来源于Bispora betulina和Nesterenkonia xinjiangensis的木聚糖酶基因克隆、表达及性质研究
|
摘要
木聚糖是一种结构复杂的多聚五碳糖分子,需要多种酶的协同作用才能完全降解。而木聚糖酶能够水解木聚糖主链的β-1,4-糖苷键,是木聚糖降解酶体系中最关键的水解酶。由于木聚糖酶在工业上的重要应用,因而对木聚糖酶的研究越来越受到关注。而选择从极端环境中筛选产酶菌株,并从中筛选到具备优良性质的酶,是目前重要的研究手段之一。
本研究以真菌Bispora betulina及嗜碱细菌Nesterenkonia xinjiangensis为实验材料,分别用加入桦木木聚糖、燕麦木聚糖或麸皮与玉米芯的培养基对实验菌株进行诱导产酶实验。经酶活力测定,这两株菌均能在诱导下产胞外木聚糖酶。
通过对木聚糖酶基因的序列分析,分别从真菌B. betulina和嗜碱细菌N.xinjiangensis中克隆得到了两个GH11木聚糖酶基因Xyn11BB与xyn11NX,通过BLAST比对分析：Xyn11BB和xyn11NX与已报道的木聚糖酶的序列的一致性均不超过70%,这表明两个基因均具有一定的新颖性。将Xyn11BB与xyn11NX分别在毕赤酵母和大肠杆菌中进行了表达,在摇床水平上重组后的木聚糖酶表达量分别为121.15和65.5 U/mL,从而验证了基因的功能。
对重组木聚糖酶rXyn11BB及rXyn11NX分别进行了纯化及酶学性质研究。其中重组酶rXyn11BB的最适pH是4.5,在pH 4.0-10.0的范围内有良好的pH稳定性,最适温度是50℃；rXyn11BB的比活为470U/mg, Km为113.06 mg/mL, Vmax为303.03μmol·min-1mg-1；它对胶原蛋白酶和胰蛋白酶及α-糜蛋白酶具有良好的抗性。而重组酶rXyn11NX的最适pH为7.0,在中性和碱性条件下有良好的pH稳定性；最适温度为55℃,此酶具有很好的热稳定性,在70℃下保温60 min后能保留近60%的剩余酶活,甚至在90℃下保温15 min后酶活还能保留40%以上。rXyn11NX的比活为2158 U/mg,Km为16.08mg/mL,Vmax为45.66μmol·min-1mg-1。同时重组酶rXyn11NX没有纤维素酶活性,这些特点使rXyn11NX在工业上尤其是造纸业有良好的应用前景。综合酶学性质表明二者可作工业应用及科学研究的良好材料。
Because of the complexity of xylan, the complete hydrolysis of xylan requires cooperation of a great variety of enzymes. Among them, xylanase plays an important role in the xylanolytic enzyme system by hydrolyzing theβ-1,4-glycoside linkages in xylan backbone. Due to the important industrial application great attention was focused on xylanase system. Nowadays, screening enzyme-producing strains from special environments and then obtaining enzymes with superior properties have been important research method.
In this study, a fungus, Bispora betulina and an alkaliphilic bacterium, Nesterenkonia xinjiangensis were used for the strains. To determine whether they could produce xylanase, mediums containing oat spelt xylan, birchwood xylan, wheat bran or corncob were used to induce them. Results showed that all the carbon sources could induce the two strains to produce xylanase.
Based on sequence analysis of xylanase, a GH11 xylanase gene, designated as Xyn11BB, was cloned from B. betulina. Meanwhile, the other GH11 xylanase gene, designated as xyn11NX, was cloned from N. xinjiangensis. By homology searches using BLAST, the sequences of nucleotides and coded amino acids of Xyn11BB or xyn11NX share the highest identities which are less than 70%among the known sequences of GH11 xylanases, respectively. The results demonstrated that both of them are novel.
Xyn11BB and xyn11NX were expressed in Pichia pastoris and Escherichia coli, respectively. The corresponding activities were 121.15 and 65.5 U/mL, which verified the function of Xyn11BB and xyn11NX.
The recombinant proteins were purified and further characterized. Xyn11BB from Pichia pastoris shows the optimal pH of 4.5, optimal temperature at 50℃, and is stable over pH 4.0-10.0; Km is 113.06 mg/mL; Vmaxis 303.03μmol-min-1mg-1; the specific activity is 470 U/mg when using oat spelt xylan as substrate; rXyn11BB has good resistance to collagenase, trypsin and a-chymotrypsin. Xyn11NX from Escherichia coli shows the optimal pH of 7.0, optimal temperature at 55℃; It is thermostable, retains nearly 60%of the initial activity after incubation at 70℃for 1 h and more than 40%of the activity at 90℃for 15 min; Km is 16.08 mg/mL; Vmaxis 45.66μmol·min-1mg-1; The specific activity is 2158 U/mg when using oat spelt xylan as substrate. In addition, the enzyme has no cellulase activity either. Above all, the characteristics make it promising for various applications, especially in pulp and paper industry. Both of two recombinant xylanases have good prospects in industrial application and scientific research due to the enzymical characterizations.
本研究以真菌Bispora betulina及嗜碱细菌Nesterenkonia xinjiangensis为实验材料,分别用加入桦木木聚糖、燕麦木聚糖或麸皮与玉米芯的培养基对实验菌株进行诱导产酶实验。经酶活力测定,这两株菌均能在诱导下产胞外木聚糖酶。
通过对木聚糖酶基因的序列分析,分别从真菌B. betulina和嗜碱细菌N.xinjiangensis中克隆得到了两个GH11木聚糖酶基因Xyn11BB与xyn11NX,通过BLAST比对分析：Xyn11BB和xyn11NX与已报道的木聚糖酶的序列的一致性均不超过70%,这表明两个基因均具有一定的新颖性。将Xyn11BB与xyn11NX分别在毕赤酵母和大肠杆菌中进行了表达,在摇床水平上重组后的木聚糖酶表达量分别为121.15和65.5 U/mL,从而验证了基因的功能。
对重组木聚糖酶rXyn11BB及rXyn11NX分别进行了纯化及酶学性质研究。其中重组酶rXyn11BB的最适pH是4.5,在pH 4.0-10.0的范围内有良好的pH稳定性,最适温度是50℃；rXyn11BB的比活为470U/mg, Km为113.06 mg/mL, Vmax为303.03μmol·min-1mg-1；它对胶原蛋白酶和胰蛋白酶及α-糜蛋白酶具有良好的抗性。而重组酶rXyn11NX的最适pH为7.0,在中性和碱性条件下有良好的pH稳定性；最适温度为55℃,此酶具有很好的热稳定性,在70℃下保温60 min后能保留近60%的剩余酶活,甚至在90℃下保温15 min后酶活还能保留40%以上。rXyn11NX的比活为2158 U/mg,Km为16.08mg/mL,Vmax为45.66μmol·min-1mg-1。同时重组酶rXyn11NX没有纤维素酶活性,这些特点使rXyn11NX在工业上尤其是造纸业有良好的应用前景。综合酶学性质表明二者可作工业应用及科学研究的良好材料。
Because of the complexity of xylan, the complete hydrolysis of xylan requires cooperation of a great variety of enzymes. Among them, xylanase plays an important role in the xylanolytic enzyme system by hydrolyzing theβ-1,4-glycoside linkages in xylan backbone. Due to the important industrial application great attention was focused on xylanase system. Nowadays, screening enzyme-producing strains from special environments and then obtaining enzymes with superior properties have been important research method.
In this study, a fungus, Bispora betulina and an alkaliphilic bacterium, Nesterenkonia xinjiangensis were used for the strains. To determine whether they could produce xylanase, mediums containing oat spelt xylan, birchwood xylan, wheat bran or corncob were used to induce them. Results showed that all the carbon sources could induce the two strains to produce xylanase.
Based on sequence analysis of xylanase, a GH11 xylanase gene, designated as Xyn11BB, was cloned from B. betulina. Meanwhile, the other GH11 xylanase gene, designated as xyn11NX, was cloned from N. xinjiangensis. By homology searches using BLAST, the sequences of nucleotides and coded amino acids of Xyn11BB or xyn11NX share the highest identities which are less than 70%among the known sequences of GH11 xylanases, respectively. The results demonstrated that both of them are novel.
Xyn11BB and xyn11NX were expressed in Pichia pastoris and Escherichia coli, respectively. The corresponding activities were 121.15 and 65.5 U/mL, which verified the function of Xyn11BB and xyn11NX.
The recombinant proteins were purified and further characterized. Xyn11BB from Pichia pastoris shows the optimal pH of 4.5, optimal temperature at 50℃, and is stable over pH 4.0-10.0; Km is 113.06 mg/mL; Vmaxis 303.03μmol-min-1mg-1; the specific activity is 470 U/mg when using oat spelt xylan as substrate; rXyn11BB has good resistance to collagenase, trypsin and a-chymotrypsin. Xyn11NX from Escherichia coli shows the optimal pH of 7.0, optimal temperature at 55℃; It is thermostable, retains nearly 60%of the initial activity after incubation at 70℃for 1 h and more than 40%of the activity at 90℃for 15 min; Km is 16.08 mg/mL; Vmaxis 45.66μmol·min-1mg-1; The specific activity is 2158 U/mg when using oat spelt xylan as substrate. In addition, the enzyme has no cellulase activity either. Above all, the characteristics make it promising for various applications, especially in pulp and paper industry. Both of two recombinant xylanases have good prospects in industrial application and scientific research due to the enzymical characterizations.
引文
[1]Prade, R. Xylanases:from biology to biotechnology. Biotechnology and Genetic Engineering Reviews,1995,13(12):101-131.
[2]Yamaura, I., Matumoto M. Purification and some properties of endo-1,3-β-D xylanase from Pseudomonas sp. PT-5. Agricultural and Biological Chemistry,1990, 54(4):921-926.
[3]谢响明,何晓青.微生物的木聚糖酶及其生物漂白研究进展.北京林业大学学报,2003,25(3)：11-116.
[4]Petit-Benvegnen, M., Saulnier L., Rouau X. Solubilition of pentosan from isolated water-unextractable and wheat flour doughs by cell wall degrading enzymes. Cereal Chemistry, 1998,75(4):551-556.
[5]Gruppen, H., Hamer R., Voragen A. Water-unextractable cell wall material from wheat flour Ⅰ. Extraction of polymers with alkali. Journal of Cereal Science,1992,16(1):41-51.
[6]Kulkarni, N., Shendye A., Rao M. Molecular and biotechnological aspects of xylanases. FEMS Microbiology Reviews,1999,23(4):411-456.
[7]Li, K.C., Azadi P., Collins R., et al. Relationships between activities of xylanases and xylan structures. Enzyme and Microbial Technology,2000,27(1-2):89-94.
[8]Sunna, A., Antranikian G. Xylanolytic enzymes from fungi and bacteria. Critical Reviews in Biotechnology,1997,17:39-67.
[9]刘瑞田,曲音波.木聚糖酶分子的结构区域.生物工程进展,1998,18(6)：26-28.
[10]Gilkes, N., Henrissat B., Kilburn DG., et al. Domains in microbial β-1,4-glycanases: Sequence conservation, function, and enzyme families. Microbiological Reviews,1991, 55(2):303-315.
[11]Van Tilbeurgh, Tomme P., Claeyssens M., et al. Limited proteolysis of the cellobiohydrolase I from Trichoderma reesei:Separation of functional domains. FEBS letters,1986,204(2):223-227.
[12]Boraston, A.B., McLean BW., Kormos JM., et al. Carbohydrate-binding modules: diversity of structure and function. In:Gilbert HJ, Davies GJ, Henrissat B, Svensson B (eds). Recent Advances in Carbohydrate Bioengineering. Cambridge:Royal Society of Chemistry,1999,202-211.
[13]Boraston, A.B., Warren RA., Kilburn DG. Glycosylation by Pichia pastoris decreases the affinity of a family 2a carbohydrate-binding module from Cellulomonas fimi:a functional and mutational analysis. Biochemical Journal,2001,358:423-430.
[14]Kleine, J. Liebl W. Comparative characterization of deletion derivatives of the modular xylanase XynA of Thermotoga maritima. Extremophiles,2006,10(5):373-381.
[15]Mamo, G., Hatti-Kaul R., Mattiasson B. Fusion of carbohydrate binding modules from Thermotoga neapolitana with a family 10 xylanase from Bacillus halodurans S7. Extremophiles,2007,11(1):169-177.
[16]Zhao, G., Ali E., Araki R., et al. Function of the family-9 and family-22 carbohydrate-binding modules in a modular β-1,3-1,4-glucanase/xylanase derived from Clostridium stercorarium Xyn10B. Bioscience, Biotechnology and Biochemistry,2005,69(8):1562-1567.
[17]Ali, E., Zhao G., Sakka M., et al. Functions of family-22 carbohydrate-binding module in Clostridium thermocellum XynlOC. Bioscience, Biotechnology and Biochemistry,2005, 69(1):160-165.
[18]Schmidt, A., Giibitz G., Kratky C. Xylan binding subsite mapping in the xylanase from Penicillium simplicissimum using xylooligosaccharides as cryo-protectant. Biochemistry, 1999,38(8):2403-2412.
[19]Fujimoto, Z., Atsushi K., Satoshi K., et al. Crystal structure of Streptomyces olivaceoviridis E-86 β-xylanase containing xylan-binding domain. Journal of Molecular Biology,2000,300(3):575-585.
[20]Black, G., Nailewood G., Xue G., et al. Xylanase B from Neocallimastix patriciarum contains a non-catalytic 455-residue linker sequence comprised of 57 repeats of an octapeptide. Biochemical Journal,1994,299(2):381-387.
[21]Fujino, T., Beguin P., Aubert JP. 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. Journal of Bacteriology,1993,175(7): 1891-1899.
[22]杨浩萌,姚斌,范云六.木聚糖酶分子结构与重要酶学性质关系的研究进展.生物工程学报,2005,21(1)：6-11.
[23]Fontes, C, Hall GAJ., Hirst BH., et al. The resistance of cellulases and xylanases to proteolytic inactivation. Applied Microbiology and Biotechnology,1995,43(1):52-57.
[24]Saul, D., Williams L., Reeves R., et al. Sequence and expression of a xylanase gene from the hyperthermophile Thermotoga sp. strain FjSS3-B.1 and characterization of the recombinant enzyme and its activity on kraft pulp. Applied and Environmental Microbiology,1995,61(11):4110-4113.
[25]Lee, Y., Lowe SE., Henrissat B., et al. Characterization of the active site and thermostability regions of endoxylanase from Thermoanaerobacterium saccharolyticum B6A-RI. Journal of Bacteriology,1993,175(18):5880-5888.
[26]Polizeli, M., Rizzatti A., Monti R., et al. Xylanases from fungi:properties and industrial applications. Applied Microbiology and Biotechnology,2005,67(5):577-591.
[27]Chavez, R., Bull P., Eyzaguirre J. The xylanolytic enzyme system from the genus Penicillium. Journal of Biotechnology,2006,123(4):413-433.
[28]罗会颖.极端嗜酸真菌Bispora sp. MEY-1胞外糖苷水解酶类的产酶分析及其相关基因的克隆与表达.中国农业科学院博士学位论文,2008,3-25.
[29]Salles, BC., Cunha RB., et al. Purification and characterization of a new xylanase from Acrophialophora nainiana. Journal of Biotechnology,2000,81(2-3):199-204.
[31]杨培龙.来源于橄榄绿链霉菌A1的高比活木聚糖酶在植物中的高效表达.中国农业科学院博士学位论文,2006,3-20.
[32]Cereghino, J., Cregg J. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiology Reviews,2000,24(1):45-66
[33]Wong, K., Tan L., Saddler J. Multiplicity of β-1,4-xylanase in microorganisms: functions and applications. Microbiology and Molecular Biology Reviews,1988,52(3): 305-317.
[34]Collins, T., Gerday C., Feller G. Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiology Reviews,2005,29(1):3-23.
[35]Henrissat, B., Claeyssens M., Tomme P., et al. Cellulase families revealed by hydrophobic cluster analysis. Gene,1989,81(1):83-95.
[36]Henrissat, B., Coutinho PM. Classification of glycoside hydrolases and glycosyltransferases from hyperthermophiles. Methods in Enzymology,2001,330:183-201.
[37]Lo Leggio, Kalogiannis S., Bhat MK., et al. High resolution structure and sequence of T. aurantiacus xylanase I:implications for the evolution of thermostability in family 10 xylanases and enzymes with (β) a-barrel architecture. Proteins,1999,36(3):295-306.
[38]Biely, P., Vrsanaka M., Tenkanen M., et al. Endo-β-1,4-xylanase families:differences in catalytic properties. Journal of Biotechnology,1997,57(1-3):151-166.
[39]Derewenda, U., Swenson L., Green R., et al. Crystal structure, at 2.6-A resolution, of the Streptomyces lividans xylanase A, a member of the F family of β-1,4-D-glycanases. Journal of Biological Chemistry,1994,269(33):20811-20814.
[40]Wouters, J., Georis J., Engher D., et al. Crystallographic analysis of family 11 endo-β-1, 4-xylanase Xyll from Streptomyces sp. S38. Acta Crystallographica Section D, Biological Crystallography,2001,57(12):1813-1819.
[41]Biely, P., Kluepfel D., Shareck F. Mode of action of three endo-β-1,4-xylanases of Streptomyces lividans. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology,1993,1162(3):246-254.
[42]Rye, C., Withers SG. Glycosidase mechanisms. Current Opinion in Chemical Biology, 2000,4(5):573-580.
[43]Zechel, D., Withers SG. Glycosidase mechanisms:anatomy of a finely tuned catalyst. Accounts of Chemical Research,2000,33(1):11-18.
[44]McCarter, J., Withers SG. Mechanisms of enzymatic glycoside hydrolysis. Current Opinion in Structural Biology,1994,4(6):885-892.
[45]Alzari, P., Souchon H., Dominguez R. The crystal structure of endoglucanase CelA, a family 8 glycosyl hydrolase from Clostridium thermocellum. Structure,1996,4(3):265-275.
[46]Guerin, D., Lascombe MB., Costabel M., et al. Atomic (0.94 A) resolution structure of an inverting glycosidase in complex with substrate. Journal of Molecular Biology,2002, 316(5):1061-1069.
[47]Ball, A.. Mc Carthy AJ. Production and properties of xylanases from actinomycetes. Journal of Applied Microbiology,1989,66(5):439-444.
[48]Okazaki, W., Akiba T., Horikoshi E. Production and properties of two types of xylanases from alkalophilic thermophilic Bacillus sp. Applied Microbiology and Biotechnology, 1984,19(5):335-340.
[49]孙振涛,赵祥颖,刘建军等.微生物木聚糖酶及其应用.生物技术,2007,17(2)：9397.
[50]Simo, R., Souza C GM., Peralta RM. The use of methylβ-D-xyloside as a substrate for xylanase production by Aspergillus tamarii. Canadian Journal of Microbiology,1997, 43(1):56-60.
[51]Ito, K., Ogasawara H., Sugimoto T., et al. Purification and properties of acid stable xylanases from Aspergillus kawachii. Bioscience, Biotechnology and Biochemistry,1992, 56(4):547-550.
[52]连惠芗,汪世华.木聚糖酶的研究与应用.武汉工业学院学报,2006,25(1)：42-46.
[53]徐君飞,张居作.木聚糖酶的应用研究进展.农产品加工,2007,8：58-59.
[54]毛连山,余世袁.木聚糖酶在纸浆漂白中应用的研究现状.中国造纸学报,2006,21(3)：93-98.
[55]Ogasawara, H., Takahashi K., Iitsuka K., et al. Contribution of Hemicellulase in Shochu kojito the Resolution of Barler in the Shochu Mash Brew. Soc. Japan,1991,86(4):304-307.
[56]Beg, Q., Kapoor M., Mahajan L., et al. Microbial xylanases and their industrial applications:a review. Applied Microbiology and Biotechnology,2001,56(3):326-338.
[57]Viikari, L., Ranva M., Kantelinen A. Bleaching with enzymes. Stockholm:Third International Conference in Biotechnology in Pulp and Paper Industry,1986,67-69.
[58]Asha Poorna, C, Prema P. Production of cellulase-free endoxylanase from novel alkalophilic thermotolerent Bacillus pumilus by solid-state fermentation and its application in wastepaper recycling. Bioresource Technology,2007,98(3):485-490.
[59]毛丹漪,戴蓓,余菁.酶在造纸工业中的应用.造纸化学品,2002,14(2)：38-41.
[60]伍安国,曾辉,苏庆平.生物技术在造纸工业中的应用研究进展.西南造纸,2005,34(2)：15-19.
[61]余有贵,蒋盛岩,梁莲花.非淀粉多糖酶改善麦汁品质的研究.酿酒科技,2005,4：65-67.
[62]Courtin, C, Delcour JA. Arabinoxylans and endoxylanases in wheat flour bread-making. Journal of Cereal Science,2002,35(3):225-243
[63]袁建国,程显好,侯永勤.食品级木聚糖酶的研究与应用.山东食品发酵,2004,135(4)：36-39.
[64]Haros, M., Rosell CM., Benedito C. Effect of different carbohydrases on fresh bread texture and bread staling. European Food Research and Technology,2002,215(5):425-430.
[65]Vazquez, M., Alonso JL., Dominguez H., et al. Xylooligosaccharides:manufacture and applications. Trends in Food Science& Technology,2000,11(11):387-393.
[66]李秀婷.微生物木聚糖酶及在食品工业中的应用.农业机械学报,2008,39(2)：175179.
[67]Burchhardt, G., Ingram L. Conversion of xylan to ethanol by ethanologenic strains of Escherichia coli and Klebsiella oxytoca. Applied and Environmental Microbiology,1992, 58(4):1128-1133.
[68]Taylor, I., Dai Z., Decker S., et al. Heterologous expression of glycosyl hydrolases in planta:a new departure for biofuels. Trends in Biotechnology,2008,26(8):413-424.
[69]Miller, G. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry,1959,31(3):426-428.
[70]李岩,蒋继志,梁宁.一种快速提取丝状真菌染色体DNA的方法.生物学杂志,2006,6(49)：52-53.
[71]Piraee, M., Vining L. Use of degenerate primers and touchdown PCR to amplify a halogenase gene fragment from Streptomyces venezuelae ISP5230. Journal of Industrial Microbiology and Biotechnology,2002,29(1):1-5.
[72]Liu, Y., Whittier R. Thermal asymmetric interlaced PCR:automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics,1995,25(3):674-681.
[73]Sumner-Smith, M., Bozzato RP., Skipper N., et al. Analysis of the inducible MEL1 gene of Saccharomyces carlsbergensis and its secreted product, alpha-galactosidase (melibiase). Gene,1985,36(3):333-340.
[74]Subramaniyan, S., Prema P. Biotechnology of microbial xylanases:enzymology, molecular biology, and application. Critical Reviews in Biotechnology,2002,22(1):33-64.
[75]侯温甫,杨文鸽.糖链及其蛋白质糖基化.生物技术通报,2005,3：14-17.
[76]Wu, S., Liu B., Zhang XB. Characterization of a recombinant thermostable xylanase from deep-sea thermophilic Geobacillus sp. MT-1 in East Pacific. Applied Microbiology and Biotechnology,2006,72(6):1210-1216.
[77]Li, N., Meng K., Wang YR., et al. Cloning, expression, and characterization of a new xylanase with broad temperature adaptability from Streptomyces sp. S9. Applied Microbiology and Biotechnology,2008,80(2):231-240.
[78]Cardoso, O., Filho EXF. Purification and characterization of a novel cellulase-free xylanase from Acrophialophora nainiana. FEMS Microbiology Letters,2006,223(2):309-314.
[79]Tsujibo, H., Miyamoto K., Kuda T., et al. Purification, properties, and partial amino acid sequences of thermostable xylanases from Streptomyces thermoviolaceus OPC-520. Applied and Environmental Microbiology,1992,58:371-375.
[80]Gessesse, A., Mamo G. Purification and characterization of an alkaline xylanase from alkaliphilic Micrococcus sp AR-135. Journal of Industrial Microbiology and Biotechnology,1998,20(3):210-214.
[81]Ruiz-Arribas, A., Fernandez-Abalos JM., Sanchez, P., et al. Overproduction, purification, and biochemical characterization of a xylanase (Xysl) from Streptomyces halstedii JM8. Applied and Environmental Microbiology,1995,61(6):2414-2419.
[82]Li, N., Yang P., Wang Y., et al. Cloning, expression, and characterization of protease-resistant xylanase from Streptomyces fradiae var. kll. Journal of Microbiology and Biotechnology,2008,18(8):410-416.
[83]Leskinen, S., Mantyla A., Fagerstrom R., et al. Thermostable xylanases, Xyn10A and Xyn11A, from the actinomycete Nonomuraea flexuosa:isolation of the genes and characterization of recombinant Xyn11A polypeptides produced in Trichoderma reesei. Applied Microbiology and Biotechnology,2005,67(4):495-505.
[84]Kluepfel, D., Vats-Mehta S., Aumont, F., et al. Purification and characterization of a new xylanase (xylanase B) produced by Streptomyces lividans 66. The Biochemical Journal, 1990,267(1):45-50.
[85]Gruninger, H., Fiechter A. A novel, highly thermostable D-xylanase. Enzyme and Microbial Technology,1986,8:309-314.
[86]Mamo, G., Hatti-Kaul R., Mattiasson B. A thermostable alkaline active endo-β-1-4-xylanase from Bacillus halodurans S7:Purification and characterization. Enzyme and Microbial Technology,2006,39(7):1492-1498.
[87]Lee, J., Park J. Y., Kwon M., et al. Purification and characterization of a thermostable xylanase from the brown-rot fungus Laetiporus sulphureus. Journal of Bioscience and Bioengineering,2009,107(1):33-37.
[88]Heck, J., Barros Soares LH., Hertz PF., et al. Purification and properties of a xylanase produced by Bacillus circulans BL53 on solid-state cultivation. Biochemical Engineering Journal,2006,32(3):179-184.
[89]Dutta, T., Sengupta R., Sahoo R., et al. A novel cellulase free alkaliphilic xylanase from alkali tolerant Penicillium citrinum:production, purification and characterization. Letters in Applied Microbiology,2006,44(2):206-211.
[90]Zhang, G., Huang J., Huang GR., et al. Molecular cloning and heterologous expression of a new xylanase gene from Plectosphaerella cucumerina. Applied Microbiology and Biotechnology,2007,74(2):339-346.
[91]Singh, S., Madlala AM., Prior B. A Thermomyces lanuginosus:properties of strains and their hemicellulases. FEMS Microbiology Reviews,2006,27(1):3-16.
[92]Subramaniyan, S., Prema P. Cellulase-free xylanases from Bacillus and other microorganisms. FEMS Microbiology Letters,2000,183(1):1-7.
[93]Ratanakhanokchai, K.., Kyu KL., Tanticharoen M. Purification and properties of a xylan-binding endoxylanase from alkaliphilic Bacillus sp. strain K-1. Applied and Environmental Microbiology,1999,65(2):694-697.
[94]Christakopoulos, Nerinckx W., Kekos D., et al. Purification and characterization of two low molecular mass alkaline xylanases from Fusarium oxysporum F3. Journal of Biotechnology,1996,51(2):181-189.
[95]Meissner, K., Wassenberg D., Liebl W. The thermostabilizing domain of the modular xylanase XynA of Thermotoga maritima represents a novel type of binding domain with affinity for soluble xylan and mixed-linkage-1,3/-1,4-glucan. Molecular Microbiology,2000,36:898-912.
[2]Yamaura, I., Matumoto M. Purification and some properties of endo-1,3-β-D xylanase from Pseudomonas sp. PT-5. Agricultural and Biological Chemistry,1990, 54(4):921-926.
[3]谢响明,何晓青.微生物的木聚糖酶及其生物漂白研究进展.北京林业大学学报,2003,25(3)：11-116.
[4]Petit-Benvegnen, M., Saulnier L., Rouau X. Solubilition of pentosan from isolated water-unextractable and wheat flour doughs by cell wall degrading enzymes. Cereal Chemistry, 1998,75(4):551-556.
[5]Gruppen, H., Hamer R., Voragen A. Water-unextractable cell wall material from wheat flour Ⅰ. Extraction of polymers with alkali. Journal of Cereal Science,1992,16(1):41-51.
[6]Kulkarni, N., Shendye A., Rao M. Molecular and biotechnological aspects of xylanases. FEMS Microbiology Reviews,1999,23(4):411-456.
[7]Li, K.C., Azadi P., Collins R., et al. Relationships between activities of xylanases and xylan structures. Enzyme and Microbial Technology,2000,27(1-2):89-94.
[8]Sunna, A., Antranikian G. Xylanolytic enzymes from fungi and bacteria. Critical Reviews in Biotechnology,1997,17:39-67.
[9]刘瑞田,曲音波.木聚糖酶分子的结构区域.生物工程进展,1998,18(6)：26-28.
[10]Gilkes, N., Henrissat B., Kilburn DG., et al. Domains in microbial β-1,4-glycanases: Sequence conservation, function, and enzyme families. Microbiological Reviews,1991, 55(2):303-315.
[11]Van Tilbeurgh, Tomme P., Claeyssens M., et al. Limited proteolysis of the cellobiohydrolase I from Trichoderma reesei:Separation of functional domains. FEBS letters,1986,204(2):223-227.
[12]Boraston, A.B., McLean BW., Kormos JM., et al. Carbohydrate-binding modules: diversity of structure and function. In:Gilbert HJ, Davies GJ, Henrissat B, Svensson B (eds). Recent Advances in Carbohydrate Bioengineering. Cambridge:Royal Society of Chemistry,1999,202-211.
[13]Boraston, A.B., Warren RA., Kilburn DG. Glycosylation by Pichia pastoris decreases the affinity of a family 2a carbohydrate-binding module from Cellulomonas fimi:a functional and mutational analysis. Biochemical Journal,2001,358:423-430.
[14]Kleine, J. Liebl W. Comparative characterization of deletion derivatives of the modular xylanase XynA of Thermotoga maritima. Extremophiles,2006,10(5):373-381.
[15]Mamo, G., Hatti-Kaul R., Mattiasson B. Fusion of carbohydrate binding modules from Thermotoga neapolitana with a family 10 xylanase from Bacillus halodurans S7. Extremophiles,2007,11(1):169-177.
[16]Zhao, G., Ali E., Araki R., et al. Function of the family-9 and family-22 carbohydrate-binding modules in a modular β-1,3-1,4-glucanase/xylanase derived from Clostridium stercorarium Xyn10B. Bioscience, Biotechnology and Biochemistry,2005,69(8):1562-1567.
[17]Ali, E., Zhao G., Sakka M., et al. Functions of family-22 carbohydrate-binding module in Clostridium thermocellum XynlOC. Bioscience, Biotechnology and Biochemistry,2005, 69(1):160-165.
[18]Schmidt, A., Giibitz G., Kratky C. Xylan binding subsite mapping in the xylanase from Penicillium simplicissimum using xylooligosaccharides as cryo-protectant. Biochemistry, 1999,38(8):2403-2412.
[19]Fujimoto, Z., Atsushi K., Satoshi K., et al. Crystal structure of Streptomyces olivaceoviridis E-86 β-xylanase containing xylan-binding domain. Journal of Molecular Biology,2000,300(3):575-585.
[20]Black, G., Nailewood G., Xue G., et al. Xylanase B from Neocallimastix patriciarum contains a non-catalytic 455-residue linker sequence comprised of 57 repeats of an octapeptide. Biochemical Journal,1994,299(2):381-387.
[21]Fujino, T., Beguin P., Aubert JP. 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. Journal of Bacteriology,1993,175(7): 1891-1899.
[22]杨浩萌,姚斌,范云六.木聚糖酶分子结构与重要酶学性质关系的研究进展.生物工程学报,2005,21(1)：6-11.
[23]Fontes, C, Hall GAJ., Hirst BH., et al. The resistance of cellulases and xylanases to proteolytic inactivation. Applied Microbiology and Biotechnology,1995,43(1):52-57.
[24]Saul, D., Williams L., Reeves R., et al. Sequence and expression of a xylanase gene from the hyperthermophile Thermotoga sp. strain FjSS3-B.1 and characterization of the recombinant enzyme and its activity on kraft pulp. Applied and Environmental Microbiology,1995,61(11):4110-4113.
[25]Lee, Y., Lowe SE., Henrissat B., et al. Characterization of the active site and thermostability regions of endoxylanase from Thermoanaerobacterium saccharolyticum B6A-RI. Journal of Bacteriology,1993,175(18):5880-5888.
[26]Polizeli, M., Rizzatti A., Monti R., et al. Xylanases from fungi:properties and industrial applications. Applied Microbiology and Biotechnology,2005,67(5):577-591.
[27]Chavez, R., Bull P., Eyzaguirre J. The xylanolytic enzyme system from the genus Penicillium. Journal of Biotechnology,2006,123(4):413-433.
[28]罗会颖.极端嗜酸真菌Bispora sp. MEY-1胞外糖苷水解酶类的产酶分析及其相关基因的克隆与表达.中国农业科学院博士学位论文,2008,3-25.
[29]Salles, BC., Cunha RB., et al. Purification and characterization of a new xylanase from Acrophialophora nainiana. Journal of Biotechnology,2000,81(2-3):199-204.
[31]杨培龙.来源于橄榄绿链霉菌A1的高比活木聚糖酶在植物中的高效表达.中国农业科学院博士学位论文,2006,3-20.
[32]Cereghino, J., Cregg J. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiology Reviews,2000,24(1):45-66
[33]Wong, K., Tan L., Saddler J. Multiplicity of β-1,4-xylanase in microorganisms: functions and applications. Microbiology and Molecular Biology Reviews,1988,52(3): 305-317.
[34]Collins, T., Gerday C., Feller G. Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiology Reviews,2005,29(1):3-23.
[35]Henrissat, B., Claeyssens M., Tomme P., et al. Cellulase families revealed by hydrophobic cluster analysis. Gene,1989,81(1):83-95.
[36]Henrissat, B., Coutinho PM. Classification of glycoside hydrolases and glycosyltransferases from hyperthermophiles. Methods in Enzymology,2001,330:183-201.
[37]Lo Leggio, Kalogiannis S., Bhat MK., et al. High resolution structure and sequence of T. aurantiacus xylanase I:implications for the evolution of thermostability in family 10 xylanases and enzymes with (β) a-barrel architecture. Proteins,1999,36(3):295-306.
[38]Biely, P., Vrsanaka M., Tenkanen M., et al. Endo-β-1,4-xylanase families:differences in catalytic properties. Journal of Biotechnology,1997,57(1-3):151-166.
[39]Derewenda, U., Swenson L., Green R., et al. Crystal structure, at 2.6-A resolution, of the Streptomyces lividans xylanase A, a member of the F family of β-1,4-D-glycanases. Journal of Biological Chemistry,1994,269(33):20811-20814.
[40]Wouters, J., Georis J., Engher D., et al. Crystallographic analysis of family 11 endo-β-1, 4-xylanase Xyll from Streptomyces sp. S38. Acta Crystallographica Section D, Biological Crystallography,2001,57(12):1813-1819.
[41]Biely, P., Kluepfel D., Shareck F. Mode of action of three endo-β-1,4-xylanases of Streptomyces lividans. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology,1993,1162(3):246-254.
[42]Rye, C., Withers SG. Glycosidase mechanisms. Current Opinion in Chemical Biology, 2000,4(5):573-580.
[43]Zechel, D., Withers SG. Glycosidase mechanisms:anatomy of a finely tuned catalyst. Accounts of Chemical Research,2000,33(1):11-18.
[44]McCarter, J., Withers SG. Mechanisms of enzymatic glycoside hydrolysis. Current Opinion in Structural Biology,1994,4(6):885-892.
[45]Alzari, P., Souchon H., Dominguez R. The crystal structure of endoglucanase CelA, a family 8 glycosyl hydrolase from Clostridium thermocellum. Structure,1996,4(3):265-275.
[46]Guerin, D., Lascombe MB., Costabel M., et al. Atomic (0.94 A) resolution structure of an inverting glycosidase in complex with substrate. Journal of Molecular Biology,2002, 316(5):1061-1069.
[47]Ball, A.. Mc Carthy AJ. Production and properties of xylanases from actinomycetes. Journal of Applied Microbiology,1989,66(5):439-444.
[48]Okazaki, W., Akiba T., Horikoshi E. Production and properties of two types of xylanases from alkalophilic thermophilic Bacillus sp. Applied Microbiology and Biotechnology, 1984,19(5):335-340.
[49]孙振涛,赵祥颖,刘建军等.微生物木聚糖酶及其应用.生物技术,2007,17(2)：9397.
[50]Simo, R., Souza C GM., Peralta RM. The use of methylβ-D-xyloside as a substrate for xylanase production by Aspergillus tamarii. Canadian Journal of Microbiology,1997, 43(1):56-60.
[51]Ito, K., Ogasawara H., Sugimoto T., et al. Purification and properties of acid stable xylanases from Aspergillus kawachii. Bioscience, Biotechnology and Biochemistry,1992, 56(4):547-550.
[52]连惠芗,汪世华.木聚糖酶的研究与应用.武汉工业学院学报,2006,25(1)：42-46.
[53]徐君飞,张居作.木聚糖酶的应用研究进展.农产品加工,2007,8：58-59.
[54]毛连山,余世袁.木聚糖酶在纸浆漂白中应用的研究现状.中国造纸学报,2006,21(3)：93-98.
[55]Ogasawara, H., Takahashi K., Iitsuka K., et al. Contribution of Hemicellulase in Shochu kojito the Resolution of Barler in the Shochu Mash Brew. Soc. Japan,1991,86(4):304-307.
[56]Beg, Q., Kapoor M., Mahajan L., et al. Microbial xylanases and their industrial applications:a review. Applied Microbiology and Biotechnology,2001,56(3):326-338.
[57]Viikari, L., Ranva M., Kantelinen A. Bleaching with enzymes. Stockholm:Third International Conference in Biotechnology in Pulp and Paper Industry,1986,67-69.
[58]Asha Poorna, C, Prema P. Production of cellulase-free endoxylanase from novel alkalophilic thermotolerent Bacillus pumilus by solid-state fermentation and its application in wastepaper recycling. Bioresource Technology,2007,98(3):485-490.
[59]毛丹漪,戴蓓,余菁.酶在造纸工业中的应用.造纸化学品,2002,14(2)：38-41.
[60]伍安国,曾辉,苏庆平.生物技术在造纸工业中的应用研究进展.西南造纸,2005,34(2)：15-19.
[61]余有贵,蒋盛岩,梁莲花.非淀粉多糖酶改善麦汁品质的研究.酿酒科技,2005,4：65-67.
[62]Courtin, C, Delcour JA. Arabinoxylans and endoxylanases in wheat flour bread-making. Journal of Cereal Science,2002,35(3):225-243
[63]袁建国,程显好,侯永勤.食品级木聚糖酶的研究与应用.山东食品发酵,2004,135(4)：36-39.
[64]Haros, M., Rosell CM., Benedito C. Effect of different carbohydrases on fresh bread texture and bread staling. European Food Research and Technology,2002,215(5):425-430.
[65]Vazquez, M., Alonso JL., Dominguez H., et al. Xylooligosaccharides:manufacture and applications. Trends in Food Science& Technology,2000,11(11):387-393.
[66]李秀婷.微生物木聚糖酶及在食品工业中的应用.农业机械学报,2008,39(2)：175179.
[67]Burchhardt, G., Ingram L. Conversion of xylan to ethanol by ethanologenic strains of Escherichia coli and Klebsiella oxytoca. Applied and Environmental Microbiology,1992, 58(4):1128-1133.
[68]Taylor, I., Dai Z., Decker S., et al. Heterologous expression of glycosyl hydrolases in planta:a new departure for biofuels. Trends in Biotechnology,2008,26(8):413-424.
[69]Miller, G. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry,1959,31(3):426-428.
[70]李岩,蒋继志,梁宁.一种快速提取丝状真菌染色体DNA的方法.生物学杂志,2006,6(49)：52-53.
[71]Piraee, M., Vining L. Use of degenerate primers and touchdown PCR to amplify a halogenase gene fragment from Streptomyces venezuelae ISP5230. Journal of Industrial Microbiology and Biotechnology,2002,29(1):1-5.
[72]Liu, Y., Whittier R. Thermal asymmetric interlaced PCR:automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics,1995,25(3):674-681.
[73]Sumner-Smith, M., Bozzato RP., Skipper N., et al. Analysis of the inducible MEL1 gene of Saccharomyces carlsbergensis and its secreted product, alpha-galactosidase (melibiase). Gene,1985,36(3):333-340.
[74]Subramaniyan, S., Prema P. Biotechnology of microbial xylanases:enzymology, molecular biology, and application. Critical Reviews in Biotechnology,2002,22(1):33-64.
[75]侯温甫,杨文鸽.糖链及其蛋白质糖基化.生物技术通报,2005,3：14-17.
[76]Wu, S., Liu B., Zhang XB. Characterization of a recombinant thermostable xylanase from deep-sea thermophilic Geobacillus sp. MT-1 in East Pacific. Applied Microbiology and Biotechnology,2006,72(6):1210-1216.
[77]Li, N., Meng K., Wang YR., et al. Cloning, expression, and characterization of a new xylanase with broad temperature adaptability from Streptomyces sp. S9. Applied Microbiology and Biotechnology,2008,80(2):231-240.
[78]Cardoso, O., Filho EXF. Purification and characterization of a novel cellulase-free xylanase from Acrophialophora nainiana. FEMS Microbiology Letters,2006,223(2):309-314.
[79]Tsujibo, H., Miyamoto K., Kuda T., et al. Purification, properties, and partial amino acid sequences of thermostable xylanases from Streptomyces thermoviolaceus OPC-520. Applied and Environmental Microbiology,1992,58:371-375.
[80]Gessesse, A., Mamo G. Purification and characterization of an alkaline xylanase from alkaliphilic Micrococcus sp AR-135. Journal of Industrial Microbiology and Biotechnology,1998,20(3):210-214.
[81]Ruiz-Arribas, A., Fernandez-Abalos JM., Sanchez, P., et al. Overproduction, purification, and biochemical characterization of a xylanase (Xysl) from Streptomyces halstedii JM8. Applied and Environmental Microbiology,1995,61(6):2414-2419.
[82]Li, N., Yang P., Wang Y., et al. Cloning, expression, and characterization of protease-resistant xylanase from Streptomyces fradiae var. kll. Journal of Microbiology and Biotechnology,2008,18(8):410-416.
[83]Leskinen, S., Mantyla A., Fagerstrom R., et al. Thermostable xylanases, Xyn10A and Xyn11A, from the actinomycete Nonomuraea flexuosa:isolation of the genes and characterization of recombinant Xyn11A polypeptides produced in Trichoderma reesei. Applied Microbiology and Biotechnology,2005,67(4):495-505.
[84]Kluepfel, D., Vats-Mehta S., Aumont, F., et al. Purification and characterization of a new xylanase (xylanase B) produced by Streptomyces lividans 66. The Biochemical Journal, 1990,267(1):45-50.
[85]Gruninger, H., Fiechter A. A novel, highly thermostable D-xylanase. Enzyme and Microbial Technology,1986,8:309-314.
[86]Mamo, G., Hatti-Kaul R., Mattiasson B. A thermostable alkaline active endo-β-1-4-xylanase from Bacillus halodurans S7:Purification and characterization. Enzyme and Microbial Technology,2006,39(7):1492-1498.
[87]Lee, J., Park J. Y., Kwon M., et al. Purification and characterization of a thermostable xylanase from the brown-rot fungus Laetiporus sulphureus. Journal of Bioscience and Bioengineering,2009,107(1):33-37.
[88]Heck, J., Barros Soares LH., Hertz PF., et al. Purification and properties of a xylanase produced by Bacillus circulans BL53 on solid-state cultivation. Biochemical Engineering Journal,2006,32(3):179-184.
[89]Dutta, T., Sengupta R., Sahoo R., et al. A novel cellulase free alkaliphilic xylanase from alkali tolerant Penicillium citrinum:production, purification and characterization. Letters in Applied Microbiology,2006,44(2):206-211.
[90]Zhang, G., Huang J., Huang GR., et al. Molecular cloning and heterologous expression of a new xylanase gene from Plectosphaerella cucumerina. Applied Microbiology and Biotechnology,2007,74(2):339-346.
[91]Singh, S., Madlala AM., Prior B. A Thermomyces lanuginosus:properties of strains and their hemicellulases. FEMS Microbiology Reviews,2006,27(1):3-16.
[92]Subramaniyan, S., Prema P. Cellulase-free xylanases from Bacillus and other microorganisms. FEMS Microbiology Letters,2000,183(1):1-7.
[93]Ratanakhanokchai, K.., Kyu KL., Tanticharoen M. Purification and properties of a xylan-binding endoxylanase from alkaliphilic Bacillus sp. strain K-1. Applied and Environmental Microbiology,1999,65(2):694-697.
[94]Christakopoulos, Nerinckx W., Kekos D., et al. Purification and characterization of two low molecular mass alkaline xylanases from Fusarium oxysporum F3. Journal of Biotechnology,1996,51(2):181-189.
[95]Meissner, K., Wassenberg D., Liebl W. The thermostabilizing domain of the modular xylanase XynA of Thermotoga maritima represents a novel type of binding domain with affinity for soluble xylan and mixed-linkage-1,3/-1,4-glucan. Molecular Microbiology,2000,36:898-912.