生物催化对乌头生物碱转化的影响研究
摘要
进入21世纪以来,随着化石资源与能源短缺、环境危机日益加剧,生物催化技术作为绿色化学与绿色化工发展的重要手段,其发展必将对医药、能源、农业、食品、生命科学等领域产生巨大影响。利用生物酶催化技术提取和分离天然产物中的有效成分是一项新兴技术。植物细胞壁是提取有效成分的主要屏障,阻碍细胞中活性成分的溶出。选用恰当的酶,通过酶催化反应温和地水解或降解植物细胞壁成分,可加速有效成分的释放提取。
本文选用纤维素酶对制草乌药材进行预处理后,用醇提法提取生物碱。考察乌头生物碱提取过程中,纤维素酶催化的适宜条件及其对产品收率的影响;比较纤维素酶催化转化乌头生物碱与其它4种提取方法,生物碱收率及纯度的大小;考察纤维素酶催化转化对传统醇提法乌头生物碱收率和纯度的影响,并初步探讨酶的催化机制。
利用纤维素酶催化转化乌头生物碱时,纤维素酶的适宜作用条件为温度:50℃,pH值:4.5,酶用量:1.0%(1 g药材中加入0. 01 g纤维素酶),作用时间:4.0 h。与其它4种提取技术相比,纤维素酶催化转化得到的乌头碱和次乌头碱收率显著增加,说明纤维素酶水化和降解制草乌细胞壁增加了药材细胞壁的通透性,减小了传质阻力,有利于生物碱成分的溶出。纤维素酶预处理制草乌药材对传统醇提法生物碱收率的提高有明显的促进作用,乌头碱收率由原来的0.00149%上升到0.00235%,次乌头碱的收率由原来的0.0233%上升到0.0386%,分别提高了57.9%和66.0%,乌头生物碱收率由0.0475%上升到0.0820%,提高72.6%。
纤维素酶催化转化得到的样品经高效液相色谱测定其中乌头碱和次乌头碱的总含量为48.9%;经紫外分光光度计测定,样品中乌头总生物碱纯度为97.9%,乌头生物碱收率为0.0820%。
Along with the petrochemical resources and the energy crisis, environment and the society crisis become worse increasingly, as the important method for develpoment of the green chemistry and green chemical engineering, the development of the biocatalysis will have significant effect on medicine, agriculture, food, life science and so on. It is a new technology to extract and separate active constituents from natural plants with enzyme catalysis. Cell walls in plants hinder the extraction of effective constituents. The cell walls will be decomposed when proper enzymes are introducted to act on natural plants, which is profitably to the extraction of effective components.
Cellulase was used to pretreat prepared CaoWu and then aconitum alkaloids were extracted by alcohol in this paper. The optimum catalysis conditions of cellulase and its effect on yield rate of aconitum alkaloids were studied. The yield rate and purity by cellulase catalysis method was compared with others. Meanwhile, the effect cellulase catalysis on conventional alcohol extraction and the mechanism of cellulase catalysis were also researched here.
The optimum conditions that cellulase catalyzed and transformed aconitum alkaloids were at 50℃, pH 4.5, dosage of cellulase 1.0% ( 0.01 g cellulase in 1 g material) and the reaction time of cellulase 4 h. Compared with the 4 other methods, the yield rates of aconitine and hypaconitine were much bigger. It indicated that the hydration and degradation on cell wall of prepared CaoWu enhanced permeation of cell wall and minished mass transfer resistance. It made aconitum alkaloids be taken out easier. The yield rate of aconitum alkaloids was improved obviously after the prepared CaoWu was pretreated by cellulase. The yield rates of aconitine and hypaconitine were improved from 0.00149% to 0.00235% and 0.0233% to 0.0386%, and rised 57.9% and 66.0% respectively. The yield rate of aconitum alkaloids was improved from 0.0475% to 0.0820%, and rised 72.6%.
The content of aconitine and hypaconitine in product by cellulase catalysis was 48.9% according to HPLC chromatogram, and the purity of aconitum alkaloids was 97.9% according to UV. And the yield rate of aconitum alkaloids was 0.0820%. This paper shows that cellulase catalysis technology will play a broad part in the extraction of active constituent from natural plants.
本文选用纤维素酶对制草乌药材进行预处理后,用醇提法提取生物碱。考察乌头生物碱提取过程中,纤维素酶催化的适宜条件及其对产品收率的影响;比较纤维素酶催化转化乌头生物碱与其它4种提取方法,生物碱收率及纯度的大小;考察纤维素酶催化转化对传统醇提法乌头生物碱收率和纯度的影响,并初步探讨酶的催化机制。
利用纤维素酶催化转化乌头生物碱时,纤维素酶的适宜作用条件为温度:50℃,pH值:4.5,酶用量:1.0%(1 g药材中加入0. 01 g纤维素酶),作用时间:4.0 h。与其它4种提取技术相比,纤维素酶催化转化得到的乌头碱和次乌头碱收率显著增加,说明纤维素酶水化和降解制草乌细胞壁增加了药材细胞壁的通透性,减小了传质阻力,有利于生物碱成分的溶出。纤维素酶预处理制草乌药材对传统醇提法生物碱收率的提高有明显的促进作用,乌头碱收率由原来的0.00149%上升到0.00235%,次乌头碱的收率由原来的0.0233%上升到0.0386%,分别提高了57.9%和66.0%,乌头生物碱收率由0.0475%上升到0.0820%,提高72.6%。
纤维素酶催化转化得到的样品经高效液相色谱测定其中乌头碱和次乌头碱的总含量为48.9%;经紫外分光光度计测定,样品中乌头总生物碱纯度为97.9%,乌头生物碱收率为0.0820%。
Along with the petrochemical resources and the energy crisis, environment and the society crisis become worse increasingly, as the important method for develpoment of the green chemistry and green chemical engineering, the development of the biocatalysis will have significant effect on medicine, agriculture, food, life science and so on. It is a new technology to extract and separate active constituents from natural plants with enzyme catalysis. Cell walls in plants hinder the extraction of effective constituents. The cell walls will be decomposed when proper enzymes are introducted to act on natural plants, which is profitably to the extraction of effective components.
Cellulase was used to pretreat prepared CaoWu and then aconitum alkaloids were extracted by alcohol in this paper. The optimum catalysis conditions of cellulase and its effect on yield rate of aconitum alkaloids were studied. The yield rate and purity by cellulase catalysis method was compared with others. Meanwhile, the effect cellulase catalysis on conventional alcohol extraction and the mechanism of cellulase catalysis were also researched here.
The optimum conditions that cellulase catalyzed and transformed aconitum alkaloids were at 50℃, pH 4.5, dosage of cellulase 1.0% ( 0.01 g cellulase in 1 g material) and the reaction time of cellulase 4 h. Compared with the 4 other methods, the yield rates of aconitine and hypaconitine were much bigger. It indicated that the hydration and degradation on cell wall of prepared CaoWu enhanced permeation of cell wall and minished mass transfer resistance. It made aconitum alkaloids be taken out easier. The yield rate of aconitum alkaloids was improved obviously after the prepared CaoWu was pretreated by cellulase. The yield rates of aconitine and hypaconitine were improved from 0.00149% to 0.00235% and 0.0233% to 0.0386%, and rised 57.9% and 66.0% respectively. The yield rate of aconitum alkaloids was improved from 0.0475% to 0.0820%, and rised 72.6%.
The content of aconitine and hypaconitine in product by cellulase catalysis was 48.9% according to HPLC chromatogram, and the purity of aconitum alkaloids was 97.9% according to UV. And the yield rate of aconitum alkaloids was 0.0820%. This paper shows that cellulase catalysis technology will play a broad part in the extraction of active constituent from natural plants.
引文
[1]王瑞玲,王宪法,李乃谦等,川乌、草乌中毒原因及救治方法,中国药学杂志,2001, 36(5):352~353
[2]王育珊,任连荣,刘忠良等,急性乌头碱中毒导致的严重心律失常与休克(附4例报告),白求恩医科大学学报,1996,22(5):521
[3]崔志高,误服含乌头碱药酒中毒16例,中国药学杂志,1995,30(5):300
[4]杨晓秋,急性乌头酒中毒致死1例,中医药学报,1992,12(5):39
[5]贝自英,酸碱中和法测定川乌、附子及炮制品中生物碱的含量,南京中医学院学报,1984,16(3):49~50
[6]陆履雯,三七伤药片中生物碱含量测定,中成药研究,1983,9(1):19
[7]颜根宝,胥法领,小活络丸中乌头总碱的分析,中成药,1989,11(3):14
[8]侯世祥,廖工铁,扬华元,复方三生注射液质量控制复方的研究,中草药,1987,18(3):13
[9]苏孝礼,中药乌头去毒指数的测定,中国药科大学学报,1992,23(4):229
[10]龙沛霞,王玉珍,一阶导数紫外分光光度法测定制川乌、制草乌、附子中生物碱含量,中草药,1996,27(9):531~533
[11]曹晖,香港市售乌头类药材及其炮制品的乌头碱含量测定,中国中药杂志,1993,18(5):279~281
[12]李焕荣,张荣华,崔健等,薄层扫描法测定镇痛宁注射液中乌头碱的含量,中草药,1996,27(1):58
[13]郑杭生,冯年平,中药饮片制川乌、制草乌中乌头碱、次乌头碱的HPLC测定,药物分析杂志, 2005,25(1):34~36
[14]张国安,陈玉定,刘力等,HPLC法测定四逆汤颗粒剂中乌头碱水解产物的含量,中成药,1995,17(12):13
[15]杨华元,彭波,刘世端,高效液相色谱法测定人参四逆汤口服液中乌头类生物碱含量,华西药学杂志,1995,10(1):9
[16]孙秀梅,用均匀设计优选川乌的“半仿生提取法”工艺条件,中药材,1999,22(12):649
[17]张兆旺,川乌两种方法提取液的成分比较,中成药,1999,21(1):5~7
[18]梁宝钻,亚东乌头总生物碱的超临界CO2萃取及含量测定,中药材,2002,25(5):332~333
[19]杨桦,邓晓静,易红,大孔树脂用于川草乌中总生物碱的分离提取,中成药, 2002,22(8):535~538
[20]黄建明,郭济贤,陈万生,大孔树脂对草乌生物碱的吸附性能及提纯工艺,复旦学报(医学版),2003,30(3):267~269
[21] WANG Zhong, SHI Zuo-Qing, WANG Rui-Fang, Studies on sorption property Forwards caffeine and theophylline by phenolic resin, Chemical Research in Chinese Universities, 2003, 24(10): 1986~1901
[22]卢定强,韦萍,周华等,生物催化与生物转化的研究进展,化工进展,2004,23(6):585~589
[23]孙志浩,生物催化工艺学,北京:化学工业出版社,2005,1~10
[24]欧阳平凯,卢定强,资源生态化利用中的生物加工过程,生物加工过程,2003,1(1):1~6
[25]帕特尔R N,立体选择性生物化学,方唯硕译,北京:化学工业出版社,2004,122~128
[26]Bommarius A S, Riebel B R, Biocatalysis, USA, Weinheim: ILEY-VCH, 2004, 478~486
[27]Helmut Uhlig, Industrial Enzymes and Their Applications, New Yoek: John Wiley & Sons Inc, 1998, 89~91
[28]Olga K, Frances H A, Directed evolution of enzyme catalysts, Trends in Biotechnology, 1997, 15(12): 523~530
[29]杜晨宇,李春,曹竹安等,工业生物技术的核心——生物催化,生物工程进展,2002,22(1):9~14
[30]何坤荣,生物催化技术前景广阔,精细与专用化学品,2002,10(22):13~14
[31]Haiyan T, Virginia W C, Milestones in directed enzyme evolution, Current Opinion in Chemical Biology, 2002, 6(6): 858~864
[32]居乃琥,酶工程研究和酶工程产业的新进展,食品与发酵工业,2000,26(13):54~62
[33]Bertus B, Extremophiles as a source for novel enzymes, Current Opinion in Microbiology, 2003, 6(3): 213~218
[34]马晓建,白净,任珂等,酶工程研究的新进展,化工进展,2003,22(8):813~817
[35]Straathof A J J, Panke S, Schmid A, The production of fine chemicals by biotransforrnations, Current Opinion in Biotechnology, 2002, 13(6): 548~556
[36]赫尔曼·舌尔,阳光经济,生态的现代战略,北京:三联书店,1999,168~176
[37] Frankel E N, Waterhouse A L, Kinsella J E, Inhibition of human LDL oxidation by resverstrol, Lancet, 1993, 341: 1103~1104
[38]侯嵘峤,孟宪纾,徐育新,以酶解法从中药及药渣中制备β-葡萄糖的研究,沈阳药学院学报,1994,11(4):289~293
[39]林桂云,生物技术与中药现代化,成都大学学报,2001,20(2):34~36
[40]胡之璧,刘涤,生物技术在中药现代化中的地位和作用,世界科学技术——中药现代化,2000,2(5):23~26
[41]赵立亚,金凤燮,鱼红闪,酶法生产稀有人参皂苷极其产物成分的分析,大连轻工业学院学报,2002,21(2):112~115
[42]张裕卿,王东青,李滨县等,阶梯生物催化协同提取盾叶薯蓣中的薯蓣皂苷元的研究,中草药, 2006,37(5): 688~691
[43]张裕卿,李滨县,改性纤维素酶生物催化酸水解薯蓣皂苷的研究,中草药,2007,38(4):527~530
[44]陶卫平,热稳定性酶,生物学通报,1998,33(1):20~21
[45]Wang Y B, Nagata S, Participation of ions and solutes on the thermostability ofα-amylase, Chinese Journal of Biotechnology, 2004, 20(1): 104~110
[46]宋小妹,唐志书,中药化学成分提取分离与制备,北京:人民卫生出版社, 2004,133~134
[47]孙文基,天然药物成分提取分离与制备(第三版),北京:中国医药科技出版社,2006,3~4
[48]Nidetzky B, Steiner S, HAYN M, et a1, Cellulose hydrolysis by the cellulases from Trichoderma Reese: a new model for synergistic interaction, Biochem J,1994, 15(298): 705~710
[49]Beguin P, Aubert J P, The biological degradation of cellulose, FEMS Microbiol Rev, 1994, 13: 25~58
[50]Lenting H B M, Warmoeskerken M M, Mechanism of interaction between cellulase action and applied shear force and hypothesis, Journal of Biotechnology, 2001, 89(2): 217~226
[51]Wang W, Gao P J, Function and mechanism of a low-molecular-weight peptide produced by Gloeophyllum trabeum J. Biotechnol, 2003, 101: 119~130
[52]Lee I, Evans B R, Woodward J, The mechanism of cellulase on cotton fibers: evidence from atomic force microscopy, Ultramicroscopy, 2000, 82: 213~221
[53]Samejima M, Eriksson K E, Comparation of the catalytic properties of cellobiose; quinone oxidoreductase and cellobiose oxidase from phanerochaete chrysosporium, Eur J Biochern, 1992, 207: 103~107
[54]Chang V S, Holtzapple M T, Fundamental factors affecting biomass enzymatic reactivity, Appl. Biochem. Biotechnol, 2000, 5~37
[55]陈洪章,纤维素生物技术,北京:化学工业出版社,2005,40~61
[56]高培基,纤维素酶降解机制及纤维素酶分子结构与功能研究进展,自然科学进展,2003,13(1):21~29
[57]Tomme P, Warren R A, Gillkes N, Cellulose hydrolysis by bacteria and fungi, Adv Microb Physiol, 1995, 37: 1~8
[58]Delong. E A, Method of rendering lignin separable from cellulose and hemicellulose in lignocellulosic material and the product so produced, Canadian Patent No 1096374, 1981, 2
[59]Diebold J P, Bridgwater A V, Overview of fast pyrolysis of biomass for the production of liguid fuels, Developments in Thermochemical Biomass Conversion, London: Blackie Academic and Professional, 1997, 5
[60]Lewis N G, Yamamoto E, lignin: occurrence, biogenesis and biodegradation, Annu Rev Plant Physiol and Plant Mol Biol, 1990, 41: 455~496
[61]Xiao Z H, Gao P J, Qu Y B, et al, Cellulose-binding domain of endoglucanaseⅢfrom Trichoderma reesei disrupting the structure of cellulose, Biotech Letter, 2001, 23: 711~715
[62]Teeri T T, Koivula A, Linder M, et al, Trichoderma reesei cellobiohydrolase: Why so efficient on crystalline cellulose? Biochemical Society Transaction, 1998, 26: 160~164
[63] W. Dan Reece, Mark Holtzapple, Concepts in Engineering, 2004, 310~365
[2]王育珊,任连荣,刘忠良等,急性乌头碱中毒导致的严重心律失常与休克(附4例报告),白求恩医科大学学报,1996,22(5):521
[3]崔志高,误服含乌头碱药酒中毒16例,中国药学杂志,1995,30(5):300
[4]杨晓秋,急性乌头酒中毒致死1例,中医药学报,1992,12(5):39
[5]贝自英,酸碱中和法测定川乌、附子及炮制品中生物碱的含量,南京中医学院学报,1984,16(3):49~50
[6]陆履雯,三七伤药片中生物碱含量测定,中成药研究,1983,9(1):19
[7]颜根宝,胥法领,小活络丸中乌头总碱的分析,中成药,1989,11(3):14
[8]侯世祥,廖工铁,扬华元,复方三生注射液质量控制复方的研究,中草药,1987,18(3):13
[9]苏孝礼,中药乌头去毒指数的测定,中国药科大学学报,1992,23(4):229
[10]龙沛霞,王玉珍,一阶导数紫外分光光度法测定制川乌、制草乌、附子中生物碱含量,中草药,1996,27(9):531~533
[11]曹晖,香港市售乌头类药材及其炮制品的乌头碱含量测定,中国中药杂志,1993,18(5):279~281
[12]李焕荣,张荣华,崔健等,薄层扫描法测定镇痛宁注射液中乌头碱的含量,中草药,1996,27(1):58
[13]郑杭生,冯年平,中药饮片制川乌、制草乌中乌头碱、次乌头碱的HPLC测定,药物分析杂志, 2005,25(1):34~36
[14]张国安,陈玉定,刘力等,HPLC法测定四逆汤颗粒剂中乌头碱水解产物的含量,中成药,1995,17(12):13
[15]杨华元,彭波,刘世端,高效液相色谱法测定人参四逆汤口服液中乌头类生物碱含量,华西药学杂志,1995,10(1):9
[16]孙秀梅,用均匀设计优选川乌的“半仿生提取法”工艺条件,中药材,1999,22(12):649
[17]张兆旺,川乌两种方法提取液的成分比较,中成药,1999,21(1):5~7
[18]梁宝钻,亚东乌头总生物碱的超临界CO2萃取及含量测定,中药材,2002,25(5):332~333
[19]杨桦,邓晓静,易红,大孔树脂用于川草乌中总生物碱的分离提取,中成药, 2002,22(8):535~538
[20]黄建明,郭济贤,陈万生,大孔树脂对草乌生物碱的吸附性能及提纯工艺,复旦学报(医学版),2003,30(3):267~269
[21] WANG Zhong, SHI Zuo-Qing, WANG Rui-Fang, Studies on sorption property Forwards caffeine and theophylline by phenolic resin, Chemical Research in Chinese Universities, 2003, 24(10): 1986~1901
[22]卢定强,韦萍,周华等,生物催化与生物转化的研究进展,化工进展,2004,23(6):585~589
[23]孙志浩,生物催化工艺学,北京:化学工业出版社,2005,1~10
[24]欧阳平凯,卢定强,资源生态化利用中的生物加工过程,生物加工过程,2003,1(1):1~6
[25]帕特尔R N,立体选择性生物化学,方唯硕译,北京:化学工业出版社,2004,122~128
[26]Bommarius A S, Riebel B R, Biocatalysis, USA, Weinheim: ILEY-VCH, 2004, 478~486
[27]Helmut Uhlig, Industrial Enzymes and Their Applications, New Yoek: John Wiley & Sons Inc, 1998, 89~91
[28]Olga K, Frances H A, Directed evolution of enzyme catalysts, Trends in Biotechnology, 1997, 15(12): 523~530
[29]杜晨宇,李春,曹竹安等,工业生物技术的核心——生物催化,生物工程进展,2002,22(1):9~14
[30]何坤荣,生物催化技术前景广阔,精细与专用化学品,2002,10(22):13~14
[31]Haiyan T, Virginia W C, Milestones in directed enzyme evolution, Current Opinion in Chemical Biology, 2002, 6(6): 858~864
[32]居乃琥,酶工程研究和酶工程产业的新进展,食品与发酵工业,2000,26(13):54~62
[33]Bertus B, Extremophiles as a source for novel enzymes, Current Opinion in Microbiology, 2003, 6(3): 213~218
[34]马晓建,白净,任珂等,酶工程研究的新进展,化工进展,2003,22(8):813~817
[35]Straathof A J J, Panke S, Schmid A, The production of fine chemicals by biotransforrnations, Current Opinion in Biotechnology, 2002, 13(6): 548~556
[36]赫尔曼·舌尔,阳光经济,生态的现代战略,北京:三联书店,1999,168~176
[37] Frankel E N, Waterhouse A L, Kinsella J E, Inhibition of human LDL oxidation by resverstrol, Lancet, 1993, 341: 1103~1104
[38]侯嵘峤,孟宪纾,徐育新,以酶解法从中药及药渣中制备β-葡萄糖的研究,沈阳药学院学报,1994,11(4):289~293
[39]林桂云,生物技术与中药现代化,成都大学学报,2001,20(2):34~36
[40]胡之璧,刘涤,生物技术在中药现代化中的地位和作用,世界科学技术——中药现代化,2000,2(5):23~26
[41]赵立亚,金凤燮,鱼红闪,酶法生产稀有人参皂苷极其产物成分的分析,大连轻工业学院学报,2002,21(2):112~115
[42]张裕卿,王东青,李滨县等,阶梯生物催化协同提取盾叶薯蓣中的薯蓣皂苷元的研究,中草药, 2006,37(5): 688~691
[43]张裕卿,李滨县,改性纤维素酶生物催化酸水解薯蓣皂苷的研究,中草药,2007,38(4):527~530
[44]陶卫平,热稳定性酶,生物学通报,1998,33(1):20~21
[45]Wang Y B, Nagata S, Participation of ions and solutes on the thermostability ofα-amylase, Chinese Journal of Biotechnology, 2004, 20(1): 104~110
[46]宋小妹,唐志书,中药化学成分提取分离与制备,北京:人民卫生出版社, 2004,133~134
[47]孙文基,天然药物成分提取分离与制备(第三版),北京:中国医药科技出版社,2006,3~4
[48]Nidetzky B, Steiner S, HAYN M, et a1, Cellulose hydrolysis by the cellulases from Trichoderma Reese: a new model for synergistic interaction, Biochem J,1994, 15(298): 705~710
[49]Beguin P, Aubert J P, The biological degradation of cellulose, FEMS Microbiol Rev, 1994, 13: 25~58
[50]Lenting H B M, Warmoeskerken M M, Mechanism of interaction between cellulase action and applied shear force and hypothesis, Journal of Biotechnology, 2001, 89(2): 217~226
[51]Wang W, Gao P J, Function and mechanism of a low-molecular-weight peptide produced by Gloeophyllum trabeum J. Biotechnol, 2003, 101: 119~130
[52]Lee I, Evans B R, Woodward J, The mechanism of cellulase on cotton fibers: evidence from atomic force microscopy, Ultramicroscopy, 2000, 82: 213~221
[53]Samejima M, Eriksson K E, Comparation of the catalytic properties of cellobiose; quinone oxidoreductase and cellobiose oxidase from phanerochaete chrysosporium, Eur J Biochern, 1992, 207: 103~107
[54]Chang V S, Holtzapple M T, Fundamental factors affecting biomass enzymatic reactivity, Appl. Biochem. Biotechnol, 2000, 5~37
[55]陈洪章,纤维素生物技术,北京:化学工业出版社,2005,40~61
[56]高培基,纤维素酶降解机制及纤维素酶分子结构与功能研究进展,自然科学进展,2003,13(1):21~29
[57]Tomme P, Warren R A, Gillkes N, Cellulose hydrolysis by bacteria and fungi, Adv Microb Physiol, 1995, 37: 1~8
[58]Delong. E A, Method of rendering lignin separable from cellulose and hemicellulose in lignocellulosic material and the product so produced, Canadian Patent No 1096374, 1981, 2
[59]Diebold J P, Bridgwater A V, Overview of fast pyrolysis of biomass for the production of liguid fuels, Developments in Thermochemical Biomass Conversion, London: Blackie Academic and Professional, 1997, 5
[60]Lewis N G, Yamamoto E, lignin: occurrence, biogenesis and biodegradation, Annu Rev Plant Physiol and Plant Mol Biol, 1990, 41: 455~496
[61]Xiao Z H, Gao P J, Qu Y B, et al, Cellulose-binding domain of endoglucanaseⅢfrom Trichoderma reesei disrupting the structure of cellulose, Biotech Letter, 2001, 23: 711~715
[62]Teeri T T, Koivula A, Linder M, et al, Trichoderma reesei cellobiohydrolase: Why so efficient on crystalline cellulose? Biochemical Society Transaction, 1998, 26: 160~164
[63] W. Dan Reece, Mark Holtzapple, Concepts in Engineering, 2004, 310~365