酶促乌头生物碱提取工艺的研究
|
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。在天然产物有效成分提取过程中,活性成分通常被植物纤维所包裹,植物细胞壁是提取有效成分的主要屏障,使有效成分不能很好的与反应体系接触,阻碍细胞中有效成分的溶出。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,可加速有效成分的溶出,提高了目标产物的提取率,降低了成本。
目前,国内外对纤维素酶辅助提取乌头生物碱的研究没有文献报道,本研究初步探索纤维素酶催化转化乌头生物碱的反应条件和反应机制。
本研究以乌头碱和乌头总碱的提取率为指标,先通过单因素实验考察纤维素酶催化对乌头生物碱提取率的影响,确定纤维素酶催化反应的温度、pH值、纤维素酶用量、作用时间等因素的范围;再通过正交试验确定纤维素酶催化转化乌头生物碱的最佳条件。并与现有的乌头生物碱提取方法:酸水提取法、大孔吸附树脂提取法、半仿生提取法、乙醇提取法作了对比实验。
实验结果表明: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:45℃,pH值:4.5,酶用量:8mg/g(1 g药材中加入0.08 g纤维素酶),作用时间:4.0 h,乌头碱和乌头总碱的提取率分别为0.002447%和0.244410%。与酸水提取法比较,酶法提取的乌头碱、乌头总碱的提取率分别提高84.4%和59.3%;与大孔吸附树脂提取比较,酶法提取的乌头碱、乌头总碱分别提高61.1%和41.4%;与半仿生提取比较,酶法提取的乌头碱、乌头总碱提取率分别提高41.0%和29.1%;与乙醇提取比较,酶法提取分别提高了30.9%和21.8%
与酸水提取法、大孔吸附树脂提取法、半仿生提取法、乙醇提取法相比,纤维素酶催化转化得到的乌头碱和乌头总碱提取率显著增加,说明采用纤维素酶对川乌药材进行预处理,酶催化破坏了植物细胞壁上的β-D-葡萄糖苷键,使包裹生物碱的纤维素大量分解,进而破坏细胞壁,增加细胞壁的通透性,减小传质阻力,从而有效提高了生物碱成分的溶出。纤维素酶催化转化法对乌头生物碱的提取率的提高有明显的促进作用,且纤维素酶催化具有条件温和、作用时间短等优点,可在实际生产中推广应用。
Owing to the deletion of petrochemical resources and the energy shortage, environment and society crisis were acceleratly deteriorating. As a promising strategy for developing green chemistry and green chemical engineering, biocatalysis technology will exert significant influence on medicine, agriculture, food, life science and so on. Biocatalysis is a new technology for extracting and separating active constituents from natural plants via enzyme catalysis. The plant cell walls usually hinder the extraction of effective constituents. If proper enzymes are employed, the cell walls will be decomposed, the extraction rate will be improved and the cost thus be reduced.
Up to now, no studies of cellulose-assisted extraction of aconitum alkaloids have been reported both at home and broad. This preliminarily study was to explore the reaction condition and underlying mechanism of aconitum alkaloids catalyzed and transformed by cellulase.
In this present study, the extraction rate of aconitine and aconitum alkaloids were regarded as indexes. The impact of aconitum alkaloids rate through enzyme-catalyzed reaction was studied by single factor experiment to determine the scope of the temperature, pH, cellulase dosage and reaction time. The optimum conditions of enzymatic extraction have been obtained by orthogonal test and compared with acid extraction, macroporous adsorbing resins extraction, semi-bionic extraction and alcohol extraction. Cellulase was recruited to pretreat the prepared Radix Aconiti, subsequently the aconitum alkaloids were extracted by alcohol. The optimum catalysis conditions of cellulase and its effect on the extraction rate of aconitum alkaloids were studied. The extraction rate and purity by cellulase catalysis synergism method was compared with others. Meanwhile, the effect of cellulase catalysis on conventional alcohol extraction and the mechanism of cellulase catalysis were also studied.
The optimum conditions that cellulase catalyzed and transformed aconitum alkaloids were at 45℃, pH 4.5, with cellulase dosage of 0.8mg/g(0.01g cellulase in 1 g material) and 4 h reaction time of cellulase. The extraction rates of aconitine and aconitum alkaloids of semi-bionic extraction were 0.002 447%, 0.244 410% respectively. Compared with the acid extraction, the extraction rates of aconitine and aconitum alkaloids of cellulose enzyme extraction increased by 84.4%, 59.3% respectively. Compared with the macroporous adsorbing resins extraction, the extraction rates of aconitine and aconitum alkaloids increased by 61.1%, 41.4%, respectively. Compared with the semi-bionic extraction, the extraction rates of aconitine and aconitum alkaloids increased by 41.0%, 29.1%, respectively. Compared with the alcohol extraction, the extraction rates of aconitine and aconitum alkaloids increased by30.9%, 21.8%, respectively. Compared with acid extraction, macroporous adsorbing resins extraction, semi-bionic extraction, alcohol extraction, the cellulose enzyme extraction rates of aconitine and aconitum alkaloids were much higher. It indicated that the enzyme catalysis destroyβ-D-glucoside bond of the cell wall and decomposed cellulose and minished mass transfer resistance. It made aconitum alkaloids be taken out easier. The yield rate of aconitum alkaloids improved obviously after the prepared radix aconiti was pretreated by cellulase. Cellulase catalysis method significantly improve the extration rate of aconitum alkaloids, which has advantages of mild conditions, short time and so on. This study suggested that the cellulase catalysis technology is promising in extracting active ingredients from natural plants and also feasible in practical production.
目前,国内外对纤维素酶辅助提取乌头生物碱的研究没有文献报道,本研究初步探索纤维素酶催化转化乌头生物碱的反应条件和反应机制。
本研究以乌头碱和乌头总碱的提取率为指标,先通过单因素实验考察纤维素酶催化对乌头生物碱提取率的影响,确定纤维素酶催化反应的温度、pH值、纤维素酶用量、作用时间等因素的范围;再通过正交试验确定纤维素酶催化转化乌头生物碱的最佳条件。并与现有的乌头生物碱提取方法:酸水提取法、大孔吸附树脂提取法、半仿生提取法、乙醇提取法作了对比实验。
实验结果表明: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:45℃,pH值:4.5,酶用量:8mg/g(1 g药材中加入0.08 g纤维素酶),作用时间:4.0 h,乌头碱和乌头总碱的提取率分别为0.002447%和0.244410%。与酸水提取法比较,酶法提取的乌头碱、乌头总碱的提取率分别提高84.4%和59.3%;与大孔吸附树脂提取比较,酶法提取的乌头碱、乌头总碱分别提高61.1%和41.4%;与半仿生提取比较,酶法提取的乌头碱、乌头总碱提取率分别提高41.0%和29.1%;与乙醇提取比较,酶法提取分别提高了30.9%和21.8%
与酸水提取法、大孔吸附树脂提取法、半仿生提取法、乙醇提取法相比,纤维素酶催化转化得到的乌头碱和乌头总碱提取率显著增加,说明采用纤维素酶对川乌药材进行预处理,酶催化破坏了植物细胞壁上的β-D-葡萄糖苷键,使包裹生物碱的纤维素大量分解,进而破坏细胞壁,增加细胞壁的通透性,减小传质阻力,从而有效提高了生物碱成分的溶出。纤维素酶催化转化法对乌头生物碱的提取率的提高有明显的促进作用,且纤维素酶催化具有条件温和、作用时间短等优点,可在实际生产中推广应用。
Owing to the deletion of petrochemical resources and the energy shortage, environment and society crisis were acceleratly deteriorating. As a promising strategy for developing green chemistry and green chemical engineering, biocatalysis technology will exert significant influence on medicine, agriculture, food, life science and so on. Biocatalysis is a new technology for extracting and separating active constituents from natural plants via enzyme catalysis. The plant cell walls usually hinder the extraction of effective constituents. If proper enzymes are employed, the cell walls will be decomposed, the extraction rate will be improved and the cost thus be reduced.
Up to now, no studies of cellulose-assisted extraction of aconitum alkaloids have been reported both at home and broad. This preliminarily study was to explore the reaction condition and underlying mechanism of aconitum alkaloids catalyzed and transformed by cellulase.
In this present study, the extraction rate of aconitine and aconitum alkaloids were regarded as indexes. The impact of aconitum alkaloids rate through enzyme-catalyzed reaction was studied by single factor experiment to determine the scope of the temperature, pH, cellulase dosage and reaction time. The optimum conditions of enzymatic extraction have been obtained by orthogonal test and compared with acid extraction, macroporous adsorbing resins extraction, semi-bionic extraction and alcohol extraction. Cellulase was recruited to pretreat the prepared Radix Aconiti, subsequently the aconitum alkaloids were extracted by alcohol. The optimum catalysis conditions of cellulase and its effect on the extraction rate of aconitum alkaloids were studied. The extraction rate and purity by cellulase catalysis synergism method was compared with others. Meanwhile, the effect of cellulase catalysis on conventional alcohol extraction and the mechanism of cellulase catalysis were also studied.
The optimum conditions that cellulase catalyzed and transformed aconitum alkaloids were at 45℃, pH 4.5, with cellulase dosage of 0.8mg/g(0.01g cellulase in 1 g material) and 4 h reaction time of cellulase. The extraction rates of aconitine and aconitum alkaloids of semi-bionic extraction were 0.002 447%, 0.244 410% respectively. Compared with the acid extraction, the extraction rates of aconitine and aconitum alkaloids of cellulose enzyme extraction increased by 84.4%, 59.3% respectively. Compared with the macroporous adsorbing resins extraction, the extraction rates of aconitine and aconitum alkaloids increased by 61.1%, 41.4%, respectively. Compared with the semi-bionic extraction, the extraction rates of aconitine and aconitum alkaloids increased by 41.0%, 29.1%, respectively. Compared with the alcohol extraction, the extraction rates of aconitine and aconitum alkaloids increased by30.9%, 21.8%, respectively. Compared with acid extraction, macroporous adsorbing resins extraction, semi-bionic extraction, alcohol extraction, the cellulose enzyme extraction rates of aconitine and aconitum alkaloids were much higher. It indicated that the enzyme catalysis destroyβ-D-glucoside bond of the cell wall and decomposed cellulose and minished mass transfer resistance. It made aconitum alkaloids be taken out easier. The yield rate of aconitum alkaloids improved obviously after the prepared radix aconiti was pretreated by cellulase. Cellulase catalysis method significantly improve the extration rate of aconitum alkaloids, which has advantages of mild conditions, short time and so on. This study suggested that the cellulase catalysis technology is promising in extracting active ingredients from natural plants and also feasible in practical production.
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[J].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)[J].白求恩医科大学学报,1996,22(5):521-523
[3]崔志高.误服含乌头碱药酒中毒16例[J].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[J].中医药学报.1992,12(5):39-41
[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[J].南京中医学院学报,1984,16(30):49-51
[6]陆履雯.三七伤药片中生物碱含量测定[J].中成药研究,1983,9(1):19-20
[7]颜根宝,胥法领.小活络丸中乌头总碱的分析[J].中成药,1989,11(3):14-15
[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[J].中草药,1987,18(3):13-15
[9]李云霞,孙照,郭艳玲.酸性染料比色法测定川乌、草乌中总乌头生物碱的含量[J].中成药,2000,22(9):662-663
[10]龙沛霞.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[J].中草药,1996,27(9):531-532
[11]赵冬霞.薄层扫描法测定通痹止痛胶囊中乌头碱的含量[J].云南中医学院学报,2003,23(2):19-21
[12]瞿发林,丁青龙,章杰兵.双波长薄层扫描法测定痛得安胶囊中新乌头碱的含量[J].中国现代应用药学杂志,2000,17(5):390-391
[13]赵永芳.生物化学技术原理及应用[M].北京:科学技术出版社,2002
[14]张春水.乌头类生物碱的检测现状[J].刑事技术,1998,(3):11-12
[15]张国红,由会玲,刘云肖.乌头生物碱含量测定方法现况[J].河北中医医学报,2000,15(1):45-47
[16]陈昕.乌头类中药的研究进展[J].时珍国医国药,2002,13(9):758-759
[17]朱正义,王白露,李君实.TLC-HPLC法测定小活络丸内中乌头碱、次乌头碱和乌头碱的含量[J].药物分析杂志,1996,16(3):154-157
[18]翁小刚,聂淑琴,黄路琦.HPLC测“半萎贝蔹攻乌”中乌头与其他诸药合煎前后次乌头碱的含量变化[J].中国药学杂志,2004,39(1):57-59
[19]王勇,刘志强,宋凤瑞等.白山草乌中二萜生物碱的电喷雾串联质谱分析[J].质谱学报,2002,23(3):160-163
[20]王勇,刘淑莹.乌头碱类生物碱的质谱研究进展[J].质谱报,2002,23(2):112-119
[21]王兆基,何绍基,徐树棋.液相色谱/质谱/质谱联用法测定乌头属药材及中成药中乌头类生物碱含量[J].分析化学,2001,29(4):391-395
[22]Wada Koji.Natural.Medcines,1997,51(1):39-39
[23]张珊亭,张兆旺,张秀梅.思维方式的转换与中药“半仿生提取法”[J].中国中药杂志,1999,22(9):542
[24]孙秀梅.用均匀设计优选川乌的“半仿生提取法”工艺条件[J].中药材,1999,22(12):649-651
[25]张兆旺.川乌两种方法提取液的成分比较[J].中成药,1999,21(1):5-7
[26]陈惠洁,王晓方,陈俊芳,等.中药“半仿生提取”的进一步仿生化构想[J].基层中药杂志,1998,12(8):18-20
[27]梁宝钻.亚东乌头总生物碱的超临界CO2萃取及含量测定[J].中药材,2002,25(5):332-333
[28]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[J].中成药,2002,22(8):535-538
[29]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[J].复旦学报(医学版),2003,30(3):267-269
[30]WANG Z,SHI Z Q,WANG R F.Studies on sorption property forwards caffeine and theophylline by phenolic resin[J].Chemical Research in Chinese Universities,2003,24(10):1986-1901
[31]卢定强,韦萍,周华,等.生物催化与生物转化的研究进展[J].化工进展,2004,23(6):585-589
[32]孙志浩.生物催化工艺学[M].北京:化学工业出版社,2005.1-10
[33]欧阳平凯,卢定强.资源生态化利用中的生物加工过程[J].生物加工过程,2003,1(1):1-6
[34]帕特尔R N.立体选择性生物化学[M].北京:化学工业出版社,2004.122-128
[35]Bommarius A S,Riebel B R,Biocatalysis[M].USA,Weinheim..ILEY-VCH,2004.478-486
[36]Helmut Uhlig.Industrial Enzymes and Their Applications[M].New York,1998.89-91
[37]Olga K,Frances H A.Directed evolution of enzyme catalysts[J].Trends in Biotechnology,1997,15(12):523-530
[38]杜晨宇,李春,曹竹安,等.工业生物技术的核心--生物催化[J].生物工程进展,2002,22(1):9-14
[39]何坤荣.生物催化技术前景广阔[J].精细与专用化学品,2002,10(22):13-14
[40]Haiyan T,Virginia W C.Milestones in directed enzyme evolution[J].Current Opinion in Chemical Biology,2002,6(6):858-864
[41]居乃琥.酶工程研究和酶工程产业的新进展[J].食品与发酵工业,2000,26(13):54-62
[42]Bertus B.Extremophiles as a source for novel enzymes[J].Current Opinion in Microbiology,2003,6(3):213-218
[43]马晓建,白净,任珂,等.酶工程研究的新进展[J].化工进展,2003,22(8):813-817
[44]Straathof A J J,Panke S,Schmid A,The production of fine chemicals by biotransformations[J].Current Opinion in Biotechnology,2002,13(6):548-556
[45]赫尔曼·舌尔.阳光经济:生态的现代战略[M].北京:三联书店,1999.168-176
[46]Frankel,E.N,Waterhouse,A.L,and Kinsella,J.E.nhibition of human LDL oxidation by resverstrol[J].Lancet,1993,341:1103-1104
[47]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[J].沈阳药学院学报,1994,11(4):289-293
[48]林桂云.生物技术与中药现代化[J].成都大学学报,2001,20(2):34-36
[49]胡之璧,刘涤.生物技术在中药现代化中的地位和作用[J].世界科学技术--中药现代化,2000,2(5):23-26
[50]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[J].大连轻工业学院学报,2002,21(2):112-115
[51]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[J].中草药,2006,37(5):688-691
[52]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[J].中草药,2007,38(4):527-530
[53]陶卫平.热稳定性酶[J].生物学通报,1998,33(1):20-21
[54]Wang Y B,Nagata S.Participation of ions and solutes on the thermostability of α-amylase[J].Chinese Journal of Biotechnology,2004,20(1):104-110
[55]宋小妹,唐志书.中药化学成分提取分离与制备[M].北京:人民卫生出版社,2004.133-134
[56]孙文基.天然药物成分提取分离与制备(第三版)[M].北京:中国医药科技出版社,2006,3-4
[57]Nidetzky B,Steiner S,Hayn M,et al.Cellulose hydrolysis by the cellulases from Trichoderma Reese:a new model for synergistic interaction[J].Biochem J,1994,15(298):705-710
[58]Beguin P,Aubert J P.The biological degradation of cellulose[J].FEMS Microbiol Rev,1994,13:25-58
[59]Lenting H B M,Warmoeskerken M M C G.Mechanism of interaction between cellulase action and applied shear force and hypothesis[J].Journal of Biotechnology,2001,89(2):217-226
[60]Wang W,Gao P J.Function and mechanism of a low-molecular-weight peptide produced by Gloeophyllum trabeum[J].Biotechnol,2003,101:119-130
[61]Lee I,Evans B R,Woodward J.The mechanism of cellulase on cotton fibers:evidence from atomic force microscopy[J].Ultramicroscopy,2000,82:213-221
[62]Samejima M,Eriksson K E.Comparation of the catalytic properties of cellobiose:quinone oxidoreductase and cellobiose oxidase from phanerochaete chrysosporium[J].Eur J Biochern,1992,207:103-107
[63]Chang V S,Holtzapple M T.Fundamental factors affecting biomass enzymatic reactivity[J].Applied Biochem and Biotechnol,2000,84:5-37
[64]陈洪章.纤维素生物技术[M].北京:化学工业出版社,2005.40-61
[65]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[J].自然科学进展,2003,13(1):21-29
[66]Tomme P,Warren R A,NGillkes N.Cellulose hydrolysis by bacteria and fungi[J].Adv Microb Physiol,1995,37:1-8
[67]Delong.E A.Method of rendering lignin separable from cellulose and hemicellulose in lignocellulosic material and the product so produced[P].Canadian Patent No 1096374,1981-2
[68]Diebold J P,Bridgwater A V.Overview of fast pyrolysis of biomass for the production of liguid fuels,Developments in Thermochemical Biomass Conversion[M].London:Blackie Academic and Professional,1997.5
[69]Lewis N G,Yamamoto E,lignin:occurrence,biogenesis and biodegradation[J].Annu Rev Plant Physiol and Plant Mol Biol,1990,41:455-496
[70]Xiao Z H,Gao P J,Qu Y B,et al.Cellulose-binding domain of endoglucanaseⅢ from Trichoderma reesei disrupting the structure of cellulose[J].Biotech Letter,2001,23:711-715
[71]Teeri T T,Koivula A,Linder M,et al.Trichoderma reesei cellobiohydrolase:Why so efficient on crystalline cellulose[J].Biochemical Society Transaction,1998,26:160-164
[72]W.Dan Reece,Mark Holtzapple.Concepts in Engineering[M].McGraw-Hill:New York,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)[J].白求恩医科大学学报,1996,22(5):521-523
[3]崔志高.误服含乌头碱药酒中毒16例[J].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[J].中医药学报.1992,12(5):39-41
[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[J].南京中医学院学报,1984,16(30):49-51
[6]陆履雯.三七伤药片中生物碱含量测定[J].中成药研究,1983,9(1):19-20
[7]颜根宝,胥法领.小活络丸中乌头总碱的分析[J].中成药,1989,11(3):14-15
[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[J].中草药,1987,18(3):13-15
[9]李云霞,孙照,郭艳玲.酸性染料比色法测定川乌、草乌中总乌头生物碱的含量[J].中成药,2000,22(9):662-663
[10]龙沛霞.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[J].中草药,1996,27(9):531-532
[11]赵冬霞.薄层扫描法测定通痹止痛胶囊中乌头碱的含量[J].云南中医学院学报,2003,23(2):19-21
[12]瞿发林,丁青龙,章杰兵.双波长薄层扫描法测定痛得安胶囊中新乌头碱的含量[J].中国现代应用药学杂志,2000,17(5):390-391
[13]赵永芳.生物化学技术原理及应用[M].北京:科学技术出版社,2002
[14]张春水.乌头类生物碱的检测现状[J].刑事技术,1998,(3):11-12
[15]张国红,由会玲,刘云肖.乌头生物碱含量测定方法现况[J].河北中医医学报,2000,15(1):45-47
[16]陈昕.乌头类中药的研究进展[J].时珍国医国药,2002,13(9):758-759
[17]朱正义,王白露,李君实.TLC-HPLC法测定小活络丸内中乌头碱、次乌头碱和乌头碱的含量[J].药物分析杂志,1996,16(3):154-157
[18]翁小刚,聂淑琴,黄路琦.HPLC测“半萎贝蔹攻乌”中乌头与其他诸药合煎前后次乌头碱的含量变化[J].中国药学杂志,2004,39(1):57-59
[19]王勇,刘志强,宋凤瑞等.白山草乌中二萜生物碱的电喷雾串联质谱分析[J].质谱学报,2002,23(3):160-163
[20]王勇,刘淑莹.乌头碱类生物碱的质谱研究进展[J].质谱报,2002,23(2):112-119
[21]王兆基,何绍基,徐树棋.液相色谱/质谱/质谱联用法测定乌头属药材及中成药中乌头类生物碱含量[J].分析化学,2001,29(4):391-395
[22]Wada Koji.Natural.Medcines,1997,51(1):39-39
[23]张珊亭,张兆旺,张秀梅.思维方式的转换与中药“半仿生提取法”[J].中国中药杂志,1999,22(9):542
[24]孙秀梅.用均匀设计优选川乌的“半仿生提取法”工艺条件[J].中药材,1999,22(12):649-651
[25]张兆旺.川乌两种方法提取液的成分比较[J].中成药,1999,21(1):5-7
[26]陈惠洁,王晓方,陈俊芳,等.中药“半仿生提取”的进一步仿生化构想[J].基层中药杂志,1998,12(8):18-20
[27]梁宝钻.亚东乌头总生物碱的超临界CO2萃取及含量测定[J].中药材,2002,25(5):332-333
[28]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[J].中成药,2002,22(8):535-538
[29]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[J].复旦学报(医学版),2003,30(3):267-269
[30]WANG Z,SHI Z Q,WANG R F.Studies on sorption property forwards caffeine and theophylline by phenolic resin[J].Chemical Research in Chinese Universities,2003,24(10):1986-1901
[31]卢定强,韦萍,周华,等.生物催化与生物转化的研究进展[J].化工进展,2004,23(6):585-589
[32]孙志浩.生物催化工艺学[M].北京:化学工业出版社,2005.1-10
[33]欧阳平凯,卢定强.资源生态化利用中的生物加工过程[J].生物加工过程,2003,1(1):1-6
[34]帕特尔R N.立体选择性生物化学[M].北京:化学工业出版社,2004.122-128
[35]Bommarius A S,Riebel B R,Biocatalysis[M].USA,Weinheim..ILEY-VCH,2004.478-486
[36]Helmut Uhlig.Industrial Enzymes and Their Applications[M].New York,1998.89-91
[37]Olga K,Frances H A.Directed evolution of enzyme catalysts[J].Trends in Biotechnology,1997,15(12):523-530
[38]杜晨宇,李春,曹竹安,等.工业生物技术的核心--生物催化[J].生物工程进展,2002,22(1):9-14
[39]何坤荣.生物催化技术前景广阔[J].精细与专用化学品,2002,10(22):13-14
[40]Haiyan T,Virginia W C.Milestones in directed enzyme evolution[J].Current Opinion in Chemical Biology,2002,6(6):858-864
[41]居乃琥.酶工程研究和酶工程产业的新进展[J].食品与发酵工业,2000,26(13):54-62
[42]Bertus B.Extremophiles as a source for novel enzymes[J].Current Opinion in Microbiology,2003,6(3):213-218
[43]马晓建,白净,任珂,等.酶工程研究的新进展[J].化工进展,2003,22(8):813-817
[44]Straathof A J J,Panke S,Schmid A,The production of fine chemicals by biotransformations[J].Current Opinion in Biotechnology,2002,13(6):548-556
[45]赫尔曼·舌尔.阳光经济:生态的现代战略[M].北京:三联书店,1999.168-176
[46]Frankel,E.N,Waterhouse,A.L,and Kinsella,J.E.nhibition of human LDL oxidation by resverstrol[J].Lancet,1993,341:1103-1104
[47]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[J].沈阳药学院学报,1994,11(4):289-293
[48]林桂云.生物技术与中药现代化[J].成都大学学报,2001,20(2):34-36
[49]胡之璧,刘涤.生物技术在中药现代化中的地位和作用[J].世界科学技术--中药现代化,2000,2(5):23-26
[50]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[J].大连轻工业学院学报,2002,21(2):112-115
[51]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[J].中草药,2006,37(5):688-691
[52]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[J].中草药,2007,38(4):527-530
[53]陶卫平.热稳定性酶[J].生物学通报,1998,33(1):20-21
[54]Wang Y B,Nagata S.Participation of ions and solutes on the thermostability of α-amylase[J].Chinese Journal of Biotechnology,2004,20(1):104-110
[55]宋小妹,唐志书.中药化学成分提取分离与制备[M].北京:人民卫生出版社,2004.133-134
[56]孙文基.天然药物成分提取分离与制备(第三版)[M].北京:中国医药科技出版社,2006,3-4
[57]Nidetzky B,Steiner S,Hayn M,et al.Cellulose hydrolysis by the cellulases from Trichoderma Reese:a new model for synergistic interaction[J].Biochem J,1994,15(298):705-710
[58]Beguin P,Aubert J P.The biological degradation of cellulose[J].FEMS Microbiol Rev,1994,13:25-58
[59]Lenting H B M,Warmoeskerken M M C G.Mechanism of interaction between cellulase action and applied shear force and hypothesis[J].Journal of Biotechnology,2001,89(2):217-226
[60]Wang W,Gao P J.Function and mechanism of a low-molecular-weight peptide produced by Gloeophyllum trabeum[J].Biotechnol,2003,101:119-130
[61]Lee I,Evans B R,Woodward J.The mechanism of cellulase on cotton fibers:evidence from atomic force microscopy[J].Ultramicroscopy,2000,82:213-221
[62]Samejima M,Eriksson K E.Comparation of the catalytic properties of cellobiose:quinone oxidoreductase and cellobiose oxidase from phanerochaete chrysosporium[J].Eur J Biochern,1992,207:103-107
[63]Chang V S,Holtzapple M T.Fundamental factors affecting biomass enzymatic reactivity[J].Applied Biochem and Biotechnol,2000,84:5-37
[64]陈洪章.纤维素生物技术[M].北京:化学工业出版社,2005.40-61
[65]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[J].自然科学进展,2003,13(1):21-29
[66]Tomme P,Warren R A,NGillkes N.Cellulose hydrolysis by bacteria and fungi[J].Adv Microb Physiol,1995,37:1-8
[67]Delong.E A.Method of rendering lignin separable from cellulose and hemicellulose in lignocellulosic material and the product so produced[P].Canadian Patent No 1096374,1981-2
[68]Diebold J P,Bridgwater A V.Overview of fast pyrolysis of biomass for the production of liguid fuels,Developments in Thermochemical Biomass Conversion[M].London:Blackie Academic and Professional,1997.5
[69]Lewis N G,Yamamoto E,lignin:occurrence,biogenesis and biodegradation[J].Annu Rev Plant Physiol and Plant Mol Biol,1990,41:455-496
[70]Xiao Z H,Gao P J,Qu Y B,et al.Cellulose-binding domain of endoglucanaseⅢ from Trichoderma reesei disrupting the structure of cellulose[J].Biotech Letter,2001,23:711-715
[71]Teeri T T,Koivula A,Linder M,et al.Trichoderma reesei cellobiohydrolase:Why so efficient on crystalline cellulose[J].Biochemical Society Transaction,1998,26:160-164
[72]W.Dan Reece,Mark Holtzapple.Concepts in Engineering[M].McGraw-Hill:New York,2004.310-365