产生多种纤维素和半纤维素酶类。所以，我们从表征其纤维素酶类的基本性质出发，开展了相关研究。广泛选取了13株纤维堆囊菌作为研究对象，对其在不同纤维素底物上的生长形态、降解纤维素特征、分化发育能力以及多种纤维素和半纤维素酶类的产生时间进行了测定和比较。通过对各菌株在滤纸纤维素底物上生长和子实体形成情况的分析，我们将其总结为两种不同的类型：一种类型是以接种点为中心，先向四周快速扩展，在扩展过程中伴随着滤纸纤维素的降解，达到一定程度以后，整个菌落的细胞同时开始聚集，并形成子实体结构，其子实体基本在生长同一时期形成，并布满整个菌落，这种类型的特点就是几乎所有的菌体细胞都处于相同的生长时期，细胞生长和分化发育具有一定的同步性；另一种类型是在菌落外围菌体细胞仍在增殖扩展时，靠近中心部位的菌体则已开始聚集和进入分化发育阶段，首先形成子实体结构，随着培养时间的延长，外围细胞也逐渐形成子实体结构，其子实体最终布满菌落中心部位或整个菌落，这种类型的特点就是菌落中不同部位的细胞所处
The myxobacteria are a special kind of Gram-negative unicellular bacteria with rod-shaped vegetative cells. Because of their gliding movement, complicated multicellular behavior and morphogenesis process, the myxobacteria are generally considered as advanced prokaryotes, which may live on the edge of eukaryotes. Another outstanding trait of the myxobacteria is that they can produce a large number of bioactive secondary metabolites with some novel structures and unique mechanism of action. Based on their specialization in degradation of biomacromolecules, they are divided into two groups, one is bacteriolytic, and the other is cellulolytic. Within cellulolytic group, there is only one genus named Sorangium, in which Sorangium cellulosum is the type species in this genus. As an important medicine microbial producer (e.g. the famous antitumor drug epothilone), S. cellulosum draws worldwide attention. However, very few studies have been focused on the activity of degrading biomacromolecular materials, such as cellulose and hemicellulose, which is the unique and basic property of S. cellulosum among the myxobacteria. In virtue of the biggest domestic myxobacteria strain bank established by our laboratory, this paper tried to explore the characteristics and mechanism of Sorangium's cellulolytic activity.
Cellulose is the most abundant and renewable organic biopolymer on earth and the degradation of cellulose is mainly carried out by microorganisms. Different microorganisms select different strategies to hydrolyze cellulose, represented by the organization and mechanism of action of their cellulolytic enzymes. Sorangium strains can grow on simple inorganic salt medium (e.g. CNST medium) with cellulose as the only carbon source by producing cellulases and hemicellulases. Accordingly, to elucidate basic property of cellulases produced by S. cellulosum was set as our research starting point. The morphologies, cellulolytic characteristics and
引文
1. Reichenbach H., Dworkin M. The Myxobacteria. In: Balow A., Truper G. H., Dworkin M., Sehleifer W. H. (eds) The prokaryotes. Springer, New York. 1992, pp3416-3487.
2. Shimkets L. J. Social and developmental biology of myxobacteria. Microbiol Rev, 1990, 54: 473-501.
3. McCurdy H. D. Order of Myxococcales. In: Stanley J. T., Bryant M. P., Pfening N., Holt J. G. (eds) Bergey's Manual of Systematic Bacteriology, 9th ed. Williams and Wilkins, Baltimore, 1989, pp2139-2170.
4. Nguimbi E., Li Y. Z., Gao B. L., Li Z. F., Wang B., Wu Z. H., Yan B. X., Qu Y. B., Gao P. J. 16S-23S ribosomal DNA intergenic spacer regions in cellulolytic myxobacteria and differentiation of closely related strains. Syst Appl Microbiol, 2003, 26(2): 262-268.
5. Yan Z. C., Wang B., Li Y. Z., Gong X., Zhang H. Q., Gao P. J. Morphologies and phylogenetic classification of cellulolytic myxobacteria. Syst Appl Microb, 2003, 26: 104-109.
6. MacRae T. H. and McCurdy H. D. Ultrastructural studies of Chondromyces crocatus vegetative cells. Can J Microbiol, 1975, 21: 1815-1826.
7．方晓梅，张利平．粘细菌生态多样性的初步研究．生物多样性，2001，9(3)：207-213．
8. Dawid W. Biology and global distribution of myxobacteria in soils. FEMS Microbiology Reviews, 2000, 24: 403-427.
9．王海英，张利平．粘细菌：一类重要的微生物资源．微生物学通报，2003，30(2)：115-116．
10. Ruckert G. Myxobakterien (Myxobacterales) auf Blattober-flachen. Z. Allg. Mikrobiol, 1981, 21: 761-763.
11. Dawid W., Gallikowski C. A. and Hirsch P. Psychrophilic myxobacteria from Antarctic soils. Polarforschung, 1988, 58: 271-278.
12. Brockman E. R. Myxobacters from arid Mexican soil. Appl Environ Microbiol, 1976, 32: 642-644.
13. Dworkin M. Recent advances in the social and developmental biology of the myxobacteria. Microbiol Rev, 1996, 60: 70-102.
14．周璐，李越中，李健．粘细菌的多细胞形态发生及其分子调控．生物化学与生物物理进展，1999，26(6)：544-547．
15. Wolgemuth C., Hoiczyk E., Kaiser D., Oster G. How myxobacteria glide. Curr Biol, 2002, 12(5): 369-377.
16. Fink J. M. and Zissler J. F. Defects in motility and development of Myxococcus xanthus lipopolysaccharide mutants. J Bacteriol, 1989, 171: 2042-2048.
17. Shimkets L. J. and Kaiser D. Induction of coordinated cell movement in Myxococcus xanthus. J Bacteriol, 1982, 152: 451-461.
18．吴斌辉，李越中，王溱圯，胡玮，候爱华．纤维堆囊菌生长及限制因素的研究．生物技术，2001，11(3)：16-18．
19. Hofle G. and Reichenbach H. Biologically active substances from microorganisms-an interdisciplinary research project at the GBF. Sci Ann Rep, 1990, pp5-22. Gesellschaft fur Biotechnolog. Forschung, Braunschweig.
20．李健，李越中，王溱圯．粘细菌生物活性物质研究进展．中国新药杂志，1998，7(2)：84-89．
21．李越中，李健，周璐，张勇，胡玮，陈琦．我国粘细菌(Myxobacteria)资源的分离与鉴定．微生物学报，2000，40(6)：652-656．
22．吴斌辉，李越中，胡玮，周璐，侯配斌．纤维堆囊菌的多细胞形态发生过程．微生物学报，2002，42(6)：657-661．
23．李越中，胡玮，吴斌辉，阎章才，陈琦．纤维堆囊菌的代谢产物及其生物学活性分析．微生物学报，2001，41(6)：716-722．
24. Domling A., Richter W. Myxobacterial epothilones and tubulysins as promising anticancer agents. Mol Divers, 2005, 9(1-3): 141-147.
25．吴斌辉，李越中，胡玮．纤维堆囊菌活性次级代谢产物的研究进展．微生物 学通报，2002，29(2)：75-78．
26．李越中，李健．粘细菌的分离纯化．微生物学通报，1997，24(4)：237-240．
27. Gardner K.H., Blackwell J. The structure of native cellulose. Biopolymer, 1974, 13: 1975-1987.
28．孟雷，陈冠军，王怡，高培基．纤维素酶的多型性．纤维素科学与技术，2002，10(2)：47-55．
29. Lynd L.R., Wyman C.E., and Gemgross T.U. Biocommodity engineering. Biotechnol Prog, 1999, 15: 777-793.
30. Marchessault R.H., and Sundararajan P.R.. Cellulose. In: Aspinall G.O. (ed.) The polysaccharides vol. 2. Academic Press, Inc., New York. 1993, pp11-95.
31. Atalla, R.H., Hackney J.M., Uhlin I., and Thompson N.S. Hemicelluloses as structure regulators in the. aggregation of native cellulose. Int J Biol Macromol, 1993, 15:109-112.
32. Lynd L.R., Weimer P.J., van Zyl W.H., and Pretorius I.S. Microbial Cellulose Utilization: Fundamentals and Biotechnology. Microbiol Mol Biol Rev, 2002, 66(3): 506-577.
33. Reese E.T. History of the cellulose program at the US army Natick development centre. Biotechnol Bioeng Symp, 1976, 6: 9-20.
34. Bhat M.K., and Bhat S. Cellulose Degrading Enzymes and Their Potential Industrial Applications. Biotechnol Adv, 1997, 15: 583-620.
35. Tomme P., Warren R.A.J., and Gilkes N.R. Cellulose hydrolysis by bacteria and fungi. Adv Microb Physiol, 1995, 37: 1-81.
36. Henrissat B. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J, 1991,280:309-316.
37. Henrissat B., Claeyssens M., Tomme P., Lemesle L., and Mornon J.-P. Cellulase families revealed by hydrophobic cluster analysis. Gene, 1989, 81: 83-95.
38. Teeri T.T. Crystalline cellulose degradation: new insight into the function of cellobiohydrolases. Trends Biotechnol, 1997, 15:160-167.
39. Din N., Damude H.G., Gilkes N.R., Miller R.C., Warren R.A.J., and Kilburn D.G. Cl-Cx revisited: intramolecular synergism in a cellulase. Proc Natl Acad Sci, 1994, 91: 11383-11387.
40. Fanutti C., Ponyi T., Black G.W., Hazlewood G.P., and Gilbert H.J. The conserved noncatalytic 40-residue sequence in cellulases and hemicellulases from anaerobic fungi functions as a protein docking domain. J Biol Chem, 1995, 270: 29314-29322.
41. Li X.-L., Chen H.Z., and Ljungdahl L.G. Monocentric and polycentric anaerobic fungi produce structurally related cellulases and xylanases. Appl Environ Microbiol, 1997, 63: 628-635.
42. Lamed R., Naimark J., Morgenstern E., and Bayer E.A. Specialized cell surface structures in cellulolytic bacteria. J Bacteriol, 1987, 169: 3792-3800.
43. Vladut-Talor M., Kauri T., and Kushner D.J. Effects of cellulose on growth, enzyme production and ultrastructure of a Cellulomonas species. Arch Microbiol, 1986, 144: 191-195.
44. Tilbeurgh H., Tomme P., Claeywwens M. Limited proteolysis of the cellobiohydrolase Ⅰ from T. reesei. FEBS Lett, 1986, 204: 223-227.
45. Abuja P.M., Pilz I., Claeywwens M. Domain structure of cellobiogydrolase Ⅱ as studied by small angle X-ray scatter: close resemblance to cellobiohydrolase Ⅰ. Biochem Biophy Res Commun, 1988, 156: 14-185.
46．阎伯旭，曲音波，高培基，孙迎庆．真菌和细菌纤维素酶的差异及内、外切酶的底物专一性．生命科学，1999，11(增)：61-64．
47．阎伯旭，齐飞，张颖舒．纤维素酶分子结构和功能研究进展．生物化学与生物物理进展，1999，26(3)：235-237．
48. Juy M., Amit G., Alzati M., et al. Crystal structure of a thermostable bacterial cellulose degrading enzyme. Nature, 1992, 357(6373): 89-91.
49．高培基，曲音波，汪天虹．微生物降解纤维素机制的分子生物学研究进展．纤维素科学与技术，1995，3(2)：1-19．
50．王春卉，汪天虹，高培基．纤维素酶分子的纤维素吸附区的研究进展．纤维素科学与技术，1997，5(4)：1-10．
51．王菁莎，王颉，刘景彬．纤维素酶的应用现状．中国食品添加剂，2005，15：107-110．
52．李燕红，赵辅昆．纤维素酶的研究进展．生命科学，17(5)：392-397．
53. Ponpium P., Ratanakhanokchai K., Kyu K.L. Isolation and properties of a cellulosome-typc multienzyme complex of the thermophilic bacteroides sp. swain P-1. Enzyme Microb Tcclmol, 2000, 26(5-6): 459-465.
54. Felix C.R., Ljungdahl L.G. The cellulosomc: the exocellular organclle of Clostridium. Annu Rev Microbiol, 1993, 47: 791-819.
55. Raghothama S., Eberhardt R.Y., Simpson P., Wigelsworth D., White P., Hazlewood G.P., Nagy T., Gilbert H.J., Williamson M.P. Characterization of a cellulosome dockerin domain from the anaerobic fungus Piromyces equi. Nat Struct Biol, 2001, 8(9): 775-778.
56. Lamed R., Setter E., and Bayer E.A. Characterization of a cellulose-binding, cellulase-containing complex in Clostridium thermocellum. J Bacteriol, 1983, 156: 825-836.
57. Bayer E.A., Kenig R., and Lamed R. Studies on the adherence of Clostridium thermocellum to cellulose. J Bacteriol, 1983, 156: 818-827.
58. Beguin P. Molecular biology of cellulose degradation. Annu Rev Microbiol, 1990, 44:219-248.
59. Hazlewood G.P., Romaniec M.P.M., Davidson K., Grepinet O., Beguin P., Millet J., Raynaud O., and Aubert J.-P. A catalogue of Clostridium thermocellum endoglucanase, β-glucosidase and xylanase genes cloned in Escherichia coli. FEMS Microbiol Lett, 1988, 51: 231-236.
60. Bayer E.A., Shimon L.J.W., Shoham Y., and Lamed R. Cellulosomes-structure and ultrastructure. J Struct Biol, 1998, 124(2-3): 221-234.
61. Kataeva I., Li X.L., Chen H., Choi S.K., Ljungdahl L.G.. Cloning and sequence analysis of a new cellulase gene encoding CelK, a major cellulosome component of Clostridium thermocellum: evidence for gene duplication and recombination. J Bacteriol, 1999, 181(17): 5288-5895.
62. Chauvaux S., Beguin P., Aubert J.-P., Bhat K.M., Gow L.A.,Wood T.M., and Bairoch A. Calcium-binding affinity and calcium-enhanced activity of Clostridium thermocellum endoglucanase D. Biochem J, 1990, 265:261-265.
63. Wang W.K., Kruus K., and Wu J.H.D. Cloning and DNA sequence of the gene coding for Clostridium thermocellum cellulase SS (CelS), a major cellulosome component. J Bacteriol, 1993,175: 1293-1302.
64. Doi R.H., Tamaru Y. The Clostridium cellulovorans cellulosome: an enzyme complex with plant cell wall degrading activity. Chem Rec, 2001, 1(1): 24-32.
65. Sara M., Sleytr U.B. S-Layer proteins. J Bacteriol, 2000,182(4): 859-868.
66. Foong F., Hamamoto T., Shoseyov O., Doi R.H. Nucleotide sequence and characteristics of endoglucanase gene engB from Clostridium cellulovorans. J Gen Microbiol, 1991, 137: 1729-1736.
67. Pohlschroder M., Canale-Parola E., and Leschine S.B. Ultrastructural diversity pf the cellulase complexes of Clostridium papyrosohens C7. J Bacteriol, 1995, 177: 6625-6629.
68. Leibovitz E., and Beguin P. A new type of cohesin domain that specifically binds the dockerin domain of the Clostridium thermocellum cellulosome-integrating protein CipA. J Bacteriol, 1996,178: 3077-3084.
69. Leibovitz E., Ohayon H., Gounon P., and Beguin P. Characterization and subcellular localization of the Clostridium thermocellum scaffoldin dockerin binding protein SdbA. J Bacteriol, 1997,179: 2519-2523.
70. Fujino T., Beguin P., and Aubert J.-P. Organization of a Clostridium thermocellum gene cluster encoding the cellulosomal scaffolding protein CipA and a protein possibly involved in attachment of the cellulosome to the cell surface. J Bacteriol, 1993,175: 1891-1899.
71. Pages S., Belaich A., Tardif C, Reverbel-Leroy C, Gaudin C, and Belaich J.-P. Interaction between the endoglucanase CelA and the scaffolding protein CipC of the Clostridium cellulolyticum cellulosome. J Bacteriol, 1996,178: 2279-2286.
72. Shoseyov O., Takagi M., Goldstein M.A., and Doi R.H. Primary sequence analysis of Clostridium cellulovorans cellulose binding protein A. Proc Natl Acad Sci, 1992, 89: 3483-3487.
73. Kakiuchi M, Isui A., Suzuki K., Fujino T., Fujino E., Kimura T., Karita S., Sakka K., and Ohmiya K. Cloning and DNA sequencing of the genes encoding Clostridium josui scaffolding protein CipA and cellulase CelD and identification of their gene products as major components of the cellulosome. J Bacteriol, 1998, 180: 4303-4308.
74．吴斌辉．硕士毕业论文：纤维堆囊菌在纤维素介质上细胞行为．山东大学，2002．5．
75．赵昕，王冬，高培基．纤维素微生物及纤维素酶技术(第三版)．山东大学微生物研究所，1994．1．
76. Teather R.M., and Wood P.J.Use of Congo Red-Polysaccharide Interactions in Enumeration and Characteriaztion of Cellulolytic Bacteria from the Bovine Rumen. Appl Environ Microbiol, 1982, 43(4): 777-780.
77. Bailey M., Peter B., Kaisa P. Interlabortory testing of methods for assay of Xylanase activity. J biotech, 1992, 23: 257-270.
78. Beguin P., Millet J., Chauvaux S., Salamitou S., Tokatlidis K., Navas J., Fujino T., Lemaire M., Raynaud O., Daniel M.K. et al. Bacterial cellulases. Biochem Soc Trans, 1992, 20: 42-46.
79. Shoham Y., Lamed R., Bayer E.A. The cellulosome concept as an efficient microbial strategy for the degradation of insoluble polysaccharides. Trends Microbiol, 1999, 7: 275-281.
80. Gal L., Pages S., Gaudin C., Belaich A., Reverbel-Leroy C., Tardif C., Belaich J.-P. Characterization of the cellulolytic complex (cellulosome) produced by Clostridium cellulolyticum. Appl Environ Microbiol, 1997, 63: 903-909.
81. Laemmli U.K. Cleavage of structural proteins during the assembly of bacteriophage T4. Nature, 1970, 227: 680-685.
82．张晓元．硕士毕业论文：纤维堆囊菌So9733-1木聚糖酶基因的克隆、表征与异源表达分析．山东大学，2005．5．
83．pET系统操作手册(第十版)．MERCK公司．
84．汪家政，范明．蛋白质技术手册．科学出版社，2000．8．
85．陶义训．免疫学和免疫学检验(第二版)．人民卫生出版社，1997．10．
86．尹学念．免疫学和免疫学检验实验指导．人民卫生出版社．1999．7．
87. Zhang H., Zhao Y.C., Zhang H.X., Feng Y.J., and Zhang C.K. Quantitative Method Measurement of Flammulin Concentration with Immunoassay. International journal of Medicinal Mushrooms, 2002,4: 237-240.
88. 于涟. 免疫学实验技术.浙江大学出版社,1992.3.
89. Hennequin C, Janoir C, Barc M.-C, Collignon A., and Karjalainen T. Identification and characterization of a fibronectinbinding protein from Clostridium difficile. Microbiology, 2003,149: 2779-2787.
90. Hennequin C, Porcheray F., Waligora-Dupriet A.-J., Collignon A., Barc M.-C, Bourlioux P., and Karjalainen T. GroEL (Hsp60) of Clostridium difficile is involved in cell adherence. Microbiology, 2001,147: 87-96.
91. Navarro A., Chebrou M.-C, Beguin P., and Aubert J.-P. Nucleotide sequence of the cellulase gene celF of Clostridium thermocellum. Res Microbiol, 1991, 142: 927-936.
92. Yague E., Beguin P., and Aubert J.-P. Nucleotide sequence and deletion analysis of the cellulase-encoding gene celH of Clostridium thermocellum. Gene, 1990, 89: 61-67.
93. Ahsan M.M., Kimura T., Karita S., Sakka K., and Ohmiya K. Cloning, DNA sequencing, and expression of the gene encoding Clostridium thermocellum cellulase CelJ, the largest catalytic component of the cellulosome. J Bacteriol, 1996, 178: 5732-5740.
94. Zverlov V.V., Velikodvorskaya G.V., Schwarz W.H., Bronnenmeier K., Kellermann J., and Staudenbauer W.L. Multidomain structure and cellulosomal localization of the Clostridium thermocellum cellobiohydrolase Cbh A. J Bacteriol, 1998,180:3091-3099.
95. Fontes CM., Hazlewood G.P., Morag E., Hall J., Hirst B.H., and Gilbert H.J. Evidence for a general role for non-catalytic thermostabilizing domains in xylanases from thermophilic bacteria. Biochem J, 1995, 307: 151-158.
96. Hayashi H., Takagi K.I., Fukumura M., Kimura T., Karita S., Sakka K., and Ohmiya K. Sequence of xynC and properties of XynC, a major component of the Clostridium thermocellum cellulosome. J Bacteriol, 1997,179: 4246-4253.
97. Swinbanks D. Government backs proteome proposal. Nature, 1995, 378: 653.
98．Bio-rad蛋白质组双向电泳实验操作手册．Bio-rad公司．
99．陈平，谢锦云，梁宋平．双向凝胶电泳银染蛋白质点的肽质谱指纹图分析．生物化学与生物物理学报，2000，32(4)：387-391．
100. Wood T.M., Mccrae S.I. Sinergism between enzymes involvement in the solubilization of native cellulose. Adv Chem Ser, 1979, 181: 181-209.
101. Berghem L.E., Petersson L.G., Axio-Fredrikwwon U.B. The mechanism of enzymatic cellulose degradation. Purification and some properties of two different 1,4-β-D-glucan glucanohydrolase from Trichoderma virid. Eur J Biochem, 1976, 61 (2): 621-630.
102. Selby K., Maitland C.C. The fractionation of Myrothecium verrucaria cellulose by gel filtration. Biochem J, 1965, 104(3): 716-724.
103. Wise M.J., Littlejohn T.G., Humphery-Smith I. Peptide-mass fingerprinting and the ideal covering set for protein characterisation. Electrophoresis, 1997, 18(8): 1399-1409.
104. Scheler C., Lamer S., Pan Z.M., Li X.P., Salnikow J., Jungblut P. Peptide mass fingerprint sequence coverage from differently stained proteins on two-dimensional electrophoresis pattems by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Electrophoresis, 1998, 19:918-927.
105. Yates J.R. Database searching using mass spectrometry data. Electrophoresis, 1998, 19: 893-900.
106. Sanz-Nebot V., Toro I., Garces A., Barbosa J. Separation and identification of peptide mixtures in a synthesis crude of carbetocin by liquid chromatography/electrospray ionization mass spectrometry. Rapid Commtm Mass Spectrom, 1999, 13: 2341-2347.
2. Shimkets L. J. Social and developmental biology of myxobacteria. Microbiol Rev, 1990, 54: 473-501.
3. McCurdy H. D. Order of Myxococcales. In: Stanley J. T., Bryant M. P., Pfening N., Holt J. G. (eds) Bergey's Manual of Systematic Bacteriology, 9th ed. Williams and Wilkins, Baltimore, 1989, pp2139-2170.
4. Nguimbi E., Li Y. Z., Gao B. L., Li Z. F., Wang B., Wu Z. H., Yan B. X., Qu Y. B., Gao P. J. 16S-23S ribosomal DNA intergenic spacer regions in cellulolytic myxobacteria and differentiation of closely related strains. Syst Appl Microbiol, 2003, 26(2): 262-268.
5. Yan Z. C., Wang B., Li Y. Z., Gong X., Zhang H. Q., Gao P. J. Morphologies and phylogenetic classification of cellulolytic myxobacteria. Syst Appl Microb, 2003, 26: 104-109.
6. MacRae T. H. and McCurdy H. D. Ultrastructural studies of Chondromyces crocatus vegetative cells. Can J Microbiol, 1975, 21: 1815-1826.
7．方晓梅，张利平．粘细菌生态多样性的初步研究．生物多样性，2001，9(3)：207-213．
8. Dawid W. Biology and global distribution of myxobacteria in soils. FEMS Microbiology Reviews, 2000, 24: 403-427.
9．王海英，张利平．粘细菌：一类重要的微生物资源．微生物学通报，2003，30(2)：115-116．
10. Ruckert G. Myxobakterien (Myxobacterales) auf Blattober-flachen. Z. Allg. Mikrobiol, 1981, 21: 761-763.
11. Dawid W., Gallikowski C. A. and Hirsch P. Psychrophilic myxobacteria from Antarctic soils. Polarforschung, 1988, 58: 271-278.
12. Brockman E. R. Myxobacters from arid Mexican soil. Appl Environ Microbiol, 1976, 32: 642-644.
13. Dworkin M. Recent advances in the social and developmental biology of the myxobacteria. Microbiol Rev, 1996, 60: 70-102.
14．周璐，李越中，李健．粘细菌的多细胞形态发生及其分子调控．生物化学与生物物理进展，1999，26(6)：544-547．
15. Wolgemuth C., Hoiczyk E., Kaiser D., Oster G. How myxobacteria glide. Curr Biol, 2002, 12(5): 369-377.
16. Fink J. M. and Zissler J. F. Defects in motility and development of Myxococcus xanthus lipopolysaccharide mutants. J Bacteriol, 1989, 171: 2042-2048.
17. Shimkets L. J. and Kaiser D. Induction of coordinated cell movement in Myxococcus xanthus. J Bacteriol, 1982, 152: 451-461.
18．吴斌辉，李越中，王溱圯，胡玮，候爱华．纤维堆囊菌生长及限制因素的研究．生物技术，2001，11(3)：16-18．
19. Hofle G. and Reichenbach H. Biologically active substances from microorganisms-an interdisciplinary research project at the GBF. Sci Ann Rep, 1990, pp5-22. Gesellschaft fur Biotechnolog. Forschung, Braunschweig.
20．李健，李越中，王溱圯．粘细菌生物活性物质研究进展．中国新药杂志，1998，7(2)：84-89．
21．李越中，李健，周璐，张勇，胡玮，陈琦．我国粘细菌(Myxobacteria)资源的分离与鉴定．微生物学报，2000，40(6)：652-656．
22．吴斌辉，李越中，胡玮，周璐，侯配斌．纤维堆囊菌的多细胞形态发生过程．微生物学报，2002，42(6)：657-661．
23．李越中，胡玮，吴斌辉，阎章才，陈琦．纤维堆囊菌的代谢产物及其生物学活性分析．微生物学报，2001，41(6)：716-722．
24. Domling A., Richter W. Myxobacterial epothilones and tubulysins as promising anticancer agents. Mol Divers, 2005, 9(1-3): 141-147.
25．吴斌辉，李越中，胡玮．纤维堆囊菌活性次级代谢产物的研究进展．微生物 学通报，2002，29(2)：75-78．
26．李越中，李健．粘细菌的分离纯化．微生物学通报，1997，24(4)：237-240．
27. Gardner K.H., Blackwell J. The structure of native cellulose. Biopolymer, 1974, 13: 1975-1987.
28．孟雷，陈冠军，王怡，高培基．纤维素酶的多型性．纤维素科学与技术，2002，10(2)：47-55．
29. Lynd L.R., Wyman C.E., and Gemgross T.U. Biocommodity engineering. Biotechnol Prog, 1999, 15: 777-793.
30. Marchessault R.H., and Sundararajan P.R.. Cellulose. In: Aspinall G.O. (ed.) The polysaccharides vol. 2. Academic Press, Inc., New York. 1993, pp11-95.
31. Atalla, R.H., Hackney J.M., Uhlin I., and Thompson N.S. Hemicelluloses as structure regulators in the. aggregation of native cellulose. Int J Biol Macromol, 1993, 15:109-112.
32. Lynd L.R., Weimer P.J., van Zyl W.H., and Pretorius I.S. Microbial Cellulose Utilization: Fundamentals and Biotechnology. Microbiol Mol Biol Rev, 2002, 66(3): 506-577.
33. Reese E.T. History of the cellulose program at the US army Natick development centre. Biotechnol Bioeng Symp, 1976, 6: 9-20.
34. Bhat M.K., and Bhat S. Cellulose Degrading Enzymes and Their Potential Industrial Applications. Biotechnol Adv, 1997, 15: 583-620.
35. Tomme P., Warren R.A.J., and Gilkes N.R. Cellulose hydrolysis by bacteria and fungi. Adv Microb Physiol, 1995, 37: 1-81.
36. Henrissat B. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J, 1991,280:309-316.
37. Henrissat B., Claeyssens M., Tomme P., Lemesle L., and Mornon J.-P. Cellulase families revealed by hydrophobic cluster analysis. Gene, 1989, 81: 83-95.
38. Teeri T.T. Crystalline cellulose degradation: new insight into the function of cellobiohydrolases. Trends Biotechnol, 1997, 15:160-167.
39. Din N., Damude H.G., Gilkes N.R., Miller R.C., Warren R.A.J., and Kilburn D.G. Cl-Cx revisited: intramolecular synergism in a cellulase. Proc Natl Acad Sci, 1994, 91: 11383-11387.
40. Fanutti C., Ponyi T., Black G.W., Hazlewood G.P., and Gilbert H.J. The conserved noncatalytic 40-residue sequence in cellulases and hemicellulases from anaerobic fungi functions as a protein docking domain. J Biol Chem, 1995, 270: 29314-29322.
41. Li X.-L., Chen H.Z., and Ljungdahl L.G. Monocentric and polycentric anaerobic fungi produce structurally related cellulases and xylanases. Appl Environ Microbiol, 1997, 63: 628-635.
42. Lamed R., Naimark J., Morgenstern E., and Bayer E.A. Specialized cell surface structures in cellulolytic bacteria. J Bacteriol, 1987, 169: 3792-3800.
43. Vladut-Talor M., Kauri T., and Kushner D.J. Effects of cellulose on growth, enzyme production and ultrastructure of a Cellulomonas species. Arch Microbiol, 1986, 144: 191-195.
44. Tilbeurgh H., Tomme P., Claeywwens M. Limited proteolysis of the cellobiohydrolase Ⅰ from T. reesei. FEBS Lett, 1986, 204: 223-227.
45. Abuja P.M., Pilz I., Claeywwens M. Domain structure of cellobiogydrolase Ⅱ as studied by small angle X-ray scatter: close resemblance to cellobiohydrolase Ⅰ. Biochem Biophy Res Commun, 1988, 156: 14-185.
46．阎伯旭，曲音波，高培基，孙迎庆．真菌和细菌纤维素酶的差异及内、外切酶的底物专一性．生命科学，1999，11(增)：61-64．
47．阎伯旭，齐飞，张颖舒．纤维素酶分子结构和功能研究进展．生物化学与生物物理进展，1999，26(3)：235-237．
48. Juy M., Amit G., Alzati M., et al. Crystal structure of a thermostable bacterial cellulose degrading enzyme. Nature, 1992, 357(6373): 89-91.
49．高培基，曲音波，汪天虹．微生物降解纤维素机制的分子生物学研究进展．纤维素科学与技术，1995，3(2)：1-19．
50．王春卉，汪天虹，高培基．纤维素酶分子的纤维素吸附区的研究进展．纤维素科学与技术，1997，5(4)：1-10．
51．王菁莎，王颉，刘景彬．纤维素酶的应用现状．中国食品添加剂，2005，15：107-110．
52．李燕红，赵辅昆．纤维素酶的研究进展．生命科学，17(5)：392-397．
53. Ponpium P., Ratanakhanokchai K., Kyu K.L. Isolation and properties of a cellulosome-typc multienzyme complex of the thermophilic bacteroides sp. swain P-1. Enzyme Microb Tcclmol, 2000, 26(5-6): 459-465.
54. Felix C.R., Ljungdahl L.G. The cellulosomc: the exocellular organclle of Clostridium. Annu Rev Microbiol, 1993, 47: 791-819.
55. Raghothama S., Eberhardt R.Y., Simpson P., Wigelsworth D., White P., Hazlewood G.P., Nagy T., Gilbert H.J., Williamson M.P. Characterization of a cellulosome dockerin domain from the anaerobic fungus Piromyces equi. Nat Struct Biol, 2001, 8(9): 775-778.
56. Lamed R., Setter E., and Bayer E.A. Characterization of a cellulose-binding, cellulase-containing complex in Clostridium thermocellum. J Bacteriol, 1983, 156: 825-836.
57. Bayer E.A., Kenig R., and Lamed R. Studies on the adherence of Clostridium thermocellum to cellulose. J Bacteriol, 1983, 156: 818-827.
58. Beguin P. Molecular biology of cellulose degradation. Annu Rev Microbiol, 1990, 44:219-248.
59. Hazlewood G.P., Romaniec M.P.M., Davidson K., Grepinet O., Beguin P., Millet J., Raynaud O., and Aubert J.-P. A catalogue of Clostridium thermocellum endoglucanase, β-glucosidase and xylanase genes cloned in Escherichia coli. FEMS Microbiol Lett, 1988, 51: 231-236.
60. Bayer E.A., Shimon L.J.W., Shoham Y., and Lamed R. Cellulosomes-structure and ultrastructure. J Struct Biol, 1998, 124(2-3): 221-234.
61. Kataeva I., Li X.L., Chen H., Choi S.K., Ljungdahl L.G.. Cloning and sequence analysis of a new cellulase gene encoding CelK, a major cellulosome component of Clostridium thermocellum: evidence for gene duplication and recombination. J Bacteriol, 1999, 181(17): 5288-5895.
62. Chauvaux S., Beguin P., Aubert J.-P., Bhat K.M., Gow L.A.,Wood T.M., and Bairoch A. Calcium-binding affinity and calcium-enhanced activity of Clostridium thermocellum endoglucanase D. Biochem J, 1990, 265:261-265.
63. Wang W.K., Kruus K., and Wu J.H.D. Cloning and DNA sequence of the gene coding for Clostridium thermocellum cellulase SS (CelS), a major cellulosome component. J Bacteriol, 1993,175: 1293-1302.
64. Doi R.H., Tamaru Y. The Clostridium cellulovorans cellulosome: an enzyme complex with plant cell wall degrading activity. Chem Rec, 2001, 1(1): 24-32.
65. Sara M., Sleytr U.B. S-Layer proteins. J Bacteriol, 2000,182(4): 859-868.
66. Foong F., Hamamoto T., Shoseyov O., Doi R.H. Nucleotide sequence and characteristics of endoglucanase gene engB from Clostridium cellulovorans. J Gen Microbiol, 1991, 137: 1729-1736.
67. Pohlschroder M., Canale-Parola E., and Leschine S.B. Ultrastructural diversity pf the cellulase complexes of Clostridium papyrosohens C7. J Bacteriol, 1995, 177: 6625-6629.
68. Leibovitz E., and Beguin P. A new type of cohesin domain that specifically binds the dockerin domain of the Clostridium thermocellum cellulosome-integrating protein CipA. J Bacteriol, 1996,178: 3077-3084.
69. Leibovitz E., Ohayon H., Gounon P., and Beguin P. Characterization and subcellular localization of the Clostridium thermocellum scaffoldin dockerin binding protein SdbA. J Bacteriol, 1997,179: 2519-2523.
70. Fujino T., Beguin P., and Aubert J.-P. Organization of a Clostridium thermocellum gene cluster encoding the cellulosomal scaffolding protein CipA and a protein possibly involved in attachment of the cellulosome to the cell surface. J Bacteriol, 1993,175: 1891-1899.
71. Pages S., Belaich A., Tardif C, Reverbel-Leroy C, Gaudin C, and Belaich J.-P. Interaction between the endoglucanase CelA and the scaffolding protein CipC of the Clostridium cellulolyticum cellulosome. J Bacteriol, 1996,178: 2279-2286.
72. Shoseyov O., Takagi M., Goldstein M.A., and Doi R.H. Primary sequence analysis of Clostridium cellulovorans cellulose binding protein A. Proc Natl Acad Sci, 1992, 89: 3483-3487.
73. Kakiuchi M, Isui A., Suzuki K., Fujino T., Fujino E., Kimura T., Karita S., Sakka K., and Ohmiya K. Cloning and DNA sequencing of the genes encoding Clostridium josui scaffolding protein CipA and cellulase CelD and identification of their gene products as major components of the cellulosome. J Bacteriol, 1998, 180: 4303-4308.
74．吴斌辉．硕士毕业论文：纤维堆囊菌在纤维素介质上细胞行为．山东大学，2002．5．
75．赵昕，王冬，高培基．纤维素微生物及纤维素酶技术(第三版)．山东大学微生物研究所，1994．1．
76. Teather R.M., and Wood P.J.Use of Congo Red-Polysaccharide Interactions in Enumeration and Characteriaztion of Cellulolytic Bacteria from the Bovine Rumen. Appl Environ Microbiol, 1982, 43(4): 777-780.
77. Bailey M., Peter B., Kaisa P. Interlabortory testing of methods for assay of Xylanase activity. J biotech, 1992, 23: 257-270.
78. Beguin P., Millet J., Chauvaux S., Salamitou S., Tokatlidis K., Navas J., Fujino T., Lemaire M., Raynaud O., Daniel M.K. et al. Bacterial cellulases. Biochem Soc Trans, 1992, 20: 42-46.
79. Shoham Y., Lamed R., Bayer E.A. The cellulosome concept as an efficient microbial strategy for the degradation of insoluble polysaccharides. Trends Microbiol, 1999, 7: 275-281.
80. Gal L., Pages S., Gaudin C., Belaich A., Reverbel-Leroy C., Tardif C., Belaich J.-P. Characterization of the cellulolytic complex (cellulosome) produced by Clostridium cellulolyticum. Appl Environ Microbiol, 1997, 63: 903-909.
81. Laemmli U.K. Cleavage of structural proteins during the assembly of bacteriophage T4. Nature, 1970, 227: 680-685.
82．张晓元．硕士毕业论文：纤维堆囊菌So9733-1木聚糖酶基因的克隆、表征与异源表达分析．山东大学，2005．5．
83．pET系统操作手册(第十版)．MERCK公司．
84．汪家政，范明．蛋白质技术手册．科学出版社，2000．8．
85．陶义训．免疫学和免疫学检验(第二版)．人民卫生出版社，1997．10．
86．尹学念．免疫学和免疫学检验实验指导．人民卫生出版社．1999．7．
87. Zhang H., Zhao Y.C., Zhang H.X., Feng Y.J., and Zhang C.K. Quantitative Method Measurement of Flammulin Concentration with Immunoassay. International journal of Medicinal Mushrooms, 2002,4: 237-240.
88. 于涟. 免疫学实验技术.浙江大学出版社,1992.3.
89. Hennequin C, Janoir C, Barc M.-C, Collignon A., and Karjalainen T. Identification and characterization of a fibronectinbinding protein from Clostridium difficile. Microbiology, 2003,149: 2779-2787.
90. Hennequin C, Porcheray F., Waligora-Dupriet A.-J., Collignon A., Barc M.-C, Bourlioux P., and Karjalainen T. GroEL (Hsp60) of Clostridium difficile is involved in cell adherence. Microbiology, 2001,147: 87-96.
91. Navarro A., Chebrou M.-C, Beguin P., and Aubert J.-P. Nucleotide sequence of the cellulase gene celF of Clostridium thermocellum. Res Microbiol, 1991, 142: 927-936.
92. Yague E., Beguin P., and Aubert J.-P. Nucleotide sequence and deletion analysis of the cellulase-encoding gene celH of Clostridium thermocellum. Gene, 1990, 89: 61-67.
93. Ahsan M.M., Kimura T., Karita S., Sakka K., and Ohmiya K. Cloning, DNA sequencing, and expression of the gene encoding Clostridium thermocellum cellulase CelJ, the largest catalytic component of the cellulosome. J Bacteriol, 1996, 178: 5732-5740.
94. Zverlov V.V., Velikodvorskaya G.V., Schwarz W.H., Bronnenmeier K., Kellermann J., and Staudenbauer W.L. Multidomain structure and cellulosomal localization of the Clostridium thermocellum cellobiohydrolase Cbh A. J Bacteriol, 1998,180:3091-3099.
95. Fontes CM., Hazlewood G.P., Morag E., Hall J., Hirst B.H., and Gilbert H.J. Evidence for a general role for non-catalytic thermostabilizing domains in xylanases from thermophilic bacteria. Biochem J, 1995, 307: 151-158.
96. Hayashi H., Takagi K.I., Fukumura M., Kimura T., Karita S., Sakka K., and Ohmiya K. Sequence of xynC and properties of XynC, a major component of the Clostridium thermocellum cellulosome. J Bacteriol, 1997,179: 4246-4253.
97. Swinbanks D. Government backs proteome proposal. Nature, 1995, 378: 653.
98．Bio-rad蛋白质组双向电泳实验操作手册．Bio-rad公司．
99．陈平，谢锦云，梁宋平．双向凝胶电泳银染蛋白质点的肽质谱指纹图分析．生物化学与生物物理学报，2000，32(4)：387-391．
100. Wood T.M., Mccrae S.I. Sinergism between enzymes involvement in the solubilization of native cellulose. Adv Chem Ser, 1979, 181: 181-209.
101. Berghem L.E., Petersson L.G., Axio-Fredrikwwon U.B. The mechanism of enzymatic cellulose degradation. Purification and some properties of two different 1,4-β-D-glucan glucanohydrolase from Trichoderma virid. Eur J Biochem, 1976, 61 (2): 621-630.
102. Selby K., Maitland C.C. The fractionation of Myrothecium verrucaria cellulose by gel filtration. Biochem J, 1965, 104(3): 716-724.
103. Wise M.J., Littlejohn T.G., Humphery-Smith I. Peptide-mass fingerprinting and the ideal covering set for protein characterisation. Electrophoresis, 1997, 18(8): 1399-1409.
104. Scheler C., Lamer S., Pan Z.M., Li X.P., Salnikow J., Jungblut P. Peptide mass fingerprint sequence coverage from differently stained proteins on two-dimensional electrophoresis pattems by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Electrophoresis, 1998, 19:918-927.
105. Yates J.R. Database searching using mass spectrometry data. Electrophoresis, 1998, 19: 893-900.
106. Sanz-Nebot V., Toro I., Garces A., Barbosa J. Separation and identification of peptide mixtures in a synthesis crude of carbetocin by liquid chromatography/electrospray ionization mass spectrometry. Rapid Commtm Mass Spectrom, 1999, 13: 2341-2347.