嗜热毛壳菌纤维素酶液体发酵条件和内切β-葡聚糖酶的分离纯化
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摘要
纤维素是目前为止世界上植物生物量中含量最丰富的碳水化合物，可以被纤维素酶转变成为葡萄糖，具有很大的开发前景。真菌的纤维素酶可分成三类：外切葡聚糖纤维二糖水解酶、内切葡聚糖酶和β-葡聚糖酶。这三类酶协同作用降解纤维素。
嗜热真菌(thermophilic fungi)产生的纤维素酶在高温条件下具有高活力和稳定性；并且与中温真菌一样，嗜热真菌产生的每类纤维素酶普遍含有多种类型的组分。嗜热真菌Chaetomium thermophile是一种腐生真菌，分布很广，能够在45℃～50℃的高温下繁殖生长，而绝大多数的真核生物若长期暴露在40℃～45℃下将不能存活。嗜热毛壳菌(Chaetomium thermophile)是一种能够产生纤维素酶的嗜热真菌，但目前关于这方面的研究还不多，特别是在国内尚未见报道。
本研究的第一部分主要对嗜热真菌Chaetomium thermophile产生纤维素酶的液体发酵条件及其滤纸酶(FPA)的特性进行了研究。采用液体发酵培养法，通过对碳源、氮源、培养时间、液体培养基的起始pH值及产酶过程中培养液中pH值和蛋白质含量变化的研究发现：在以2％纤维素、1％可溶性淀粉为碳源，2.0％KNO_3+0.2％酵母为氮源，液体培养基的起始pH值为6.5，50℃下液体发酵培养8d～9d后，各种酶活力最高。发酵过程中，培养液的pH值和蛋白质的含量均在前3d下降，后升高，其变化与菌丝的生长和产酶在时间上相一致。FPA的反应最适温度和pH值分别为60℃和5.5～6.0。FPA具有较高的热稳定性，在50℃～65℃下保温1h，活性没有损失；80℃下保温1h，仍有5％的相对酶活。FPA耐酸性，在pH4.5～6.0范围内稳定。
本研究第二部分主要探讨了液体发酵嗜热真菌Chaetomium thermophile产生的内切β-葡聚糖酶的分离纯化及特性。粗酶液经硫酸铵分级沉淀、DEAE-Sepharose阴离子层析、Phenyl-Sepharose疏水层析、SephacrylS-100分子筛层析等步骤获得凝胶电泳均一的内切β-葡聚糖酶。经12.5％SDS-聚丙烯酰胺凝胶电泳和凝胶过滤层析法分别测得所分离纯化酶蛋白的分子量约为67.8kDa和69.8kDa，并且通过糖染色反应确定该酶蛋白为一种糖蛋白。由芬兰Turku生物技术中心完成该酶的晶体结晶，为今后该酶三维空间结构的分析和同类型酶蛋白的结晶打下了基础。内切β-葡聚糖酶反应的最适温度和pH分别为60℃和4.0～4.5，并且具有很高的热稳定性和耐酸性。在pH5.0条件下，该酶在60℃下稳定；70℃保温1h后，仍保留30％的活性；在80℃的半衰期为25min。在pH4.0～5.0间该酶的相对活性在65％～100％之间。金属离子对内切β-葡聚糖酶的活性影响较大，其中Na~+对酶有激活作用；Fe~(2+)、Ag~+、Cu~(2+)、Ba~(2+)、Zn~(2+)等对酶有抑制作用。薄层层析和酶的底物特异性实验表明该酶不能水解纤维二糖，对含有β-1，4-糖苷键的分子化合物具有专化性，其水解微晶纤维素的产物为寡糖(C3或C4或C5)；并且对结晶纤维素也没有水解能力，而对羧甲基纤维素具有很高的活性。
Cellulose is by far the most abundant carbohydrate available from plant biomass, and can be converted into glucose by cellulase. The cellulase system in fungi is considered to comprise three hydrolytic enzymes: cellobiohydrolase, endocellulase, glucosidase. The hydrolyzation of cellulose is the cooperative action of the three types of enzyme.
Cellulases from the thermophiJic fungi have reported to be stable at high temperature and like mesophilic fungi, the thermophilic fungi produce multiple froms of the cellulase components.
C. thermophile, one of the thermophilic fungi, is a metatrophic soil fungus, and canbe thrive at temperature between 45 and 50, while most fungi will die above 40. Reporters about cellulase from C. thermophile have been few in the word, especially in China. C. thermophile used in the present study was isolated in China.
In the first part of the article, we did some basal studies on the Studies on liquid-state fermentation for and properties of cellulase from C. thermophile. By the liquid-state fermentation, the best factors for cellulase from C. thermophile were investigated. The factors including carbon sources, nitrogen sources, the original pH of medium and varieties of pH and the protein concentration during the cultural process were tested .The result showed that when the medium contained 2% celluse , 2.0% KNO3, 0.2%Yeast and 1% dissolvable starch, the original pH was 6.5,and the optimum time was 9 days under the temperature of 50, activities of cellulase were high. During the first three days, either pH or protein decreased, but after then the both raised. The optimum temperature and pH of FPA were 60 and 5.5-6.0, respectively. FPA could endure high temperature (50 -65) and low pH (4.5-6.0).
In the second part of the article, an endocellulase from culture supernatant of a thermophlic fungus C. thermophile was purifided to homogeneity, by using ammonium sulfate fraction, DEAE-Sepharose chromatography, Phenyl-Sepharose chromatography and Sephacryl S-100 chromatography. The enzyme was a glycoprotein with an apparent molecular weight of 67.8kDa and 69.8kDa, as determinded by 12.5% SDS-PAGE and gel
filtration, respectively. The crystalline body of the enzyme had been attained in the Turku biological technology center, Finland. The endocellulase was optimally active at pH 4.0-4.5 and 60. It was thermostable at 60 and retained 30% activity after 60min at 70 . The half life time of the enzyme at 80 was 25min. Different metal ions showed different effects on the endocellulase activity. Na+ enhanced the enzyme activity, whereas Fe2 Ag Cu2, Ba2 Zn2+cause obvious inhibition. The enzyme did not hydrolyze cellobiose, and was special for molecules containing p-l,4-glycosidic linkages.The products of the endocellulase hydrolying microcrystalline cellulose are oligosaccharies (C3 or C4 or C5) . It also didn't work on crystalline celllose and showed high activity towards CMC.
嗜热真菌(thermophilic fungi)产生的纤维素酶在高温条件下具有高活力和稳定性；并且与中温真菌一样，嗜热真菌产生的每类纤维素酶普遍含有多种类型的组分。嗜热真菌Chaetomium thermophile是一种腐生真菌，分布很广，能够在45℃～50℃的高温下繁殖生长，而绝大多数的真核生物若长期暴露在40℃～45℃下将不能存活。嗜热毛壳菌(Chaetomium thermophile)是一种能够产生纤维素酶的嗜热真菌，但目前关于这方面的研究还不多，特别是在国内尚未见报道。
本研究的第一部分主要对嗜热真菌Chaetomium thermophile产生纤维素酶的液体发酵条件及其滤纸酶(FPA)的特性进行了研究。采用液体发酵培养法，通过对碳源、氮源、培养时间、液体培养基的起始pH值及产酶过程中培养液中pH值和蛋白质含量变化的研究发现：在以2％纤维素、1％可溶性淀粉为碳源，2.0％KNO_3+0.2％酵母为氮源，液体培养基的起始pH值为6.5，50℃下液体发酵培养8d～9d后，各种酶活力最高。发酵过程中，培养液的pH值和蛋白质的含量均在前3d下降，后升高，其变化与菌丝的生长和产酶在时间上相一致。FPA的反应最适温度和pH值分别为60℃和5.5～6.0。FPA具有较高的热稳定性，在50℃～65℃下保温1h，活性没有损失；80℃下保温1h，仍有5％的相对酶活。FPA耐酸性，在pH4.5～6.0范围内稳定。
本研究第二部分主要探讨了液体发酵嗜热真菌Chaetomium thermophile产生的内切β-葡聚糖酶的分离纯化及特性。粗酶液经硫酸铵分级沉淀、DEAE-Sepharose阴离子层析、Phenyl-Sepharose疏水层析、SephacrylS-100分子筛层析等步骤获得凝胶电泳均一的内切β-葡聚糖酶。经12.5％SDS-聚丙烯酰胺凝胶电泳和凝胶过滤层析法分别测得所分离纯化酶蛋白的分子量约为67.8kDa和69.8kDa，并且通过糖染色反应确定该酶蛋白为一种糖蛋白。由芬兰Turku生物技术中心完成该酶的晶体结晶，为今后该酶三维空间结构的分析和同类型酶蛋白的结晶打下了基础。内切β-葡聚糖酶反应的最适温度和pH分别为60℃和4.0～4.5，并且具有很高的热稳定性和耐酸性。在pH5.0条件下，该酶在60℃下稳定；70℃保温1h后，仍保留30％的活性；在80℃的半衰期为25min。在pH4.0～5.0间该酶的相对活性在65％～100％之间。金属离子对内切β-葡聚糖酶的活性影响较大，其中Na~+对酶有激活作用；Fe~(2+)、Ag~+、Cu~(2+)、Ba~(2+)、Zn~(2+)等对酶有抑制作用。薄层层析和酶的底物特异性实验表明该酶不能水解纤维二糖，对含有β-1，4-糖苷键的分子化合物具有专化性，其水解微晶纤维素的产物为寡糖(C3或C4或C5)；并且对结晶纤维素也没有水解能力，而对羧甲基纤维素具有很高的活性。
Cellulose is by far the most abundant carbohydrate available from plant biomass, and can be converted into glucose by cellulase. The cellulase system in fungi is considered to comprise three hydrolytic enzymes: cellobiohydrolase, endocellulase, glucosidase. The hydrolyzation of cellulose is the cooperative action of the three types of enzyme.
Cellulases from the thermophiJic fungi have reported to be stable at high temperature and like mesophilic fungi, the thermophilic fungi produce multiple froms of the cellulase components.
C. thermophile, one of the thermophilic fungi, is a metatrophic soil fungus, and canbe thrive at temperature between 45 and 50, while most fungi will die above 40. Reporters about cellulase from C. thermophile have been few in the word, especially in China. C. thermophile used in the present study was isolated in China.
In the first part of the article, we did some basal studies on the Studies on liquid-state fermentation for and properties of cellulase from C. thermophile. By the liquid-state fermentation, the best factors for cellulase from C. thermophile were investigated. The factors including carbon sources, nitrogen sources, the original pH of medium and varieties of pH and the protein concentration during the cultural process were tested .The result showed that when the medium contained 2% celluse , 2.0% KNO3, 0.2%Yeast and 1% dissolvable starch, the original pH was 6.5,and the optimum time was 9 days under the temperature of 50, activities of cellulase were high. During the first three days, either pH or protein decreased, but after then the both raised. The optimum temperature and pH of FPA were 60 and 5.5-6.0, respectively. FPA could endure high temperature (50 -65) and low pH (4.5-6.0).
In the second part of the article, an endocellulase from culture supernatant of a thermophlic fungus C. thermophile was purifided to homogeneity, by using ammonium sulfate fraction, DEAE-Sepharose chromatography, Phenyl-Sepharose chromatography and Sephacryl S-100 chromatography. The enzyme was a glycoprotein with an apparent molecular weight of 67.8kDa and 69.8kDa, as determinded by 12.5% SDS-PAGE and gel
filtration, respectively. The crystalline body of the enzyme had been attained in the Turku biological technology center, Finland. The endocellulase was optimally active at pH 4.0-4.5 and 60. It was thermostable at 60 and retained 30% activity after 60min at 70 . The half life time of the enzyme at 80 was 25min. Different metal ions showed different effects on the endocellulase activity. Na+ enhanced the enzyme activity, whereas Fe2 Ag Cu2, Ba2 Zn2+cause obvious inhibition. The enzyme did not hydrolyze cellobiose, and was special for molecules containing p-l,4-glycosidic linkages.The products of the endocellulase hydrolying microcrystalline cellulose are oligosaccharies (C3 or C4 or C5) . It also didn't work on crystalline celllose and showed high activity towards CMC.
引文
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38． Bao, W., S. N. Usha, and V. Renganathan. Purification and characterization of celiobiose Dehydrogenase, a Novel Extracellular Hemoflavoenzyme from the White-rot fungus Phanerochaete chrysosporium [J] . Arch. Biochem. Biophys. 300 (2) : 705-713．
39． Beguin P. Molecular biology of cellular degradation [J] . Annu. Rcv. Microbiol. 1990,44:219-248．
40． Bhat, K. M., and S.Bhat. Cellulose degrading enzymes and their potential industrial applications [J] . Biotachnol. Adv. 1997,15: 583-620．
41． Bhat, K. M., J. S. Gaikwad, and R. Maheshwari. Purification and characterization of extracellular β-glucosidase from the thermophilic fungus Sporotrichum thermophile and its influence on cellulase activity [J] . J. General. Microbiol. 1993, 139: 2825-2832．
42． Bisaria, V. S., S. Mishra. Regluatory aspects of cellulase biosynthesis and secretion [J] . Critical reviews in Biotechnology. 1989, 9 (2) : 61-106．
43． Bok, J. D., D. A. Yernool, and D. E. Eveleigh. Purification, characterization, and molecular analysis of thermostable cellulases CelA and CelB from Thermotoga neapolitana [J] . 1998, 64 (12) : 4774-4781．
44． Castanon, M., and C. R. Wilke. Adsorption and recovery of cellulases during hydrolysis of newspaper [J]. Biotechnol. Bioeng. 1980, 22: 1037．
45． Chapman, C. M., J. R. Lowenberg., and M. J. Schaller. Ultrastructural localization of cellulase in T. reesei using immunochemistry and enzyme cytochemistry [J] . J. Histochem. Cytochem. 1983, 31: 1363．
46． Cheorl H K. Characterization and substrate specificity of an endo-β-1,4-D-glucanase I (Avicelase I ) from an extracellular multienzyme complex of Bacillus circulans [J] . Applied and Environment Microbiology, 1995, 61 (3) : 959-965．
47． Cooney, D. G., and R. Emerson. Thermophilic Fungi. An account of their biology, activities and classification [J]. W. H. Freeman & Co., San Francisco, 1964, 27-42．
48． Coughlan, M. P., and A. McHafe. Purification of β-glucosidase glucohydrolases of Talaromyces emersonii [J] . Methods Enzymol. 1988,160: 437-443．
49． Coughlan, M. P., and L. G. Ljungdahl. Comparative biochemistry of fungal and bacterial cellulolytic enzyme systems [J] . FEMS Symp. 43． London: Academic, 1988, 11-30．
50． Fierobe, H. P., B. T. Chantal, and C. Gaudin., et al. Purification and characterization of endoglucannase C from Clostridium cellulolyticum catalytic comparison with endoglucanase A [J]. Bur. Biochem. 1993,217: 557-565．
51． Filho, E. X. F. Purification and characterization of a β-glucosidase from solid-state cultures of Humicolagrisea var.thermoidea [J]. 1996, Can. J.Microbiol. 42:1-5．
Ferment. Bioeng. 1989, 67:380-386．
38． Bao, W., S. N. Usha, and V. Renganathan. Purification and characterization of cellobiose Dehydrogenase, a Novel Extracellular Hemoflavoenzyme from the White-rot fungus Phanerochaete chrysosporium [J] . Arch. Biochem. Biophys. 300 (2) : 705-713．
39． Beguin P. Molecular biology of cellular degradation [J] . Annu. Rev. Microbiol. 1990, 44: 219-248．
40． Bhat, K. M., and S.Bhat. Cellulose degrading enzymes and their potential industrial applications [J]. Biotachnol. Adv. 1997,15: 583-620．
41． Bhat, K. M., J. S. Gaikwad, and R. Maheshwari. Purification and characterization of extracellular β-glucosidase from the thermophilic fungus Sporotrichum thermophile and its influence on cellulase activity [J] . J. General. Microbiol. 1993, 139: 2825-2832．
42． Bisaria, V. S., S. Mishra. Regluatory aspects of cellulase biosynthesis and secretion [J]. Critical reviews in Biotechnology. 1989, 9 (2) : 61-106．
43． Bok, J. D., D. A. Yernool, and D. E. Eveleigh. Purification, characterization, and molecular analysis of thermostable cellulases CelA and CelB from Thermotoga neapolitana [J]. 1998, 64 (12) :4774-4781．
44． Castanon, M., and C. R. Wilke. Adsorption and recovery of cellulases during hydrolysis of newspaper [J]. Biotechnol. Bioeng. 1980, 22: 1037．
45． Chapman, C. M., J. R. Lowenberg., and M. J. Schaller. Ultrastructural localization of cellulase in T. reesei using immunochemistry and enzyme cytochemistry [J] . J. Histochem. Cytochem. 1983,31: 1363．
46． Cheorl H K. Characterization and substrate specificity of an endo-β-1,4-D-glucanase I (Avicelase I ) from an extracellular multienzyme complex of Bacillus circulans [J] . Applied and Environment Microbiology, 1995, 61 (3) : 959-965．
47． Cooney, D. G., and R. Emerson. Thermophilic Fungi. An account of their biology, activities and classification [J]. W. H. Freeman & Co., San Francisco, 1964, 27-42．
48． Coughlan, M. P., and A. McHale. Purification of β-glucosidase glucohydrolases of Talaromyces emersonii [J] . Methods Enzymol. 1988, 160: 437-443．
49． Coughlan, M. P., and L. G. Ljungdahl. Comparative biochemistry of fungal and bacterial cellulolytic enzyme systems [J]. FEMS Symp. 43． London: Academic, 1988, 11-30．
50． Fierobe, H. P., B. T. Chantal, and C. Gaudin., et al. Purification and characterization of endoglucannase C from Clostridium cellulolyticum catalytic comparison with endoglucanase A [J]. Bur. Biochem. 1993, 217: 557-565．
51． Filho, E. X. F. Purification and characterization of a β-glucosidase from solid-state cultures of Humicola grisea var.thermoidea [J] . 1996, Can. J .Microbiol. 42:1-5．
exoglucanase activity from a newly isolated Streptomyces sp. LX [J] . The Society for Applied Microbiology. 1998, 85: 347-356．
66． Li, X., P. Gao. CMC-liquefying enzyme, a low molecular mass initial cellulose-decomposing cellulase responsible for fragmentation from Streoptomyces sp. LX [J]. The Society for Applied Bacteriology. 1997, 83: 59-66．
67． Macris, B. J. Production and characterization of cellulase and β-glucosidase from a mutant of Alternaria alternata [J] . Appl. Environ. Microbiol. 1984, 47 (3) : 560-565．
68． Madarro, A., J. L. Pena, and J. L. Lequerica., et al. Partial purification and characterization of the cellulase from Clostridium cellulolyticum H10 [J] . J. Chem. Tech. Biotechnol. 1991, 52: 393-406．
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