糖蜜酒精废液诱导康氏木霉产纤维素酶的研究
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摘要
糖蜜酒精废液是糖厂的主要污染源之一，是一种深褐色、高酸性、高浓度有机废水。广西大学食品与发酵工程所研究其综合治理时，发现糖蜜酒精废液有促进康氏木霉产纤维素酶的作用，本论文为此作了深入研究。
论文研究了糖蜜酒精废液诱导下的纤维素酶酶学特性：FP，EG，BG，CBH酶的最适pH分别为4.8，4.8，4.8，5.0；最适作用温度分别为50℃，70℃，60℃，55℃；测定了CBH，EG，BG酶的米氏常数；探讨了底物浓度、稀释倍数、金属离子等因素对FP，EG，BG，CBH酶的影响。
论文分离纯化了纤维素酶：EG，BG酶经DEAE C52，CL-6B柱层析分离，酶活力最终回收率分别为2.06％，65.11％，纯化倍数分别为24.55、169.84，比活力分别达189.48IU／mg蛋白，656.28IU／mg蛋白。CBH酶经DEAE-C52分离，回收率和纯化倍数分别为85.61％，31.56。
论文研究分析了糖蜜酒精废液的诱导机理：表明糖蜜酒精废液对纤维素酶的产生具有诱导效应，而非营养因素的作用；其FP酶活力是Mandels营养液的4.8倍；EG，BG，CBH酶活力是Mandels营养液的3～5倍。经电泳测定，糖蜜酒精废液诱导康氏木霉大量表达胞外蛋白p~(17.8)，p~(72.9)；胞外蛋白p~(17.8)，p~(72.9)不具有EG，BG，CBH酶活性，即无纤维素酶活性。
The waste molasses of ethanol, deep brow, high acid, condensed waste organic water, is one of the main puollution sourses from sugar refineries. During the comprehensive disposal of the waste performed by Food and Fermentation engineering Institute of Guangxi University, the waste molasses of ethanol was found to be helpful to the excretion of cellulase from Trichoderma Knoningii. The further studies on cellulase from Trichoderma Knoningii affected by the waste are achieved in this thesis.
Firstly, the enzymic characteristics of cellulase from waste molasses medium, including FP, EG, BG and CBH are determined in this thesis. The optimum pH for catalyzed reactions of FP, EG, BG and CBH are 4.8, 4.8, 4.8, 5.0, respectively. The optimum temperature for catalyzed reactions of FP, EG, BG and CBH are 50℃, 70℃, 60℃, 55℃, respectively. The michaelis constant of EG, BG and CBH, as well as the effects of substrates, dilutions and ions on FP, EG, BG and CBH are assayed.
Secondly, purification and isolation of cellulase are performed in the thesis. By means of ammonium sulphate precipitation, anion exchange chromatography column DEAE cellulose 52 and DEAE sepharose CL-6B, the recoveries of EG and BG reach 2.06% and 65.11%, purity of EG and BG reach 24.55 times and 169.84 times, specfic activities of EG and BG reach
189.48IU per mg protein and 656.28IU per mg protein, respectively.
Thirdly, the mechanism of cellulase induced by waste molasses of ethanol from Trichoderma Knoningii is investigated. It's the function of inducement, not function of nourishment that cellulase is excreted excessively from Trichoderma Knoningii on the medium of waste molasses. The activity of FP is 4.8 times as much as that of Mandels medium, comparative with EG, BG and CBH are 3 to 5 times as much as that of Mandels medium. Analyzed by SDS-PAGE, waste molasses of ethanol can induce two proteins over-Expression from Trichoderma Knoningi. These proteins, named P17.8and
P72.9 according to molecular weight 17.8KD, 72.9KD respectively, have no activities of EG, BG and CBH. So P17.8and P72.9 have not activity of cellulase. They are not cellulase.
论文研究了糖蜜酒精废液诱导下的纤维素酶酶学特性：FP，EG，BG，CBH酶的最适pH分别为4.8，4.8，4.8，5.0；最适作用温度分别为50℃，70℃，60℃，55℃；测定了CBH，EG，BG酶的米氏常数；探讨了底物浓度、稀释倍数、金属离子等因素对FP，EG，BG，CBH酶的影响。
论文分离纯化了纤维素酶：EG，BG酶经DEAE C52，CL-6B柱层析分离，酶活力最终回收率分别为2.06％，65.11％，纯化倍数分别为24.55、169.84，比活力分别达189.48IU／mg蛋白，656.28IU／mg蛋白。CBH酶经DEAE-C52分离，回收率和纯化倍数分别为85.61％，31.56。
论文研究分析了糖蜜酒精废液的诱导机理：表明糖蜜酒精废液对纤维素酶的产生具有诱导效应，而非营养因素的作用；其FP酶活力是Mandels营养液的4.8倍；EG，BG，CBH酶活力是Mandels营养液的3～5倍。经电泳测定，糖蜜酒精废液诱导康氏木霉大量表达胞外蛋白p~(17.8)，p~(72.9)；胞外蛋白p~(17.8)，p~(72.9)不具有EG，BG，CBH酶活性，即无纤维素酶活性。
The waste molasses of ethanol, deep brow, high acid, condensed waste organic water, is one of the main puollution sourses from sugar refineries. During the comprehensive disposal of the waste performed by Food and Fermentation engineering Institute of Guangxi University, the waste molasses of ethanol was found to be helpful to the excretion of cellulase from Trichoderma Knoningii. The further studies on cellulase from Trichoderma Knoningii affected by the waste are achieved in this thesis.
Firstly, the enzymic characteristics of cellulase from waste molasses medium, including FP, EG, BG and CBH are determined in this thesis. The optimum pH for catalyzed reactions of FP, EG, BG and CBH are 4.8, 4.8, 4.8, 5.0, respectively. The optimum temperature for catalyzed reactions of FP, EG, BG and CBH are 50℃, 70℃, 60℃, 55℃, respectively. The michaelis constant of EG, BG and CBH, as well as the effects of substrates, dilutions and ions on FP, EG, BG and CBH are assayed.
Secondly, purification and isolation of cellulase are performed in the thesis. By means of ammonium sulphate precipitation, anion exchange chromatography column DEAE cellulose 52 and DEAE sepharose CL-6B, the recoveries of EG and BG reach 2.06% and 65.11%, purity of EG and BG reach 24.55 times and 169.84 times, specfic activities of EG and BG reach
189.48IU per mg protein and 656.28IU per mg protein, respectively.
Thirdly, the mechanism of cellulase induced by waste molasses of ethanol from Trichoderma Knoningii is investigated. It's the function of inducement, not function of nourishment that cellulase is excreted excessively from Trichoderma Knoningii on the medium of waste molasses. The activity of FP is 4.8 times as much as that of Mandels medium, comparative with EG, BG and CBH are 3 to 5 times as much as that of Mandels medium. Analyzed by SDS-PAGE, waste molasses of ethanol can induce two proteins over-Expression from Trichoderma Knoningi. These proteins, named P17.8and
P72.9 according to molecular weight 17.8KD, 72.9KD respectively, have no activities of EG, BG and CBH. So P17.8and P72.9 have not activity of cellulase. They are not cellulase.
引文
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[4]．李壮，叶洪武，周小群等，糖蜜酒精废液燃烧处理技术研究，广西轻工业，2001(1)26～32
[5]．徐文，李蘅，张生炎等，糖蜜酒精废液生化法的研究进展，矿产与地质，2002(6)，375～380
[6]．孟芙蓉，利用糖蜜酒精废液生产单细胞蛋白，全国配合饲料资源调查协作组论文集，1990
[7]．陈红征，刘勇，申彤等，甜菜糖蜜酒精废液生产菌体蛋白饲料初探，新疆大学学报(自然科学版)，2002(3)，330～332
[8]．周素华，日本糖蜜酒精废液处理技术，酿酒科技，2003(3)，90～92
[9]．周桂，邓光辉，何子平，糖蜜酒精废液综合利用进展，广西民族学院学报(自然科学版)，2000(2)，111～114
[10]．吴忠铨，梁建生，谭礼宁，糖蜜酒精废液处理的探讨，广西蔗糖2000(3)，44～47
[11]．杨斌，高孔荣，蔗渣糖化和转化为乙醇的进展。食品与发酵工业，1995(6)，16～19
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[17]. Selby K. A dv in Chem Ser, 1969 (95), 34～50
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[23]．钟发刚，王新华，饲用纤维素酶研究进展，中国微生态学杂志，2002(5)，308～309
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[30]. Reese E T. B io techno 1B ioeng Sympo sium, 1976 (6): 9～20
[31]．汪天虹，吴静，邹玉霞，氏木霉分子生物学研究进展[J]，菌物系统，2000，19(1)，147-152
[32]. Kim JO, Park SR,Lim WJ, et al. Cloning and characterization of thermostable endoglucanase (Ce18Y) from the hypert thermophilic Aquifex aeolicus VF5 [J]. Biochemical & Biophysical Research Communications, 2000,279(2): 420 - 4261
[33]. Hakamada Y, Endo K,Takizawa S, et al. Enzymatic properties, crystallization, and deduced amino acid sequence of an alkaline endoglucanase from Bacil.lus circulans [J]. Biochimica et Biophysica Acta, 2002,1570: 174 - 1801
[34]. Shou Takashima. Overproduction of recombinant Trichoderma reesei cellulases by Aspergillus oryzae and their enzymatic properties. Journal of Biotechnology 65 (1998) 163-171
[35]．肖志壮，王婷，汪天虹，等，瑞氏木霉内切葡聚糖酶Ⅲ基因的克隆及在酿酒酵母中的表达[J]，微生物学，2001，41(4)，391-3961
[35]. M.K. Bhat. Cellulases and related enzymes in biotechnology. Biotechnology Advances 18 (2000) 355-383
[36]．周正红，贾宗剑，高孔荣，纤维素酶在食品发酵工业的应用及前景，暨南大学学报，1998(5)，125-130
[37]．云秀芳，纤维素酶在酱油生产中的应用研究，中国调味品，2001(3)，15-19
[38]．朱元招，刘亚力，陈宏等，补饲不同配方酶制剂对断奶犊牛生产性能的影响，中国草食动物，2001(3)
[39]．席兴军，韩鲁佳，原慎一郎等，添加乳酸菌和纤维素酶对玉米秸秆青贮饲料品质的影响，中国农业大学学报，2003，8(2)，21～24
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[42]．赵玉林，陈中豪，王福君，纤维素酶在造纸工业中的应用研究进展纤维素酶在造纸工业中的应用研究进展，生物技术，2003(3)，64-66
[43]．陈冠军，秦梦华，曲音波等，纤维素酶脱墨机理的研究进展，生物工程进展，2001(3)，17-22
[44]．邹水洋，甘蔗糖厂废弃物饲料化途径的研究，广西大学论文集，2000．5
[45]．李军辉，糖蜜酒精废液诱导产生纤维素酶及其诱导活性因子的分离鉴定，广西大学论文集，2001．5
[46]．杜宗军．黑色葡萄状穗霉纤维素酶的纯化和性质[J]，青岛海洋大学学报，2002，32(1)，79-84
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[2]．黄志甲，张旭，糖蜜酒精废液处理技术，工业水处理，2002，第22卷第10期，19～25
[3]．黄伟添、陈新军，甘蔗糖蜜废液综合利用的探索与实践，甘蔗糖业，1997(5)，41～45
[4]．李壮，叶洪武，周小群等，糖蜜酒精废液燃烧处理技术研究，广西轻工业，2001(1)26～32
[5]．徐文，李蘅，张生炎等，糖蜜酒精废液生化法的研究进展，矿产与地质，2002(6)，375～380
[6]．孟芙蓉，利用糖蜜酒精废液生产单细胞蛋白，全国配合饲料资源调查协作组论文集，1990
[7]．陈红征，刘勇，申彤等，甜菜糖蜜酒精废液生产菌体蛋白饲料初探，新疆大学学报(自然科学版)，2002(3)，330～332
[8]．周素华，日本糖蜜酒精废液处理技术，酿酒科技，2003(3)，90～92
[9]．周桂，邓光辉，何子平，糖蜜酒精废液综合利用进展，广西民族学院学报(自然科学版)，2000(2)，111～114
[10]．吴忠铨，梁建生，谭礼宁，糖蜜酒精废液处理的探讨，广西蔗糖2000(3)，44～47
[11]．杨斌，高孔荣，蔗渣糖化和转化为乙醇的进展。食品与发酵工业，1995(6)，16～19
[12]. M eisel C et al. Food B io techno logy, 1989 (3), 145
[13]．高洁等，纤维素科学，北京，科学出版社，1996
[14]. Percival E G V. St ructural Carbohydrate Chem, London, J, Gamet Miller Ltd, 1962,1～63
[15]. Duret te P Let al. Advances in Carbohydrate Chem, 1971 (26), 490
[16]. M archessault R H et al. Advances in Carbohydrate Chem, 1967 (22), 421
[17]. Selby K. A dv in Chem Ser, 1969 (95), 34～50
[18]. Wood T M et al. B io techno 1B ioeng Symp, 1975, 5, 111～137
[19]. Wood T Metal. BiochemJ, 1972(128), 1183～1192
[20]. Erikaaon K E et al. Biotechno 12B ioeng, 1978(20), 317～332
[21]. Reese E T. B io techno 1B ioeng Symposium, 1976 (6), 9～20
[22]．冯骉，业纤维素酶的测定和比较，无锡轻工大学学报，994(4)，323～330
[23]．钟发刚，王新华，饲用纤维素酶研究进展，中国微生态学杂志，2002(5)，308～309
[24]. Pettersson L. Axio2Frederksson U. - B. and Berghen L. E.. Proceedings of the 4th International Fermentation Symposium:Fermentation technology,Japan. 1972:727～729
[25]. Mandels,M.J.Weber and R.Parizek:Appl.Microbiol.,21:1971,152
[26]. Mandels M,Sternberg D,Andreotti RE.Growth and Cellulase Production by Trichoderma, Bailey M.Enavi T.L.M. ends.symposium on enzymatic Hydrolysis of cellullulose.Helsinki: Technical Research center, 1975:81-110
[27]. Mandels M. et al. Growth and cellulase production by Trichoderma,
[28]. Wood,T.M.:Biotechnol.Bioemg.symp.No.5:1975,111-137. symposium on enzymatic Hydrolysis of cellullulose.Aulanko,Helsinki,Finland. 1975,p. 155
[29]．中国科学院微生物所纤维素酶组．微生物学报，1976(3)，240-248
[30]. Reese E T. B io techno 1B ioeng Sympo sium, 1976 (6): 9～20
[31]．汪天虹，吴静，邹玉霞，氏木霉分子生物学研究进展[J]，菌物系统，2000，19(1)，147-152
[32]. Kim JO, Park SR,Lim WJ, et al. Cloning and characterization of thermostable endoglucanase (Ce18Y) from the hypert thermophilic Aquifex aeolicus VF5 [J]. Biochemical & Biophysical Research Communications, 2000,279(2): 420 - 4261
[33]. Hakamada Y, Endo K,Takizawa S, et al. Enzymatic properties, crystallization, and deduced amino acid sequence of an alkaline endoglucanase from Bacil.lus circulans [J]. Biochimica et Biophysica Acta, 2002,1570: 174 - 1801
[34]. Shou Takashima. Overproduction of recombinant Trichoderma reesei cellulases by Aspergillus oryzae and their enzymatic properties. Journal of Biotechnology 65 (1998) 163-171
[35]．肖志壮，王婷，汪天虹，等，瑞氏木霉内切葡聚糖酶Ⅲ基因的克隆及在酿酒酵母中的表达[J]，微生物学，2001，41(4)，391-3961
[35]. M.K. Bhat. Cellulases and related enzymes in biotechnology. Biotechnology Advances 18 (2000) 355-383
[36]．周正红，贾宗剑，高孔荣，纤维素酶在食品发酵工业的应用及前景，暨南大学学报，1998(5)，125-130
[37]．云秀芳，纤维素酶在酱油生产中的应用研究，中国调味品，2001(3)，15-19
[38]．朱元招，刘亚力，陈宏等，补饲不同配方酶制剂对断奶犊牛生产性能的影响，中国草食动物，2001(3)
[39]．席兴军，韩鲁佳，原慎一郎等，添加乳酸菌和纤维素酶对玉米秸秆青贮饲料品质的影响，中国农业大学学报，2003，8(2)，21～24
[40]．林开江，阮丽娟，王龙英等，纤维素酶及其在生物抛光上的应用，生物技术，1996(3)，45-48
[41]．费云山，梅卓然，王荣生．酶洗涤中纤维素酶的作用方式与途径的探讨，印染，1994(9)，5-10
[42]．赵玉林，陈中豪，王福君，纤维素酶在造纸工业中的应用研究进展纤维素酶在造纸工业中的应用研究进展，生物技术，2003(3)，64-66
[43]．陈冠军，秦梦华，曲音波等，纤维素酶脱墨机理的研究进展，生物工程进展，2001(3)，17-22
[44]．邹水洋，甘蔗糖厂废弃物饲料化途径的研究，广西大学论文集，2000．5
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