植物细胞壁降解真菌及酶的研究
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摘要
对实验室收集的50余株青藏高原的真菌资源进行筛选，获得4株高效崩解植物细胞壁真菌中，并对该4株真菌进行了分子及形态学鉴定，分别为镰刀菌Q7-31（Fusarium sp.Accession No:FJ646593）；木霉菌M50537（Trichoderma sp.AccessionNo: GQ249380）拟盘多毛孢菌50647（Pestacotiopsis sp. Accession No:GQ249381），单色革盖菌Cerrena unicolor Ref1(Accession No: EU834831)根据菌种的特性，分别进行这些菌株的木聚糖酶的分离纯化、酶学特性、基因克隆与表达、生物信息学分析、植物细胞降解等协同作用等方面的研究。
对Fusarium sp.Q7-31，用1％的植物秸秆粉末作底物与粗酶液反应可产生还原糖高达265ug min~(-1)mL~(-1)，这种酶在25℃、40℃放置三天依然能保持初始状态酶活性的75%。水稻粉末诱导物培养Fusarium sp. Q7-31产生的粗酶液还可以降解其他秸秆粉末如大麦、小麦、玉米、燕麦、黑麦。Fusarium sp. Q7-31在植物细胞壁降并转换成为生物乙醇方面具有较好的潜力。Fusarium sp.Q7-31粗酶液降解水稻T-DNA突变株进行筛选试验，结果：选出降解活性最高的4株水稻T-DNA突变株，并对其基因突变点进行定位，这四4株编号及突变位点分别为：1B-0G636：xyloglucanendotransglucosylase/hydrolase protein1precursor；1B-08331：cellulose synthase；2D-00494：CESA5-cellulose synthase；3A-50357：xyloglucanase inhibitor，水稻突变株对Fusarium sp. Q7-31产生的酶对上述4株突变株降解能力比野生菌株的提高了3-5倍。通过单因素实验和正交实验对菌株Fusarium sp. Q7-31的产酶培养基组分进行了优化，确定了生产木聚糖酶的最佳培养基成分为：玉米芯0.5%，NaNO30.6%，K2HPO40.1%，MgSO4·7H2O0.05%，KCl0.05%，FeSO4·7H2O0.001%，pH5.0。装液量50/150mL，接种量7%，振荡培养7天。经优化后Fusarium sp. Q7-31产生的木聚糖酶活力从初始的3.145U/mL上升到11.72U/mL，提高了3.73倍。
对单色革盖菌Cerrena unicolor Ref1的培养特性、产木聚糖酶条件和酶学特性进行了初步研究。结果表明：该菌为低温型菌株，其最佳生长条件为pH6、20℃和酵母膏作为氮源；最佳产酶条件为pH3-7、15℃及以酵母膏氮源；条件优化后产酶可达118.7u/mL，可溶蛋白含量可达到60g/mL，酶溶液的比活可达到1250U/mg蛋白质；该木聚糖酶的最适反应温度和pH分别为50℃和5，金属离子Mg~(2+)、Na~+和8mmol/L的Fe~(2+)、Cu~(2+)、Zn~(2+)等对木聚糖酶的活性有抑制作用，而Ca~(2+)、4mmol/L的Fe~(2+)、Cu~(2+)、Zn~(2+)和8mmol/L的Mn~(2+)等对该酶反应则有促进作用；该木聚糖酶在保温2h后在15-40℃范围内能保持80%以上的酶活性，在50℃时能保持68%的酶活性；用lineweaver-Burk作图法（双倒数作图法）求得该酶的最大反应速度Vmax和Km值分别为163.38mmol/mg/min和0.75mg/mL。
研究协同作用的四株真菌(镰刀菌属Fusarium sp.、木霉属Trichoderma sp.、曲霉属Aspergillus sp.、拟盘多毛孢属Pestalotiopsis sp.)液体发酵所产粗酶液单独及混合降解水稻秸秆活性比较研究，结果表明：无论是CMC酶活、木聚糖酶活，还是对水稻秸秆的降解活性，各菌株粗酶液混合后普遍表现出不同程度的协同作用。如木霉菌与曲霉菌粗酶液混合液对水稻秸秆的降解效果较两株菌粗酶液单独降解效果之和提高了85.60%。值得注意的是，各菌株中，拟盘多毛孢菌的粗酶液的CMC酶活、木聚糖酶活都最高(分别达到了0.3149U/mL和38.4817U/mL),对水稻秸秆的降解能力也不错，为28.9490μg/mL，该方面的研究为进一步揭示植物细胞壁降解机制的研究有较重要的意义。
此外，以Fusarium sp Q7-31为实验材料克隆的木聚糖酶基因xyn8，经生物信息学分析：该基因可编码一个分子量为25.7KDa的木聚糖酶，理论等电点为6.9，并含有一个信号肽序列，序列为MVSFKFLLVAASAITGALA。该木聚糖酶属于G11家族。将木聚糖酶基因xyn8连入质粒pGEX-5x-1，构建表达载体pGEX-5x-1-xyn8，转化大肠杆菌BL21(DE3)。异源表达蛋白通过SDS-PAGE和Western blot进行鉴定，亲和层析纯化，并且对重组木聚糖酶的酶学特性进行了研究。木聚糖酶基因xyn8在大肠杆菌BL21(DE3)中表达，以GST融合蛋白形式存在于胞内，其分子量约52kDa，大小与预测相符。底物特异性结果表明：重组木聚糖酶能特异性降解木聚糖，但是对淀粉和CMC没有降解活性。其最适pH为6.0，最适反应温度为40℃。
植物细胞壁这一巨大可再生资源利用的成功与否，很大程度上取决于植物中纤维素骨架的彻底降解，木聚糖酶将会起到非常关键的作用。因此，植物细胞壁降解酶生物学特性的研究，为生物能源的开发，具有重要的意义。很多的植物病原菌中主要的致病因子为木聚糖降解酶，因此，如何利用植物病原菌中的高产木聚糖酶基因，在一定的宿主中表达或共表达，构建出植物细胞壁降解高效降解菌种，也将会将为是植物细胞壁利用的新途径。为植物细胞壁中的纤维素、半纤维素等地安全、有效的降解转化利用，为人类的可持续发展，为环境问题的解决及变废为宝的工程提供了重要的技术手段。
We screened50fungi strains of which isolated from the Qinghai–Tibet Plateau andstored in Qin-Hai University Lab. Among them,4strains had higher capacity of degradingplant cell wall were identified. Based on the different characteristics of these strains,xylanase characteristics,xylanase gene cloning,bioinformatics analysis, synergisticfunction were studied in this paper.
We screened fungal isolates of Q7-31for higher,more stable,and more comprehensiveenzyme activity toward powder from rice cell walls. Degrading enzyme was induced byculturing each strain for3d in a liquid medium supplemented with0.3%powder as thesole carbon source. The culture supernatant as crude enzyme was immediately used forassaying cell wall-degrading activity via the dinitrosalicylic acid method. Among ourscreened fungal strains,the crude enzyme of Fusarium sp. Q7-31hydrolyzed the ricepowder to reducing sugars at a rate of265g min~(-1)mL~(-1). To compare their stability, crudeenzymes of Fusarium sp. Q7-21and Q7-31strains were stored at25or40oC. Regardlessof temperature, the latter strain remained stable, with at least75%of its initial activity,for3d.Furthermore, this Q7-31enzyme showed even higher degradabilities toward othercereal plant powders, including local varieties of barley, wheat, corn, oats, and rye, thantoward rice. These results suggest that Fusarium sp. Q7-31is capable of producinghighly stable enzymes to degrade the polysaccharides within cereal cell walls. Thus,it hasgreat potential for cost-efficient bioethanol production from such feedstocks.
The rice mutants of T-DNA were screened by using the crude Enzyme of Fusariumsp. Q7-31to degrading the cell wall of rice mutants, it showed that:four rice mutantstrains were selected based on the highest reducing sugar production. The geneticmutation of the strains were identified.1B-0G636:xyloglucan endotransglucosylase/hydrolase protein1precursor;1B-08331:cellulose synthase;2D-00494:CESA5-cellulosesynthase;3A-50357:xyloglucanase inhibitor. After degrading, the capacity of the mutantincreased3-5time compared with the wild type of the rice. Through single factor analysisand orthogonal experiments, effects of carbon source, nitrogen source and other factorson xylanase production from Fusarium sp. Q7-31were studied. The optimal mediumcomposition for the xylanase production was: corn steep0.5%, NaNO30.6%, K2HPO4 0.1%, MgSO4·7H2O0.05%, KCl0.05%, FeSO4·7H2O0.001%, and the pH was5.0. Afterscreening, the xylanase activity increased by3.73times from3.145U/mL to11.72U/mL.
The characteristics of culture, xylanase producing condition and characteristics ofenzyme of Ref1were studied. The result showed that Ref1was a psychrophile, and thebest growing conditions were pH6,20℃and yeast extract as nitrogen sourcewhile thebest xylanase producing conditions were pH3-7,15℃and yeast extract as nitrogensource. Xylanase activity of optimized Ref1reached118.7u/mL and soluble protein60ug/mL; the specific activity of the crude enzyme was1250U/mg protein. Thexylanase has an optimal activity at50℃and pH5.0and the relative enzyme activity ofxylanase remains80%while keeping at the temperatures of15℃-40℃. Mg~(2+), Na+and8mmol/L Fe~(2+), Cu~(2+), Zn~(2+)were strong inhibitors, while Ca~(2+),4mmol/L Fe~(2+), Cu~(2+), Zn~(2+)and8mmol/L Mn~(2+)were stimulators of the enzyme. The maximum velocity of reactionand the Kmof the xylanase were163.38mmol/mg/min and0.75mg/mL, respectively.
Non-mixed and mixed enzymes produced by liquid state fermentation of fourdifferent fungi which are come from Fusarium sp., Trichoderma sp., Aspergillus sp. andPestacotiopsis sp. were used to degrading rice straw. The degrading effect which isexpressed by amounts of reduced sugar (μg.min~(-1).mL~(-1)) was tested by DNS method.Meanwhile CMCase and Xylanase of the non-mixed and mixed enzymes weredetermined respectively. The results showed that more or less synergistic effects areubiquity in mixed enzymes on digesting rice straw, CMC and Xylan. For example, thedegrading effects of mixed enzymes of Trichoderma sp..and Aspergillus sp.improved85.60%on digesting Rice straw. To be mentioned, in these strains, CMCase and Xylanaseof enzyme of Pestacotiopsis sp. are the highest, which is up to0.3149U/mL and38.4817U/mL respectively. It’s digesting effect on Rice straw is also not bad which is28.9490μg/mL.
The xlanase gene of xyn8which was cloned by our laboratory was cloned fromcDNA of Fusarium sp. Q7-31. Xyn8encodes a xylanase with a calculated molecularweight of25.7KD. The theoretical pI is6.9. The protein has a signal peptide with thesequence MVSFKFLLVAASAITGALA. The xylanase Xyl8was belonging to G11xylanase family. Construct the recombinant expression vector of pGEX_(5x-1-xyn)8. Thexylanase gene xyn8was expressed in Ecoli.BL21(DE3). The production of recombine xylanase was identified by SDS-PAGE and Western blot. Recombine xylanase waspurified by affinity chromatography. The characteristics of expressed Xyl8were studied.
The study of xylanase of deferent fungi has very important role in utilization of plantcell wall.
对Fusarium sp.Q7-31，用1％的植物秸秆粉末作底物与粗酶液反应可产生还原糖高达265ug min~(-1)mL~(-1)，这种酶在25℃、40℃放置三天依然能保持初始状态酶活性的75%。水稻粉末诱导物培养Fusarium sp. Q7-31产生的粗酶液还可以降解其他秸秆粉末如大麦、小麦、玉米、燕麦、黑麦。Fusarium sp. Q7-31在植物细胞壁降并转换成为生物乙醇方面具有较好的潜力。Fusarium sp.Q7-31粗酶液降解水稻T-DNA突变株进行筛选试验，结果：选出降解活性最高的4株水稻T-DNA突变株，并对其基因突变点进行定位，这四4株编号及突变位点分别为：1B-0G636：xyloglucanendotransglucosylase/hydrolase protein1precursor；1B-08331：cellulose synthase；2D-00494：CESA5-cellulose synthase；3A-50357：xyloglucanase inhibitor，水稻突变株对Fusarium sp. Q7-31产生的酶对上述4株突变株降解能力比野生菌株的提高了3-5倍。通过单因素实验和正交实验对菌株Fusarium sp. Q7-31的产酶培养基组分进行了优化，确定了生产木聚糖酶的最佳培养基成分为：玉米芯0.5%，NaNO30.6%，K2HPO40.1%，MgSO4·7H2O0.05%，KCl0.05%，FeSO4·7H2O0.001%，pH5.0。装液量50/150mL，接种量7%，振荡培养7天。经优化后Fusarium sp. Q7-31产生的木聚糖酶活力从初始的3.145U/mL上升到11.72U/mL，提高了3.73倍。
对单色革盖菌Cerrena unicolor Ref1的培养特性、产木聚糖酶条件和酶学特性进行了初步研究。结果表明：该菌为低温型菌株，其最佳生长条件为pH6、20℃和酵母膏作为氮源；最佳产酶条件为pH3-7、15℃及以酵母膏氮源；条件优化后产酶可达118.7u/mL，可溶蛋白含量可达到60g/mL，酶溶液的比活可达到1250U/mg蛋白质；该木聚糖酶的最适反应温度和pH分别为50℃和5，金属离子Mg~(2+)、Na~+和8mmol/L的Fe~(2+)、Cu~(2+)、Zn~(2+)等对木聚糖酶的活性有抑制作用，而Ca~(2+)、4mmol/L的Fe~(2+)、Cu~(2+)、Zn~(2+)和8mmol/L的Mn~(2+)等对该酶反应则有促进作用；该木聚糖酶在保温2h后在15-40℃范围内能保持80%以上的酶活性，在50℃时能保持68%的酶活性；用lineweaver-Burk作图法（双倒数作图法）求得该酶的最大反应速度Vmax和Km值分别为163.38mmol/mg/min和0.75mg/mL。
研究协同作用的四株真菌(镰刀菌属Fusarium sp.、木霉属Trichoderma sp.、曲霉属Aspergillus sp.、拟盘多毛孢属Pestalotiopsis sp.)液体发酵所产粗酶液单独及混合降解水稻秸秆活性比较研究，结果表明：无论是CMC酶活、木聚糖酶活，还是对水稻秸秆的降解活性，各菌株粗酶液混合后普遍表现出不同程度的协同作用。如木霉菌与曲霉菌粗酶液混合液对水稻秸秆的降解效果较两株菌粗酶液单独降解效果之和提高了85.60%。值得注意的是，各菌株中，拟盘多毛孢菌的粗酶液的CMC酶活、木聚糖酶活都最高(分别达到了0.3149U/mL和38.4817U/mL),对水稻秸秆的降解能力也不错，为28.9490μg/mL，该方面的研究为进一步揭示植物细胞壁降解机制的研究有较重要的意义。
此外，以Fusarium sp Q7-31为实验材料克隆的木聚糖酶基因xyn8，经生物信息学分析：该基因可编码一个分子量为25.7KDa的木聚糖酶，理论等电点为6.9，并含有一个信号肽序列，序列为MVSFKFLLVAASAITGALA。该木聚糖酶属于G11家族。将木聚糖酶基因xyn8连入质粒pGEX-5x-1，构建表达载体pGEX-5x-1-xyn8，转化大肠杆菌BL21(DE3)。异源表达蛋白通过SDS-PAGE和Western blot进行鉴定，亲和层析纯化，并且对重组木聚糖酶的酶学特性进行了研究。木聚糖酶基因xyn8在大肠杆菌BL21(DE3)中表达，以GST融合蛋白形式存在于胞内，其分子量约52kDa，大小与预测相符。底物特异性结果表明：重组木聚糖酶能特异性降解木聚糖，但是对淀粉和CMC没有降解活性。其最适pH为6.0，最适反应温度为40℃。
植物细胞壁这一巨大可再生资源利用的成功与否，很大程度上取决于植物中纤维素骨架的彻底降解，木聚糖酶将会起到非常关键的作用。因此，植物细胞壁降解酶生物学特性的研究，为生物能源的开发，具有重要的意义。很多的植物病原菌中主要的致病因子为木聚糖降解酶，因此，如何利用植物病原菌中的高产木聚糖酶基因，在一定的宿主中表达或共表达，构建出植物细胞壁降解高效降解菌种，也将会将为是植物细胞壁利用的新途径。为植物细胞壁中的纤维素、半纤维素等地安全、有效的降解转化利用，为人类的可持续发展，为环境问题的解决及变废为宝的工程提供了重要的技术手段。
We screened50fungi strains of which isolated from the Qinghai–Tibet Plateau andstored in Qin-Hai University Lab. Among them,4strains had higher capacity of degradingplant cell wall were identified. Based on the different characteristics of these strains,xylanase characteristics,xylanase gene cloning,bioinformatics analysis, synergisticfunction were studied in this paper.
We screened fungal isolates of Q7-31for higher,more stable,and more comprehensiveenzyme activity toward powder from rice cell walls. Degrading enzyme was induced byculturing each strain for3d in a liquid medium supplemented with0.3%powder as thesole carbon source. The culture supernatant as crude enzyme was immediately used forassaying cell wall-degrading activity via the dinitrosalicylic acid method. Among ourscreened fungal strains,the crude enzyme of Fusarium sp. Q7-31hydrolyzed the ricepowder to reducing sugars at a rate of265g min~(-1)mL~(-1). To compare their stability, crudeenzymes of Fusarium sp. Q7-21and Q7-31strains were stored at25or40oC. Regardlessof temperature, the latter strain remained stable, with at least75%of its initial activity,for3d.Furthermore, this Q7-31enzyme showed even higher degradabilities toward othercereal plant powders, including local varieties of barley, wheat, corn, oats, and rye, thantoward rice. These results suggest that Fusarium sp. Q7-31is capable of producinghighly stable enzymes to degrade the polysaccharides within cereal cell walls. Thus,it hasgreat potential for cost-efficient bioethanol production from such feedstocks.
The rice mutants of T-DNA were screened by using the crude Enzyme of Fusariumsp. Q7-31to degrading the cell wall of rice mutants, it showed that:four rice mutantstrains were selected based on the highest reducing sugar production. The geneticmutation of the strains were identified.1B-0G636:xyloglucan endotransglucosylase/hydrolase protein1precursor;1B-08331:cellulose synthase;2D-00494:CESA5-cellulosesynthase;3A-50357:xyloglucanase inhibitor. After degrading, the capacity of the mutantincreased3-5time compared with the wild type of the rice. Through single factor analysisand orthogonal experiments, effects of carbon source, nitrogen source and other factorson xylanase production from Fusarium sp. Q7-31were studied. The optimal mediumcomposition for the xylanase production was: corn steep0.5%, NaNO30.6%, K2HPO4 0.1%, MgSO4·7H2O0.05%, KCl0.05%, FeSO4·7H2O0.001%, and the pH was5.0. Afterscreening, the xylanase activity increased by3.73times from3.145U/mL to11.72U/mL.
The characteristics of culture, xylanase producing condition and characteristics ofenzyme of Ref1were studied. The result showed that Ref1was a psychrophile, and thebest growing conditions were pH6,20℃and yeast extract as nitrogen sourcewhile thebest xylanase producing conditions were pH3-7,15℃and yeast extract as nitrogensource. Xylanase activity of optimized Ref1reached118.7u/mL and soluble protein60ug/mL; the specific activity of the crude enzyme was1250U/mg protein. Thexylanase has an optimal activity at50℃and pH5.0and the relative enzyme activity ofxylanase remains80%while keeping at the temperatures of15℃-40℃. Mg~(2+), Na+and8mmol/L Fe~(2+), Cu~(2+), Zn~(2+)were strong inhibitors, while Ca~(2+),4mmol/L Fe~(2+), Cu~(2+), Zn~(2+)and8mmol/L Mn~(2+)were stimulators of the enzyme. The maximum velocity of reactionand the Kmof the xylanase were163.38mmol/mg/min and0.75mg/mL, respectively.
Non-mixed and mixed enzymes produced by liquid state fermentation of fourdifferent fungi which are come from Fusarium sp., Trichoderma sp., Aspergillus sp. andPestacotiopsis sp. were used to degrading rice straw. The degrading effect which isexpressed by amounts of reduced sugar (μg.min~(-1).mL~(-1)) was tested by DNS method.Meanwhile CMCase and Xylanase of the non-mixed and mixed enzymes weredetermined respectively. The results showed that more or less synergistic effects areubiquity in mixed enzymes on digesting rice straw, CMC and Xylan. For example, thedegrading effects of mixed enzymes of Trichoderma sp..and Aspergillus sp.improved85.60%on digesting Rice straw. To be mentioned, in these strains, CMCase and Xylanaseof enzyme of Pestacotiopsis sp. are the highest, which is up to0.3149U/mL and38.4817U/mL respectively. It’s digesting effect on Rice straw is also not bad which is28.9490μg/mL.
The xlanase gene of xyn8which was cloned by our laboratory was cloned fromcDNA of Fusarium sp. Q7-31. Xyn8encodes a xylanase with a calculated molecularweight of25.7KD. The theoretical pI is6.9. The protein has a signal peptide with thesequence MVSFKFLLVAASAITGALA. The xylanase Xyl8was belonging to G11xylanase family. Construct the recombinant expression vector of pGEX_(5x-1-xyn)8. Thexylanase gene xyn8was expressed in Ecoli.BL21(DE3). The production of recombine xylanase was identified by SDS-PAGE and Western blot. Recombine xylanase waspurified by affinity chromatography. The characteristics of expressed Xyl8were studied.
The study of xylanase of deferent fungi has very important role in utilization of plantcell wall.
引文
[1] Ahmed, S., S. Riaz and A. Jamil."Molecular cloning of fungal xylanases: an overview."(2009)Appl Microbiol Biotechnol Vol:84(1) pp:19-35.
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[3] Blanco A, Vidal T, Colon J F, et al. Purification and properties of xylanase a from alkali-tolerantBacillus sp. Strain BP-23[J]. Appl Environ Microbiol,1995,61:4468-4470.
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[5] Collins T, Gerday C, Feller G. Xylanases, xylanase families and extremophilic xylanases[J].FEMS Microbiology Reviews,2005,29:3–23.
[6] Chadha,B.S.,Ajay B.K.,Mellon F.,et al.Two endoxylanases active and stable at alkaline pH fromthe newly isolated thermophilic fungus.Myceliophthorasp. IMI387099.Jorumal ofBiotechnologoy.2004,109:227～237.
[7] Chen,C.,Chen,J-L.,Lin,T-Y. Purification and characterization of a xylanase from Trichodermalongibrachiatum for xylooligosaccharide production[J].Enzyme and MicrobialTechnology.1997,21:91-96.
[8] Deepak S.,Shulin C.,2008.The white-rot fungus Phanerochaete chrysosporium:conditions for theproduction of lignin-degrading enzymes,Appl Microbiol Biotechnol DOI10.1007/s00253-008-1706-9.
[9] Debeire P.,Priem B.,Strecker G.,et al.Purification and properties of an endo-1,4-xylanase excretedby a hydrolytic thermophilic anaerobe,Clostridium thermolacticum[J]. Eur JBiochem,1990,V187(3):573-580.
[10] Dow JM, Clarke BR, Milligan DE, Tang J-L, Daniels MJ (1990) Extracellular proteasesfrom Xanthomonas campestris pv. campestris, the black rot pathogen. Appl EnvironMicrobiol562994-2998.
[11] Ferreira L.M.A.,Hazlewood G.P.,Barker P.J.,et al.The cellodextrinase from Pseudomonasfluorescens Subsp.Cellulosa consists of multiple functional domains[J].BiochemJ,1991,V276(1):261-264.
[12] Fischer K.,Akhtar M.,Messner K.,et al.Pitch reduction with the white-rot fungus Ceriporiopsissubvermispora[A].in E.Srebotnik and K.Messner(eds.) Biotechnology in the Pulp and PaperIndustry[C].Facultas-Unlversitatsverlag, Vienna.1995:193-198.
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[14] Fujimoto H, Ooi T, Wang S-L, et al. Purification and properties of three xylanases fromAspergillus aculeatus[J]. Biosci Biotechnol Biochem,1995,59:538-540.
[15] Gilbert H.J.,Hazlewood G..P.Bacterial cellulases and xylanases[J].J GenMicrobiol,1993,V139(2):187-194.
[16] Gilkes NR，Henrissat B, Kilbmu DG, Miller RC Jr, Warren R.A. Domains in microbialbeta-l,4-glycanses: sequence conservation function and enzyme families[J]. Microbiol Rev,2002,55:303～315
[17] Lee Y.,Lowe S.E.,Henrissat B.,etal. Characterization of the active site and thermostability regionsof endoxylanase from Thermoanaerobacterium saccharolyticum B6A-RI.[J].JBacteriol,1993,V175(18):5890-5898.
[18] Henrissat B, Bairoch A. New families in the classification of glycosyl hydrolases based on aminoacid sequence similarities. Biochem J,1993,293:781-788.
[19] Hardy L.W.,Poteete A.R.Reexamination of the role of Asp20in catalysis by bacteriophage T4lysozyme[J].Biochemistry,1991,V30(39):9457-9463
[20] Irwin D,Jung E,Wilson D.Characterization and sequence of a Thermomonospora fuscaxylanase.Apple Environ Microbiol,1994,60:762～770.
[21] Ito Y, Tomita T, Roy N, et al. Cloning, expression and cellsurface localization ofPaenibacilluss.strain W-61xylanase5,a multidomain xylanase[J]. Appl Environ Microbiol,2003,69(12):6969～6978.
[22] Johnson K.G.,Ross N.W.,Enzyme and Microbiol Technol,1990,12:960-964.
[23] Kolattukudy PE (1985) Enzymatic penetration of the plíint cuticle by fungal pathogens. AnnuRev Phytopathol23:233-238
[24] Krengel et al.(1996). Acta Crystallogr. sect. D,52,571-576.
[25] Knox (1997) Int.Rev.Cytol.171:79-120.
[26] P. Christakopoulos，W.Nerinckx, D.Kekos, Purification and characteristics of two low molecularmass alkaline xylanaases from fusarium oxysporum F3[J].Journal of biotechnology（1996）51:181-189.
[27] Polizeli M. L. T. M., Rizzatti A. C. S.,. Monti R., et al. Xylanases from fungi: properties andindustrial applications[J]. Appl Microbiol Biotechnol,2005,67:577-591.
[28] Qureshy A F, Khan L A, Khanna S. Expression of BacilluscirculansTeri-42xylanase gene inBacillus subtilis [J]. Enzyme Microb Technol,2000,27(3-5):227～233.
[29] Ried JL, Collmer A (1988) Construction and characterization of an Eminia chrysanthemi mutantwith directed deletion in a11of the pectate lyase structural genes. Mo1Plant Microbe Interact1:261:1032-103532-38.
[30] Robinson DG (1991) What is a plant cell? The last word. Plant Cell3:1145-1146.
[31] Stahl DJ, Schafer W (1992) Cutinase is not required for fungal pathogenicity onpea. Plant Cell4621-629.
[32] Sapag A,Johan W, Christophe L, etal. The endoxylanases from family11:computer analysis ofprotein sequence reveals important structure and phylogenetic relationships.JBiotechnol,2002,95:109～131.
[33] Sundberg M.,Poutanen K.,Bitechnol And Applied Biochem,1991,12:1-11.
[34] Srivastava R, Ali S S, Srivastava B S. Cloning of xylanase gene of Streptomyces flavogriseusinEscherichia coli and baterio phageλ-induced lysis for the release of cloned enzyme[J].FEMSMicrobiology Letters,1991,78:201～206.
[35] Tenkanen M.Binding of hemicellulases on isolated Polysaccharide substrates.Enzyme andMicrobialTechnology，1995，17(6):499～505.
[36] Torronen, A.&Rouvinen, J.(1995). Structural comparison of two major endo-1,4-xylanasesfrom Trichoderma reesei. Biochemistry,34,847–856.
[37] Tony C ollins,Charles Gerday,Georges Feller.Xylanases, xylanase families and extremophilicxylanases [J].FEMS Microbiology Reviews,2005,29:3～23.
[38] Walton, J.D.(1994). Deconstructing the plant cell wall. Plant Physiol.04:1113-1118. JonathanD. W. Deconstructing the Cell Wall，Plant Physiol.(1994)104:1113-1118.
[39] Winterhalter C,Heinrich P,Candussio G,Wich G,Liebl W.Indentification of a novelcellulose-binding domain within the multidomain120kDa xylanase xynA of thehyperthermoohilic bacterium Thermotoga maritima.Mol Microbiol,1995,15(3):431～444.
[40] Wong K.K.Y.,Tan L.U.L.,Saddler J.N.Multiplicity of β-1,4-xylanase in microorganisms:functionand applications Microbiol Mol Biol Rev,1998,V52(3):305-317.
[41] Zhan-ling X. Cloning of a novel xylanase gene from a newly isolated Fusarium sp. Q7-31and itsexpression in Escherichia coli, Brazail Journal.(2011)108:15-19.
[42]刘亮伟.木聚糖酶蛋白质序列分析、分子进化和分子模拟(2005）江南大学,无锡(博士论文).
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