一、纤维堆囊菌So9733-1木聚糖酶XynB结构域功能研究 二、四种人源肝脏蛋白的异源表达、纯化和结晶
|
摘要
本研究分为两大内容:一、纤维堆囊菌So9733-1木聚糖酶XynB结构域功能研究;二、四种人源肝脏蛋白的异源表达纯化和晶体生长。重点是XynB结构域功能研究。
一、纤维堆囊菌So9733-1木聚糖酶XynB结构域功能研究
纤维堆囊菌不但可以产生多种有活性的次级代谢产物,而且还可以有效降解纤维素,但目前关于其纤维素降解机制还未有报道。在本实验室的前期研究中,已经克隆获得了编码纤维堆囊菌So9733-1木聚糖酶XynB的完整基因。该基因全长1197bp,编码398个氨基酸的多肽链。研究表明,该酶为内切木聚糖酶。但进一步的序列分析显示,该酶N端大约100个氨基酸与数据库中的现有序列没有任何同源性,功能未知。
在本文中,我们重点研究了纤维堆囊菌木聚糖酶XvnB N端结构域的功能。通过生物信息学方法对木聚糖酶XynB进行了保守结构域预测,结果显示XynB中氨基酸114-398为糖基水解酶家族10保守区,氨基酸1-113功能未知。在此基础上,我们成功构建了木聚糖酶XynB N端结构域、C端结构域和完整XynB的异源表达质粒,并在大肠杆菌中实现了异源表达。通过亲和层析和凝胶过滤层析,分离纯化了XynB C端结构域的重组蛋白GST-C和XynB的重组蛋白15b-XynB。其中15b-XynB表达形成包涵体,随后对其进行了复性,获得了有活性的木聚糖酶。接下来,我们对15b-XynB与GST-C在酶活力、Km值、最适反应温度和最适反应pH值等方面进行了比较分析。结果表明,二者在上述酶学性质方面没有明显差异。因此,我们推测,XynB N端结构域的缺失对XynB与底物的结合能力和降解木聚糖的能力没有显著的影响,XvnB的N端结构域不具有底物结合区等与木聚糖降解有关的功能。随后,我们又比较了15b-C和15b-XynB在其异源表达菌株中与细胞膜的结合能力。结果显示,15b-XynB可以与细胞膜结合,而15b-C不能与细胞膜结合。因此,我们推测,XynB N端结构域的缺失使蛋白丧失了与细胞膜结合的能力,XvnB N端结构域与XynB在细胞中的定位有关。这为更加深入地研究XynB N端结构域功能打下基础。
二、四种人源肝脏蛋白的异源表达、纯化和结晶
本文的第二部分研究内容是四种人源肝脏蛋白的异源表达、纯化和结晶,是人类肝脏结构基因组计划中的一部分。人类肝脏结构基因组计划旨在通过实验方式(X-射线晶体衍射、核磁共振质谱)或计算方式(Homology Modeling),对肝脏中一些具有重要生物学意义的生物大分子(特别是蛋白质分子)的三维结构进行规模化测定。在参加人类肝脏结构基因组研究工作的一年中,主要参与了80多个基因的克隆、表达和纯化筛选工作,从中获得了一定数量的目的蛋白纯品。对其中GA3、GA8、GA17、GA19四个蛋白进行了结晶条件摸索并获得微晶,为相关工作的深入开展奠定了基础。
During the study for my master's dissertation,my research has been focusing on the functional characterization of the domains of xylanase XynB from Sorangium cellulosum So9733-1.Meanwhile,I have conducted my research concerning the overexpression,purification and crystallization of four human liver proteins.
Sorangium cellulosum not only generates a spectrum of secondary metabolism products with enzymatic activity,it also mediates the degradation of cellulose effectively.However,the underlying mechanisms concerning its role in cellulose degradation has not been reported yet.Our preliminary data indicate that the full length of xylanase XynB cloning from Sorangium cellulosum So9733-1 comprise 1197bp nucleotides encoding for a peptide with 398 amino acids.Emerging evidence suggests that this enzyme belongs to endo-xylanase family.Furthermore,sequencing analysis showed that the most N-terminal 100 amino acids have no genetic homology to the reported sequences from data base,and no function about the novel fragment has been reported yet.
The undergoing study is designated to explore the function of the domain of xylanase XynB from Sorangium cellulosum So9733-1.After analyzing the conserved sequence of xylanase XynB,we found that the amino acids fragment(114-398)of xylanase XynB belongs to glycosyl hydrolase family concerved sequence,whereas the function of amino acids fragment(1-113)is unknown.Accordingly,we had constructed three plasmids that can heterogenously overexpress the full-length, C-terminal domain or N-terminal structure domain of xylanase XynB,respectively. Resorting to affinity and gel filtration chromatography,we had purified C-terminal domain recombinant protein of xylanase XynB(GST-C)and XynB recombinant protein(15b-XynB).Particularly,XynB recombinant protein(15b-XynB)forms inclusion body,whose xylanase activity can be recovered.Furthermore,we had investigated the differences of 15b-XynB and GST-C recombinant protein in enzyme activity,Km value,and the optimal reaction temperature and pH value.Our data indicate that the loss of XynB N-terminal domain does not exert significant effects on the substrates binding ability and catalytic capability of XynB.Instead,the N-terminal domain of XynB may be involved in the cellular location of XynB within Sorangium cellulosum So9733-1,which sheds lights on the further expatiation of its function within XynB protein.
The remaining part of the undergoing study is related to the expression, purification and crystallization of four human liver proteins,which is part of human liver structural genomic project.The human liver structural genomic project is designated to applying experiment modeling(X-ray crystal diffraction analysis and nuclear magnetic resonance mass spectrometry)and Homology Modeling to analyzing three-dimensional structure of the macro-molecules(especially protein molecules).Within this project,I was actively involved in cloning,expressing,and purifying more than eighty genes,and finally obtained certain purified proteins.In particular,after optimize the condition of crystallization,I had got micro-crystals from four proteins,namely GA3、GA8、GA17、GA19,which provides solid platform for following up research.
一、纤维堆囊菌So9733-1木聚糖酶XynB结构域功能研究
纤维堆囊菌不但可以产生多种有活性的次级代谢产物,而且还可以有效降解纤维素,但目前关于其纤维素降解机制还未有报道。在本实验室的前期研究中,已经克隆获得了编码纤维堆囊菌So9733-1木聚糖酶XynB的完整基因。该基因全长1197bp,编码398个氨基酸的多肽链。研究表明,该酶为内切木聚糖酶。但进一步的序列分析显示,该酶N端大约100个氨基酸与数据库中的现有序列没有任何同源性,功能未知。
在本文中,我们重点研究了纤维堆囊菌木聚糖酶XvnB N端结构域的功能。通过生物信息学方法对木聚糖酶XynB进行了保守结构域预测,结果显示XynB中氨基酸114-398为糖基水解酶家族10保守区,氨基酸1-113功能未知。在此基础上,我们成功构建了木聚糖酶XynB N端结构域、C端结构域和完整XynB的异源表达质粒,并在大肠杆菌中实现了异源表达。通过亲和层析和凝胶过滤层析,分离纯化了XynB C端结构域的重组蛋白GST-C和XynB的重组蛋白15b-XynB。其中15b-XynB表达形成包涵体,随后对其进行了复性,获得了有活性的木聚糖酶。接下来,我们对15b-XynB与GST-C在酶活力、Km值、最适反应温度和最适反应pH值等方面进行了比较分析。结果表明,二者在上述酶学性质方面没有明显差异。因此,我们推测,XynB N端结构域的缺失对XynB与底物的结合能力和降解木聚糖的能力没有显著的影响,XvnB的N端结构域不具有底物结合区等与木聚糖降解有关的功能。随后,我们又比较了15b-C和15b-XynB在其异源表达菌株中与细胞膜的结合能力。结果显示,15b-XynB可以与细胞膜结合,而15b-C不能与细胞膜结合。因此,我们推测,XynB N端结构域的缺失使蛋白丧失了与细胞膜结合的能力,XvnB N端结构域与XynB在细胞中的定位有关。这为更加深入地研究XynB N端结构域功能打下基础。
二、四种人源肝脏蛋白的异源表达、纯化和结晶
本文的第二部分研究内容是四种人源肝脏蛋白的异源表达、纯化和结晶,是人类肝脏结构基因组计划中的一部分。人类肝脏结构基因组计划旨在通过实验方式(X-射线晶体衍射、核磁共振质谱)或计算方式(Homology Modeling),对肝脏中一些具有重要生物学意义的生物大分子(特别是蛋白质分子)的三维结构进行规模化测定。在参加人类肝脏结构基因组研究工作的一年中,主要参与了80多个基因的克隆、表达和纯化筛选工作,从中获得了一定数量的目的蛋白纯品。对其中GA3、GA8、GA17、GA19四个蛋白进行了结晶条件摸索并获得微晶,为相关工作的深入开展奠定了基础。
During the study for my master's dissertation,my research has been focusing on the functional characterization of the domains of xylanase XynB from Sorangium cellulosum So9733-1.Meanwhile,I have conducted my research concerning the overexpression,purification and crystallization of four human liver proteins.
Sorangium cellulosum not only generates a spectrum of secondary metabolism products with enzymatic activity,it also mediates the degradation of cellulose effectively.However,the underlying mechanisms concerning its role in cellulose degradation has not been reported yet.Our preliminary data indicate that the full length of xylanase XynB cloning from Sorangium cellulosum So9733-1 comprise 1197bp nucleotides encoding for a peptide with 398 amino acids.Emerging evidence suggests that this enzyme belongs to endo-xylanase family.Furthermore,sequencing analysis showed that the most N-terminal 100 amino acids have no genetic homology to the reported sequences from data base,and no function about the novel fragment has been reported yet.
The undergoing study is designated to explore the function of the domain of xylanase XynB from Sorangium cellulosum So9733-1.After analyzing the conserved sequence of xylanase XynB,we found that the amino acids fragment(114-398)of xylanase XynB belongs to glycosyl hydrolase family concerved sequence,whereas the function of amino acids fragment(1-113)is unknown.Accordingly,we had constructed three plasmids that can heterogenously overexpress the full-length, C-terminal domain or N-terminal structure domain of xylanase XynB,respectively. Resorting to affinity and gel filtration chromatography,we had purified C-terminal domain recombinant protein of xylanase XynB(GST-C)and XynB recombinant protein(15b-XynB).Particularly,XynB recombinant protein(15b-XynB)forms inclusion body,whose xylanase activity can be recovered.Furthermore,we had investigated the differences of 15b-XynB and GST-C recombinant protein in enzyme activity,Km value,and the optimal reaction temperature and pH value.Our data indicate that the loss of XynB N-terminal domain does not exert significant effects on the substrates binding ability and catalytic capability of XynB.Instead,the N-terminal domain of XynB may be involved in the cellular location of XynB within Sorangium cellulosum So9733-1,which sheds lights on the further expatiation of its function within XynB protein.
The remaining part of the undergoing study is related to the expression, purification and crystallization of four human liver proteins,which is part of human liver structural genomic project.The human liver structural genomic project is designated to applying experiment modeling(X-ray crystal diffraction analysis and nuclear magnetic resonance mass spectrometry)and Homology Modeling to analyzing three-dimensional structure of the macro-molecules(especially protein molecules).Within this project,I was actively involved in cloning,expressing,and purifying more than eighty genes,and finally obtained certain purified proteins.In particular,after optimize the condition of crystallization,I had got micro-crystals from four proteins,namely GA3、GA8、GA17、GA19,which provides solid platform for following up research.
引文
1.Reichenbach,H.and Hofle,G..(1999).Myxobacteria as producers of secondary metabolites.In:Grabley S,Thiericke R(eds)Drug discovery from nature.Springer,Berlin Heidelberg New York,pp 149-179.
2.Dworkin,M.(2000).Introduction to the myxobacteria.In:Brun YV,Shimkets LJ (eds)Prokaryotic development.American Society of Microbiology,Washington,D.C.,pp 221-242.
3.Shimkets,L.J.(1990)Social and developmental biology of myxobacteria.Microbiol Rev,54:473-501.
4.Dawid,W.2000.Biology and global distribution of myxobacteria in soils.FEMS Microbiol.Rev.24.403-427.
5.Gerth,K.,Pradella,S.,Perlova,O.,Beyer,S.,and Muller,R.(2003).Myxobacteria:proficient producers of novel natural products with various biological activities--past and future biotechnological aspects with the focus on the genus Sorangium.J.Biotechnol.106:233-253.
6.Pradella,S.,Hans,A.,Sproer,C.,Reichenbach,H.,Gerth,K.,and Beyer,S.(2002).Characterisation,genome size and genetic manipulation of the myxobacterium Sorangium cellulosum So ce56.Arch.Microbiol.178:484-492.
7.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.
8.Hofle G.and Reichenbach H.(1990)Biologically active substances from microorganisms - an interdisciplinary research project at the GBF.Sci Ann Rep,pp5-22.Gesellschaft fur Biotechnolog Forschung,Braunschweig.
9.Reichenbach,H.(2005).The Myxococcales.In:Garrity GM.Bergey's Manual of Systematic Bacteriology,Part 3:The Alpha-,Beta-,Delta-,and Epsilon-proteobacteria.2rid ed,New York:Springer-Verlag,pp 1059-1143.
10.Reichenbach,H.and Dworkin,M.(1992).The myxobacteria.In:Balows A, Truper HG,Dworkin M et aL The Prokaryotes.2nd ed.New York:Springer-Verlag
11.Krzemieniewska,H.and Krzemieniewski,S.(1927b).Rozsiedlenie miksobakteryj(Uber die Verbreitung der Myxobakterien im Boden)[in Polish,with German summary.].Acta Soc Botan Polon 5,102-139.
12.McCurdy H.D.(1989)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,pp2139-2170.
13.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.(2003)16S-23S ribosomal DNA intergenic spacer regions in cellulolytic myxobacteria and differentiation of closely related strains.Syst Appl Microbiol,26(2):262-268.
14.Yan,Z.C.,Wang,B.,Li,Y.Z.,Gong,X.,Zhang,H.Q,Gao,P.J.(2003)Morphologies and phylogenetic classification of cellulolytic myxobacteria.Syst Appl Microb,26:104-109.
15.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.
16.Pradella,S.,Hans,A.,Sproer,C.,Reichenbach,H.,Gerth,K.,and Beyer,S.(2002).Characterisation,genome size and genetic manipulation of the myxobacterium Sorangium cellulosum So ce56.Arch.Microbiol.178:484-492.
17.Kopp,M.,Irschik,H.,Pradella,S.,and Muller,R..(2005).Production of the tubulin destabilizer disorazol in Sorangium cellulosum:biosynthetic machinery and regulatory genes.Chembiochem.6:1277-1286.
18.Perlova,O.,Gerth,K.,Kaiser,O.,Hans,A.,and Muller,R.(2006).Identification and analysis of the chivosazol biosynthetic gene cluster from the myxobacterial model strain Sorangium cellulosum So ce56.J Biotechnol.121:174-191.
19.Julien,B.,Shah,S.,Ziermann,R,,Goldman,R.,Katz,L.,and Khosla,C.(2000).Isolation and characterization of the epothilone biosynthetic gene cluster from Sorangium cellulosum.Gene 249:153-160.
20.Gerth,K.,Bedorf,N.,Irschik,H.,Hofle,G,and Reichenbach,H.(1994).The soraphens:a family of novel antifungal compounds from Sorangium cellulosum (Myxobacteria).Ⅰ.Soraphen Al alpha:fermentation,isolation,biological properties.J Antibiot.(Tokyo)47:23-31.
21.Gerth.K.,Washausen,P.,Hofle,G,Irschik,H.,and Reichenbach,H.(1996).The jerangolids:A family of new antifungal compounds from Sorangium cellulosum (Myxobacteria).Production,physico-chemical and biological properties of jerangolid A.J Antibiot.(Tokyo)49:71-75.
22.Irschik,H.,Jansen,R.,Gerth,K.,Hofle,G..and Reichenbach,H.(1995).Sorangiolid A,a new antibiotic isolated from the myxobacterium Sorangium cellulosum So ce 12.J Antibiot.(Tokyo)48:886-887.
23.Gerth,K.,Schummer,D.,Hofle,G,Irschik,H.,and Reichenbach,H..(1995).Ratjadon:a new antifungal compound from Sorangium cellulosum(myxobacteria)production,physio-chemical and biological properties.J Antibiot.(Tokyo)48:973-976.
24.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.
25.Joseleau,J.P.,Comtat,J.,Ruel,K.(1992)Chemical structure of xylans and their interactions in the plant cell walls.In:Visser J,Beldman G,vanSomeren MAK,Voragen AGJ(eds)Xylans and xylanases.Elsevier,Amsterdam,pp 1-15
26.Whistler,R.L.,Richards,E.L.(1970)Hemicelluloses.In:Pigman W,Horton D (eds)The carbohydrates.Academic Press,New York,pp 447-469
27.Biely,P.,Puls,J.,Schneider,H.(1985)Acetyl xylan esterases in fungal cellulolytic systems.FEBS Lett 186:80-84
28.Chanda,S.K.,Hirst,E.L.,Jones,J.K.N.,Percival,E.G.V.(1950)The constitution ofxylan from esparto grass(Stipa tenacissima).J Chem Soc 50:1287-1289
29.Eda,S.,Ohnishi,A.,Kato,K.(1976)Xylan isolated from the stalks of Nicotiana tabacum.Agric Biol Chem 40:359-364
30.Montgomery,R.,Smith,F.,Srivastava,H.C.(1956)Structure of cornhull hemicellulose.Ⅰ.Partial hydrolysis and identification of 2-0-(α-D-glucopyranosyluronic acid)-D-xylopyranose.J Am Chem Soc 78:2837-2839
31.Dekker,R.F.H.,Richards,G.N.(1976)Hemicellulases,their occurrence,purification,properties and mode of action.Adv Carbohydr Chem Biochem 32:277-352
32.Barry,V.C.,Dillon,T.(1940)Occurrence of xylans in marine algae.Nature 146:620
33.Biely,P.,Mackenzie,C.R.,Puls,J.,Schneider,H(1986)Cooperativity of esterases and xylanases in the enzymatic degradation of acetyl xylan.Biotechnology 4:731-733
34.Sunna,A.and Antranikian,G.(1997)Xylanolytic enzymes from fungi and bacteria.Crit.Rev.Biotechnol.17,39-67.
35.Dekker,R.F.H and Richards,G.N.(1975)Purification,properties and mode of action of hemicellulase-produced by Ceratocystis paradoxa.Carbohydr.Res.39,97-114
36.Ito,K.,Ogassawara,J.,Sugimoto,T.and Ishikawa,T.(1992)Purification and properties of acid stable xylanases form Aspergillus kawachii.Biosci.Biotechnol.Biochem.56,547-550
37.Funaguma,T.,Naito,S.,Morita,M.,Okumara,M.,Sugiura,M.and Hara,A.(1991)Purification and some properties of xylanase from Penicillium herquei Banier and Sartory.Agric.Biol.Chem.55,1163-1165.
38.Spurway,T.D.,Morland,C.,Cooper,A.,Sumner,I.,Hazlewood,G.P.,O'Donnell,A.G.,Pickersgill,R.W.and Gilbert,H.J.(1997)Calcium protects a mesophilic xylanase from proteinase inactivation and thermal unfolding.J.Biol.
39.江正强.基因工程极耐高温木聚糖酶的提纯和酶学性质:博士学位论文.北京:中国农业大学,2001.
40.Wakarchuk,W.W.,Campbell,R.L.,Sung,W.L.,Davoodi,J.and Yaguchi,M.(1994)Mutational and crystallographic analysis of the active residues of the Bacillus circulans xylanase.Protein Sci.3,467-475
41.Torronen,A.,Harkki,A.and Rouvinen,J.(1994)Threedimensional structure of endo-1,4-L-xylanase Ⅱ from Trichoderma reesei:two conformational states in the active site. EMBO J. 13, 2493-2501.
42. Katusbe, Y., Hata, Y., Yamaguchi, H., Moriyama, H., Shinmyo, A. and Okada, H. (1990) Estimation of the xylanase active site from crystalline structure. Proc. Protein Eng. 3, 289-290.
43. Campbell, R.L., Rose, D.R, Wakarchuk, W.W., To, R., Sung, W. and Yaguchi, M. (1993) A comparison of the structure of the 20 kD xylanase from Trichoderma harzianum and Bacillus circulans. Proceedings of the second TRICEL symposium on Trichoderma reesei cellulases of other hydrolases. Espoo 8, 63-72.
44. Pickersgill, R.W., Debeire, P., Debeire-Gosselin, M. and Jenkins, J.A. (1993) Crystallization and preliminary X-ray analysis of a thermophilic Bacillus xylanase. J. Mol. Biol. 230, 664-666.
45. Anna, T., Hadar, F., Rotem, G., Aviva, L., Adra, M., Vivian, S., Capel, M., Shoham, Y. and Shoham, G. (1997) Crystallization and preliminary X-ray analysis of the thermostable alkaline xylanase from Bacillus stearothermophilus. Acta Crystallogr. D53, 608-611.
46. Durrand, R., Rascle, C. and Fevre, M. (1996) Molecular characterization of Xyn 3, a member of the endoxylanase multigene family of the rumen anaerobic fungus Neocallimastix frontalis. Curr. Genet. 30, 531-540
47. Morosoli, R., Roy, C. and Yaguchi, M. (1986) Isolation and partial primary' sequence of a xylanase from the yeast Cryptococcus albidus. Biochim. Biophys. Acta 870, 473-478.
48. Grapinet, O., Chebrou, M.C. and Beguin, P. (1988) Nucleotide sequence and deletion analysis of the xylanase gene (XylZ) of Clostridium thermocellum. J. Bacteriol. 170,4582-4588.
49. Black, G.W., Hazlewood, G.P., Millward-Sadler, J. and Lauirie, J.I. (1995) A modular xylanase containing a novel catalytic xylan-speci(?)c binding domain. Biochem. J. 307, 191-195
50. Winterhalter, C, Heinrich, P., Candussio, A., Wich, G. and Liebl, W. (1995) Identification of a novel cellulose-binding domain within the multidomain 120 kDa xylanase XynA of the hyperthermophilic bacterium Thermotoga maritima. Mol. Microbiol. 15, 431-444.
51. Hayashi, H., Takagi, K.I., Fukumura, M., Kimura, T., Karita, S., Sakka, K. and Ohmiya, K. (1997) Sequence of xynC and properties of XynC, a major component of the Clostridium thermocellum cellulosome. J. Bacteriol. 179, 4246-4253
52. Millward-Sadler, S.J., Poole, D.M., Henrissat, B., Hazlewood, G.P., Clarke, J.H. and Gilbert, H.J. (1994) Evidence for a general role for high affinity non-catalytic cellulose binding domains in microbial plant cell wall hydrolases. Mol. Microbiol. 11,375-382.
53. Kulkarni, N, Shendye, A., Rao, M. (1999) Molecular and biotechnological aspects of xylanases. FEMS Microbiol Rev 23:411-456
54. Bernier, R. Jr., Driguez, H., Desrochers, M. Molecular cloning of a Bacillus subtilis xylanase gene in Escherichia coli. Gene 1983, 26(1):59-65.
55. Kellett, L.E., Poole, D.M., Ferreira, L.M.A., Durrant, A.J., Hazlewood, G.P. and Gilbert, H.J. (1990) Xylanase B and an arabinofuranosidase from Psuedomonas fluorescens subsp. cellulosa contain identical cellulose binding domains and are encoded by adjacent genes. Biochem. J. 272, 369-376.
56. Gosalves, M.J., Perez-Gonzalez, J.A., Gonzalez, R. and Navarro, A. (1991) Two L-glycanase genes are clustered in Bacillus polymyxa: Molecular cloning expression and sequence analysis of genes encoding a xylanase and an endo-L-(1,3)-(M)-glucanase. J. Bacteriol. 173, 7705-7710
57. Bajpai, P. (1999) Application of enzymes in the pulp and paper industry. Biotechnol Prog 15:147-157
58. Bajpai, P., Bhardwaj, N.K., Bajpai, P.K., Jauhari, M.B. (1994) The impact of xylanases on bleaching of eucalyptus kraft pulp. J Biotechnol 38:1-6
59. Srinivasan, M.C., Rele. M.V. (1999) Microbial xylanases for paper industry. Curr Sci 77:137-142
60. Viikari, L., Kantelinen, A., Sandquist, J., Linko, M. (1994) Xylanases in bleaching: from an idea to the industry. FEMS Microbiol Rev 13:338-350
61. Bedford, M.R., Classen, H.L. (1992) The influence of dietary xylanase on intestinal viscosity and molecular weight distribution of carbohydrates in rye-fed broiler chick. In: Visser J, Beldman G, vanSomeren MAK, Voragen AGJ (eds) Xylans and xylanases, Elsevier, Amsterdam, pp 361-370
62. Vanparidon, P.A., Booman, J.C.P., Selten, G.C.M., Geerse, C, Barug, D., deBot, P.H.M., Hemke G (1992) Application of fungal endoxylanase in poultry diets In: Visser J, Beldman G, vanSomeran MAK, Voragen, AGJ (eds) Xylans and xylanases. Elsevier, Amsterdam, pp 371-378
63. Rani, S., Nand, K. (1996) Development of cellulase-free xylanase producing anaerobic consortia for the use of lignocellulosic wastes. Enzyme Microb Technol 18:23-28
64. Dominguez, J.M. (1998) Xylitol production by free and immobilized Debaryomyces hansenii. Biotechnol Lett 20:53-56
65. Khanna, S., Gauri, P. (1993) Regulation, purification and properties of xylanase from Cellulomonas fimi.Enzyme Microb Technol 15:990-995
66. Olsson, L., Hahn-Hagerdal, B. (1996) Fermentation of lignocellulosic hydrolysates for ethanol production. Enzyme Microb Technol 18:312-331
67. Maat, J., Roza, M., Verbakel, J., Stam, H., daSilra, M.J.S., Egmond, M.R., Hagemans, M.L.D., vanGarcom, R.F.M., Hessing, J.G.M., vanDerhondel, C.A.M.J.J., vanRotterdam, C. (1992) Xylanases and their application in bakery. In: Visser J, Beldman G, vanSomeren MAK, Voragen AGJ (eds) Xylans and xylanases. Elsevier, Amsterdam, pp 349-360
68. Kulkarni, N. and Shendye, A. (1999) Molecular and biotechnological aspects of xylanases FEMS Microbiology Reviews 23:411-456
69. Derewenda, U., Swenson, L., Green, R., Wei, Y., Morosoli, R., Shareck, F., Kluepfel, D. and Derewenda, Z.S. (1994) Crystal structure, at 2.6-A resolution, of the Streptomyces lividans xylanase A, a member of the F family of beta-1,4-D-glycanases. J. Biol. Chem. 269, 20811-20814
70. Torronen, A., Harkki, A. and Rouvinen, J. (1994) Threedimensional structure of endo-1,4-beta-xylanase II from Trichoderma reesei: two conformational states in the active site.EMBO J.13,2493-2501.
71.饶子和 结构基因组学的影响、进展与期待(2007)2007科学发展报告 中国科学院
72.Mcpherson,A.Crystallization of biological macromolecules
2.Dworkin,M.(2000).Introduction to the myxobacteria.In:Brun YV,Shimkets LJ (eds)Prokaryotic development.American Society of Microbiology,Washington,D.C.,pp 221-242.
3.Shimkets,L.J.(1990)Social and developmental biology of myxobacteria.Microbiol Rev,54:473-501.
4.Dawid,W.2000.Biology and global distribution of myxobacteria in soils.FEMS Microbiol.Rev.24.403-427.
5.Gerth,K.,Pradella,S.,Perlova,O.,Beyer,S.,and Muller,R.(2003).Myxobacteria:proficient producers of novel natural products with various biological activities--past and future biotechnological aspects with the focus on the genus Sorangium.J.Biotechnol.106:233-253.
6.Pradella,S.,Hans,A.,Sproer,C.,Reichenbach,H.,Gerth,K.,and Beyer,S.(2002).Characterisation,genome size and genetic manipulation of the myxobacterium Sorangium cellulosum So ce56.Arch.Microbiol.178:484-492.
7.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.
8.Hofle G.and Reichenbach H.(1990)Biologically active substances from microorganisms - an interdisciplinary research project at the GBF.Sci Ann Rep,pp5-22.Gesellschaft fur Biotechnolog Forschung,Braunschweig.
9.Reichenbach,H.(2005).The Myxococcales.In:Garrity GM.Bergey's Manual of Systematic Bacteriology,Part 3:The Alpha-,Beta-,Delta-,and Epsilon-proteobacteria.2rid ed,New York:Springer-Verlag,pp 1059-1143.
10.Reichenbach,H.and Dworkin,M.(1992).The myxobacteria.In:Balows A, Truper HG,Dworkin M et aL The Prokaryotes.2nd ed.New York:Springer-Verlag
11.Krzemieniewska,H.and Krzemieniewski,S.(1927b).Rozsiedlenie miksobakteryj(Uber die Verbreitung der Myxobakterien im Boden)[in Polish,with German summary.].Acta Soc Botan Polon 5,102-139.
12.McCurdy H.D.(1989)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,pp2139-2170.
13.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.(2003)16S-23S ribosomal DNA intergenic spacer regions in cellulolytic myxobacteria and differentiation of closely related strains.Syst Appl Microbiol,26(2):262-268.
14.Yan,Z.C.,Wang,B.,Li,Y.Z.,Gong,X.,Zhang,H.Q,Gao,P.J.(2003)Morphologies and phylogenetic classification of cellulolytic myxobacteria.Syst Appl Microb,26:104-109.
15.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.
16.Pradella,S.,Hans,A.,Sproer,C.,Reichenbach,H.,Gerth,K.,and Beyer,S.(2002).Characterisation,genome size and genetic manipulation of the myxobacterium Sorangium cellulosum So ce56.Arch.Microbiol.178:484-492.
17.Kopp,M.,Irschik,H.,Pradella,S.,and Muller,R..(2005).Production of the tubulin destabilizer disorazol in Sorangium cellulosum:biosynthetic machinery and regulatory genes.Chembiochem.6:1277-1286.
18.Perlova,O.,Gerth,K.,Kaiser,O.,Hans,A.,and Muller,R.(2006).Identification and analysis of the chivosazol biosynthetic gene cluster from the myxobacterial model strain Sorangium cellulosum So ce56.J Biotechnol.121:174-191.
19.Julien,B.,Shah,S.,Ziermann,R,,Goldman,R.,Katz,L.,and Khosla,C.(2000).Isolation and characterization of the epothilone biosynthetic gene cluster from Sorangium cellulosum.Gene 249:153-160.
20.Gerth,K.,Bedorf,N.,Irschik,H.,Hofle,G,and Reichenbach,H.(1994).The soraphens:a family of novel antifungal compounds from Sorangium cellulosum (Myxobacteria).Ⅰ.Soraphen Al alpha:fermentation,isolation,biological properties.J Antibiot.(Tokyo)47:23-31.
21.Gerth.K.,Washausen,P.,Hofle,G,Irschik,H.,and Reichenbach,H.(1996).The jerangolids:A family of new antifungal compounds from Sorangium cellulosum (Myxobacteria).Production,physico-chemical and biological properties of jerangolid A.J Antibiot.(Tokyo)49:71-75.
22.Irschik,H.,Jansen,R.,Gerth,K.,Hofle,G..and Reichenbach,H.(1995).Sorangiolid A,a new antibiotic isolated from the myxobacterium Sorangium cellulosum So ce 12.J Antibiot.(Tokyo)48:886-887.
23.Gerth,K.,Schummer,D.,Hofle,G,Irschik,H.,and Reichenbach,H..(1995).Ratjadon:a new antifungal compound from Sorangium cellulosum(myxobacteria)production,physio-chemical and biological properties.J Antibiot.(Tokyo)48:973-976.
24.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.
25.Joseleau,J.P.,Comtat,J.,Ruel,K.(1992)Chemical structure of xylans and their interactions in the plant cell walls.In:Visser J,Beldman G,vanSomeren MAK,Voragen AGJ(eds)Xylans and xylanases.Elsevier,Amsterdam,pp 1-15
26.Whistler,R.L.,Richards,E.L.(1970)Hemicelluloses.In:Pigman W,Horton D (eds)The carbohydrates.Academic Press,New York,pp 447-469
27.Biely,P.,Puls,J.,Schneider,H.(1985)Acetyl xylan esterases in fungal cellulolytic systems.FEBS Lett 186:80-84
28.Chanda,S.K.,Hirst,E.L.,Jones,J.K.N.,Percival,E.G.V.(1950)The constitution ofxylan from esparto grass(Stipa tenacissima).J Chem Soc 50:1287-1289
29.Eda,S.,Ohnishi,A.,Kato,K.(1976)Xylan isolated from the stalks of Nicotiana tabacum.Agric Biol Chem 40:359-364
30.Montgomery,R.,Smith,F.,Srivastava,H.C.(1956)Structure of cornhull hemicellulose.Ⅰ.Partial hydrolysis and identification of 2-0-(α-D-glucopyranosyluronic acid)-D-xylopyranose.J Am Chem Soc 78:2837-2839
31.Dekker,R.F.H.,Richards,G.N.(1976)Hemicellulases,their occurrence,purification,properties and mode of action.Adv Carbohydr Chem Biochem 32:277-352
32.Barry,V.C.,Dillon,T.(1940)Occurrence of xylans in marine algae.Nature 146:620
33.Biely,P.,Mackenzie,C.R.,Puls,J.,Schneider,H(1986)Cooperativity of esterases and xylanases in the enzymatic degradation of acetyl xylan.Biotechnology 4:731-733
34.Sunna,A.and Antranikian,G.(1997)Xylanolytic enzymes from fungi and bacteria.Crit.Rev.Biotechnol.17,39-67.
35.Dekker,R.F.H and Richards,G.N.(1975)Purification,properties and mode of action of hemicellulase-produced by Ceratocystis paradoxa.Carbohydr.Res.39,97-114
36.Ito,K.,Ogassawara,J.,Sugimoto,T.and Ishikawa,T.(1992)Purification and properties of acid stable xylanases form Aspergillus kawachii.Biosci.Biotechnol.Biochem.56,547-550
37.Funaguma,T.,Naito,S.,Morita,M.,Okumara,M.,Sugiura,M.and Hara,A.(1991)Purification and some properties of xylanase from Penicillium herquei Banier and Sartory.Agric.Biol.Chem.55,1163-1165.
38.Spurway,T.D.,Morland,C.,Cooper,A.,Sumner,I.,Hazlewood,G.P.,O'Donnell,A.G.,Pickersgill,R.W.and Gilbert,H.J.(1997)Calcium protects a mesophilic xylanase from proteinase inactivation and thermal unfolding.J.Biol.
39.江正强.基因工程极耐高温木聚糖酶的提纯和酶学性质:博士学位论文.北京:中国农业大学,2001.
40.Wakarchuk,W.W.,Campbell,R.L.,Sung,W.L.,Davoodi,J.and Yaguchi,M.(1994)Mutational and crystallographic analysis of the active residues of the Bacillus circulans xylanase.Protein Sci.3,467-475
41.Torronen,A.,Harkki,A.and Rouvinen,J.(1994)Threedimensional structure of endo-1,4-L-xylanase Ⅱ from Trichoderma reesei:two conformational states in the active site. EMBO J. 13, 2493-2501.
42. Katusbe, Y., Hata, Y., Yamaguchi, H., Moriyama, H., Shinmyo, A. and Okada, H. (1990) Estimation of the xylanase active site from crystalline structure. Proc. Protein Eng. 3, 289-290.
43. Campbell, R.L., Rose, D.R, Wakarchuk, W.W., To, R., Sung, W. and Yaguchi, M. (1993) A comparison of the structure of the 20 kD xylanase from Trichoderma harzianum and Bacillus circulans. Proceedings of the second TRICEL symposium on Trichoderma reesei cellulases of other hydrolases. Espoo 8, 63-72.
44. Pickersgill, R.W., Debeire, P., Debeire-Gosselin, M. and Jenkins, J.A. (1993) Crystallization and preliminary X-ray analysis of a thermophilic Bacillus xylanase. J. Mol. Biol. 230, 664-666.
45. Anna, T., Hadar, F., Rotem, G., Aviva, L., Adra, M., Vivian, S., Capel, M., Shoham, Y. and Shoham, G. (1997) Crystallization and preliminary X-ray analysis of the thermostable alkaline xylanase from Bacillus stearothermophilus. Acta Crystallogr. D53, 608-611.
46. Durrand, R., Rascle, C. and Fevre, M. (1996) Molecular characterization of Xyn 3, a member of the endoxylanase multigene family of the rumen anaerobic fungus Neocallimastix frontalis. Curr. Genet. 30, 531-540
47. Morosoli, R., Roy, C. and Yaguchi, M. (1986) Isolation and partial primary' sequence of a xylanase from the yeast Cryptococcus albidus. Biochim. Biophys. Acta 870, 473-478.
48. Grapinet, O., Chebrou, M.C. and Beguin, P. (1988) Nucleotide sequence and deletion analysis of the xylanase gene (XylZ) of Clostridium thermocellum. J. Bacteriol. 170,4582-4588.
49. Black, G.W., Hazlewood, G.P., Millward-Sadler, J. and Lauirie, J.I. (1995) A modular xylanase containing a novel catalytic xylan-speci(?)c binding domain. Biochem. J. 307, 191-195
50. Winterhalter, C, Heinrich, P., Candussio, A., Wich, G. and Liebl, W. (1995) Identification of a novel cellulose-binding domain within the multidomain 120 kDa xylanase XynA of the hyperthermophilic bacterium Thermotoga maritima. Mol. Microbiol. 15, 431-444.
51. Hayashi, H., Takagi, K.I., Fukumura, M., Kimura, T., Karita, S., Sakka, K. and Ohmiya, K. (1997) Sequence of xynC and properties of XynC, a major component of the Clostridium thermocellum cellulosome. J. Bacteriol. 179, 4246-4253
52. Millward-Sadler, S.J., Poole, D.M., Henrissat, B., Hazlewood, G.P., Clarke, J.H. and Gilbert, H.J. (1994) Evidence for a general role for high affinity non-catalytic cellulose binding domains in microbial plant cell wall hydrolases. Mol. Microbiol. 11,375-382.
53. Kulkarni, N, Shendye, A., Rao, M. (1999) Molecular and biotechnological aspects of xylanases. FEMS Microbiol Rev 23:411-456
54. Bernier, R. Jr., Driguez, H., Desrochers, M. Molecular cloning of a Bacillus subtilis xylanase gene in Escherichia coli. Gene 1983, 26(1):59-65.
55. Kellett, L.E., Poole, D.M., Ferreira, L.M.A., Durrant, A.J., Hazlewood, G.P. and Gilbert, H.J. (1990) Xylanase B and an arabinofuranosidase from Psuedomonas fluorescens subsp. cellulosa contain identical cellulose binding domains and are encoded by adjacent genes. Biochem. J. 272, 369-376.
56. Gosalves, M.J., Perez-Gonzalez, J.A., Gonzalez, R. and Navarro, A. (1991) Two L-glycanase genes are clustered in Bacillus polymyxa: Molecular cloning expression and sequence analysis of genes encoding a xylanase and an endo-L-(1,3)-(M)-glucanase. J. Bacteriol. 173, 7705-7710
57. Bajpai, P. (1999) Application of enzymes in the pulp and paper industry. Biotechnol Prog 15:147-157
58. Bajpai, P., Bhardwaj, N.K., Bajpai, P.K., Jauhari, M.B. (1994) The impact of xylanases on bleaching of eucalyptus kraft pulp. J Biotechnol 38:1-6
59. Srinivasan, M.C., Rele. M.V. (1999) Microbial xylanases for paper industry. Curr Sci 77:137-142
60. Viikari, L., Kantelinen, A., Sandquist, J., Linko, M. (1994) Xylanases in bleaching: from an idea to the industry. FEMS Microbiol Rev 13:338-350
61. Bedford, M.R., Classen, H.L. (1992) The influence of dietary xylanase on intestinal viscosity and molecular weight distribution of carbohydrates in rye-fed broiler chick. In: Visser J, Beldman G, vanSomeren MAK, Voragen AGJ (eds) Xylans and xylanases, Elsevier, Amsterdam, pp 361-370
62. Vanparidon, P.A., Booman, J.C.P., Selten, G.C.M., Geerse, C, Barug, D., deBot, P.H.M., Hemke G (1992) Application of fungal endoxylanase in poultry diets In: Visser J, Beldman G, vanSomeran MAK, Voragen, AGJ (eds) Xylans and xylanases. Elsevier, Amsterdam, pp 371-378
63. Rani, S., Nand, K. (1996) Development of cellulase-free xylanase producing anaerobic consortia for the use of lignocellulosic wastes. Enzyme Microb Technol 18:23-28
64. Dominguez, J.M. (1998) Xylitol production by free and immobilized Debaryomyces hansenii. Biotechnol Lett 20:53-56
65. Khanna, S., Gauri, P. (1993) Regulation, purification and properties of xylanase from Cellulomonas fimi.Enzyme Microb Technol 15:990-995
66. Olsson, L., Hahn-Hagerdal, B. (1996) Fermentation of lignocellulosic hydrolysates for ethanol production. Enzyme Microb Technol 18:312-331
67. Maat, J., Roza, M., Verbakel, J., Stam, H., daSilra, M.J.S., Egmond, M.R., Hagemans, M.L.D., vanGarcom, R.F.M., Hessing, J.G.M., vanDerhondel, C.A.M.J.J., vanRotterdam, C. (1992) Xylanases and their application in bakery. In: Visser J, Beldman G, vanSomeren MAK, Voragen AGJ (eds) Xylans and xylanases. Elsevier, Amsterdam, pp 349-360
68. Kulkarni, N. and Shendye, A. (1999) Molecular and biotechnological aspects of xylanases FEMS Microbiology Reviews 23:411-456
69. Derewenda, U., Swenson, L., Green, R., Wei, Y., Morosoli, R., Shareck, F., Kluepfel, D. and Derewenda, Z.S. (1994) Crystal structure, at 2.6-A resolution, of the Streptomyces lividans xylanase A, a member of the F family of beta-1,4-D-glycanases. J. Biol. Chem. 269, 20811-20814
70. Torronen, A., Harkki, A. and Rouvinen, J. (1994) Threedimensional structure of endo-1,4-beta-xylanase II from Trichoderma reesei: two conformational states in the active site.EMBO J.13,2493-2501.
71.饶子和 结构基因组学的影响、进展与期待(2007)2007科学发展报告 中国科学院
72.Mcpherson,A.Crystallization of biological macromolecules