摘要
在木质纤维素生产燃料乙醇的研究中,选用更加廉价、易收集处理的原料,提高预处理物料的酶解率和得糖率,是极为有效降低生产成本的途径之一。纤维素酶在生物质转化过程中的重要作用与地位毋庸置疑,设法提高纤维素酶的酶活也是降低生产成本的最重要途径之一。因此,在本次研究课题中,拟选用葵花籽壳为原材料,纤维素酶为研究对象,以磁性Fe_3O_4与游离纤维素酶的生物磁效应机理为指导,探索磁性Fe_3O_4在纤维素酶酶水解中的应用,同时在提高纤维素酶的活力方面进行主要研究,从而达到降低纤维素酶生产成本的目的。
本论文的主要研究内容是将磁化后的Fe_3O_4微粒作为一种添加剂,考察其在葵花籽壳的酶解过程中,对纤维素酶的酶活、还原糖浓度、纤维素的转化、水解液的表面张力和pH值的影响和作用。此外,还将以磁性Fe_3O_4微粒作为载体,通过明胶包埋法,吸附固定化纤维素酶,研究磁性固定化酶的酶水解性质,以及添加表面活性剂后对磁性固定化纤维素酶酶活的影响,并初步分析和阐述产生这些变化的原因。
研究结果表明,磁性Fe_3O_4添加量为0.5 g/L~2.0 g/L时,对游离纤维素酶酶活的提高﹑还原糖浓度的增加和纤维素的转化率在48 h后表现出较明显的促进作用。磁性Fe_3O_4添加量为2 g/L时,纤维素酶60 h酶活最高可提高25.9%。添加量为1.5 g/L时,还原糖浓度从6.950 mg/mL提高到8.775 mg/mL;同时,其纤维素的最高转化率达60.531%,明显高于不添加Fe_3O_4样品的纤维素转化率47.932%。此外,分次添加磁性Fe_3O_4的酶活稳定性要优于一次添加。在相同水解条件下,与游离纤维素酶相比,磁性固定化纤维素酶能将还原糖的最高产量从8.775 mg/mL提高到9.162 mg/mL,相比提高4.4%。特别是添加表面活性剂后,还原糖的最高值产量可达9.975 mg/mL,相比提高13.68%。
While the technology of transforming lignocellulose into ethanol is studied, choosing low-cost, easily collective and processing raw materials to improve hydrolysate rate and sugar yields, is one of the most effective ways to reduce production costs. In another way, the status of enzymes in the technology of transforming cellulase into ethanol was proved beyond the shadow of a doubt. The technology of improving the cellulase activity also played an important part in the cutting off the production costs. In the paper, refering the sunflower seed hull as the raw materials, the enzymes as the object of study, the magnetic mechanisms of cellulase and cellulase immobilized by Fe_3O_4, the improvement of the cellulase activity were investigated to cut off the cellulase produxtion costs, and to explore the utilization of magnetic Fe_3O_4 particles in the enzymatic hydrolysis.
The chief topic of the paper is adding magnetic Fe_3O_4 particles on cellulase in the enzymatic hydrolysis of sunflower seedhull. In the process of enzymatic hydrolysis of sunflower seed hull, the variations of cellulase activity,reducing sugar concentration, cellulose conversion, the analysis of pH and surface tension of hydrolysate were evaluated. In additon, the utilization of magnetic Fe_3O_4 particles as the carrier of cellulase Immobilized by Gelatin adsorption, was used to study the enzymatic hydrolysis quality of cellulase Immobilized by Magnetic Gelatin, to analysis the variations and expatiat on the reasons.
The results indicated that after adding magnetic Fe_3O_4, the cellulase activity, reducing sugar concentration and conversion of cellulose had an increased between the 0.5 g/L and 2.0 g/L cases after 48 h. When the additive amount of magnetic Fe_3O_4 was 2 g/L, the cellulase activity at 60 h was improved significantly by 25.9%. It was found that the concentration of reducing sugar was increased from 6.950 mg/mL to 8.775 mg/mL with magnetic Fe_3O_4 1.5 g/L. Simultaneously,compared with the blank, which the conversion of cellulose was 47.932%, the maximum cellulose conversion of samples with adding magnetic Fe_3O_4 was 60.531%. Besides, the stability of cellulase activity adding in times was better than in one time. It was approved that the cellulase Immobilized by Magnetic Gelatin adsorption had increased the maximum reducing sugar from 8.775 mg/mL to 9.162 mg/mL., which was 4.4% higher compared with the free cellulose under similar hydrolysis conditions.After adding biosurfactant, the maximum reducing sugar was 9.975 mg/mL, which was 13.68% higher than the blank especially.
引文
[1] Lynd L R , Wermer P J, Vanzy W H, et al. Microbial cellulose utilization:fundamentals and Biotechnology. Microbiol Mol Biol Rev, 2002, 66(3): 506-577
[2]曲音波,高培基.造纸厂废物发酵生产纤维素酶、酒精和酵母综合工艺的研究进展.食品与发酵工业, 1993, 6(3): 62
[3]曲音波,高培基,赵昕,等.纤维素类废弃物生物转化技术研究进展.纤维素科学与技术, 1997, 5(2): 1-9
[4] Wheals A E, Basso L C, Alves D M G, et al.Feul ethanol after 25 years. Trend in Biotechnology, 1999, 17(3/4): 482
[5] Mielenz J R. Ethanol production from biomass:technology and commercialization status. Curr Opin Microbiol, 2001, 4(3): 324
[6]陈洪章.纤维素生物技术.北京:化学工业出版社, 2004:8-69.
[7] Sun Y, Cheng J. Hydrolysis of linfnocellulosic materials for ethanol production: a review. Bioreseource Technology, 2002, 83(10): 1
[8] Lynd L R, Wyman C E, Gerngross T U, Biocommodity engineering. Biotechnol Prog, 1999, 15(7): 777
[9]高培基,许平.资源环境微生物技术.北京:化学工业出版社, 2004:20-117
[10] Keller F A. Integrated bioprocess development for bioethanol production. In: Wyman C E (ed) Handbook on bioethanol: production and utilization. Bristol:Taylor & Francis, 1996: 351-379
[11] M Galbe, G. Zacchi. A review of the production of ethanol from softwood. Appl Microbiol Biotechnol, 2002, 59(1): 618-628
[12] Katzen R A. 60 year journey through bioconversion of biomass to ethanol. In: Ramos L P (ed) Proceedings of the Fifth Brazilian symposium on the chemistry of lignins and other wood components, Curitiba& Brazil:P R, 1997(1):334-339
[13] Jones J L,Semrau K T. Wood hydrolysis for ethanol production-previous experience and the economics of selected processes. Biomass. 1984, 5(2): 109-135
[14] Larsson S, Palmqvist E, Hahn-H?gerdal B, et al. The generation of fermentation inhibitors during dilute acid hydrolysis of softwood. Enzyme Microb Technol. 1999, 24(13): 151-159
[15] Nguyen Q A, Tucker M P, Keller F A, et al. Dilute acid hydrolysis of softwoods. ApplBiochem Biotechnol. 1999, 77(79): 133-142
[16] O Boyle A, Good D, Potts D,et al. The Swedish Ethanol Foundation (Bioalcohol Fuel Foundation), Sweden: The Cash process, 1999:33-66
[17] Wyman C E.Production of low cost sugars from biomass: progress, opportunities, and challenges. In:Overend R P,Cornet E (eds) Biomass– a growth opportunity in green energy and valueadded products.Proceedings of the 4th Biomass Conference of the Americas, Oxford :vol 1 Pergamon, 1999: 867–872
[18] Duff.S J B, Murray.W D.Bioconversion of forest products industry waste cellulosics to fuel ethanol a review. Bioresour Technol. 1996, 55(1), 1–33
[19] Saddler J N,Gregg D J.Ethanol production from forest product wastes. In: Bruce A, Palfreyman J W (eds) Forest products biotechnology. London:Taylor Francis, 1998:56
[20] Tengborg C, Stenberg K, Galbe M, et al. Comparison of SO2 and H2SO4 impregnation of softwood prior to steam pretreatment on ethanol production. Appl Biochem Biotechnol. 1998, 70(72): 3–15
[21] Converse.A O, Matsuno R, Tanaka M, et al. A model for enzyme adsorption and hydrolysis of microcrystalline cellulose with slow deactivation of the adsorbed enzyme. Biotechnol Bioeng. 1988, 32(1), 38–45
[22] Prosen E M, Radlein D, Piskorz J, et al. Microbial Utilization of Levoglucosan in Wood Pyrolysate as a Carbon and Energy Source. Biotech. Bioeng, 1993, 42(1): 538-541
[23] Bonn G..Analytical Determination of 1,6-anhydro-b-D-glucopyranose and Kinetic Studies under Hydrothermal Conditions. Carbohyd. Chem, 1985, 4(3): 405-419
[24] Bozell J J. Chemicals and Materials from Renewable Resources. Oxford:Oxford University Press, 2001: 122-132
[25]陈坚.环境生物技术.北京:中国轻工业出版社, 1999:242
[26] Lynd L R. Annual Review of Energy and the Environment, 1996, 27(21): 403-465
[27]王健,袁永俊,张弛松.纤维素发酵产酒精研究进展.中国酿造, 2006, 4(6): 9-13
[28]张树政.酶制剂工业.北京:科学出版社, 1984:595
[29]商丹,张朝平,等.纳米磁性四氧化三铁的制备方法比较及应用研究.贵州大学,硕士学位论文, 2006, 5
[30]尚荣,徐芬,韦桂哗.磁性皮内针内关穴防治室上性心率失常38例.中医研究, 2001, 14(4): 62
[31]黄飚,梁长虹,曾辉,等.超顺磁氧化铁诊断肝脏小病灶的临床应用价值初探.中国放射学杂志, 2001, 35(12): 929
[32]徐光炜,林言蔑,陈道达.提高进展期胃癌疗效的研究.医学研究通讯, 2002, 31(3): 11
[33]韩晓红,石远凯,张伟.免疫磁性细胞分选技术净化乳腺癌自体造血干细胞的实验研究.中华医学杂志, 2002, 82(3): 203
[34] Lubbe A S, Bergemann C, Huhnt W, et al.PrecIinical experiences with magnetic grug targeting tolerance and efficancy. Cancer Res. 1996, 56(20): 4694-4701
[35] Lubbe A S, Bergemann C, Riess H, et al. ClinicaI experiences with magnetic drug targeting-Aphase I study with 4’-epidoxorubicin in l4 patients with advanced solid tumors. Cancer Res, 1996, 56(20): 4686-4693
[36]范崇政,肖建平,丁延伟,等纳米TiO制备与光催化反应研究进展.科学通报, 2001, 46(4): 256-273
[37]徐翠香,张明,等.聚合物包裹纳米磁性微球的制备及对生物物质的吸附影响因素研究.扬州:扬州大学, 2006:5
[38]罗付生,李凤生.超微粒子设计技术及应用.材料科学与工程, 2001, 19(2): 115-118.
[39]闰翠娥,徐章煌,程时远,等.生物活性高分子微球.高分子材料科学与工程, 1999, 15(1): 1-3
[40]徐辉,张国亮,张凤宝,等.免疫磁性微球的研究进展.化学工业与工程, 2003, 20(l): 27-33
[41]魏衍超,杨连生.生物高分子磁性微球的制备、结构、性质和应用.磁性材料及器件, 1999, 6(30): 18-21
[42]李凤生,罗付生,杨毅,等.磁响应纳米四氧化三铁/壳聚糖复合微球的制备及特性.磁性材料及器件, 2002, 33(6): 14-17
[43] Enkbas E B, Kilicay E, Birlikseven C, et al. Magneticchitosanmicrospheres: Preparation and haracterization. Reactive & Functional polymers, 2002, 50(3): 225-232
[44]常铮,郭灿雄,李峰,等.新型磁性纳米固体酸催化剂ZrO2/Fe_3O_4的制备及表征.化学学报, 2002, 60(2): 298-304
[45]邱星屏.四氧化三铁磁性纳米粒子的合成及表征.厦门大学学报(自然科学版), 1999, 38(5): 711-715
[46]陈盛,罗志敏,马秀玲.磁性壳聚糖微球.化学通报, 2003, 66(26): 1-6
[47]邓勇辉,汪长春,杨武利,等.磁性聚合物微球研究进展.高分子通报,2006. 5(2): 27-33
[48]程彬,朱玉瑞.无机高分子磁性复合粒子的制备与表征.化学物理学报, 2000, 3(13): 359-362
[49]马秀玲,黄丽梅,郑思宁,等.磁性微球的制备及研究进展.广州化学, 2003, 28(3): 58-64
[50]成当效.载药磁性微球一种新的靶向给药系统.国外医学肿瘤学分册, 1995, 2(22): 96-98
[51]严希康,朱留沙,董建春.聚合物粒子在生物化学与生物医学中的应用.功能高分子学报, 1997, 10(1): 128-129
[52]王延梅,封麟先.磁性高分子微球的研究进展.高分子材料科学与工程, 1998, 14(1): 5-8
[53]常兰,李玲,等.核壳型磁性高分子微球的制备及应用进展.暨南大学学报(自然科学版), 2004, 25(3): 375– 376
[54]步星耀,于耀宇,赵红卫,等.磁导向下磁性药物载体的脑定位分布及对肝、肾、骨髓的影响.河南医学研究, 2001, 10(4): 289– 292
[55]佟哓冬,薛博,白姝,等.聚乙烯醇亲和磁性载体的吸附性能研究.天津大学报, 2001, 34(1): 111-114
[56]邱广亮,李咏兰,丁炜.磁性导向柔红霉素白蛋白微球的研究.精细化工,2001,18 (3):141 -143
[57]邱广亮,李咏兰,斯日古楞,等.靶向性药物载体-磁性淀粉微球的制备及性质.化工科技, 2001, 9 (4): 15-18
[58]常津.医用磁性纳米材料的制备.中国生物医学工程学报, 1996, 15(4): 378-381
[59] Gordula Grüttner, Sandra Rudershausen, Joachim Teller. Improved properties of materials as particle matrix. Magn Magn Mater, 2001, 225(1): 1-7
[60] Li Guiyin, Jiang Yuren, Huang Kelong, et al. Preparation and properties of magnetic Fe_3O_4–chitosan nanoparticles. Journal of Alloys and Compounds, 2008, 466(1-2): 451-456
[61] Saiyed Z M, Sharma S, Godawat R, et al. Activity and stability of alkaline phosphatase (ALP)immobilized onto magnetic nanoparticles (Fe_3O_4). Journal of Biotechnology , 2007, 131(3): 240-244
[62] Lei Zhongli, Bi Shuxian, Hu Bing, et al. Combined magnetic and chemical covalent immobilization of pectinase on composites membranes improves stability and activity. Food Chemistry, 2007, 105(3): 889-896
[63] Horst F, Rueda E H, Ferreira M L, et al. Activity of magnetite-immobilized catalase in hydrogen peroxide decomposition.Enzyme and Microbial Technology, 2006, 38(7): 1005-1012.
[64]王山杉,李琳,李冰.磁场对酶学效应影响的研究进展.现代生物医学进展, 2006, 6(10): 111-114.
[65]邱广亮,栗淑媛,金志兰,等.磁性琼脂糖复合微球的制备和性质.精细化工, 1999, 16 (1): 38
[66] Lee Josephon. Magnetic particle for use in separation .US: 4672040 ,1987:1-3
[67]陆彬.药物新剂型与新技术.北京:人民卫生出版社, 1998, 536
[68]邱广明,孙宗华.磁性微球表面固定中性蛋白酶.生物医学工程杂志, 1995, 12(3): 209
[69]邱广亮,张艳茹.磁性载体用于酶固定化方面的研究.内蒙古师范大学报, 1998, 27(2): 129
[70] Rittich B, Spanova A, Ohlashennyy Y, et al. Characterization of deoxyribonuclease I immobilized on magnetic hydrophilic polymer particles. Journal of Chromatography B, 2002, 774(1): 25–31
[71]邱广亮,李咏兰.纳米级磁性微粒的制备及固定化纤维素酶的研究.药物生物技术, 2001, 8(4): 197
[72]王玫,宋芳,汪世龙,等.磁性纳米颗粒Fe_3O_4固定化纤维素酶的光谱学研究.光谱学与光谱分析, 2006, 26(5): 895-898
[73]周建芹,朱忠奎.固定化纤维素酶催化反应动力学模型研究.农业工程学报, 2006, 22(52): 14—18
[74]袁勤生,赵健.酶与酶工程.上海:华东理工大学出版社, 2005: 225-226
[75]李冰,邵海员,黎锡流,等.磁性固定化纤维素酶的交联法制备及其磁致酶学性质,河南工业大学学报(自然科学版), 2006, 27(6): 10-14
[76] Wu J, Ju. L K. Enhancing enzymatic saccharification of waste newsprint by surfactant addition. Biotechnol Prog. 1998, 14(4), 649-652
[77] Park. J W, Takahata. Y, Kajiuchi. T, Akehata. T. Effects of nonionic surfactant on enzymatic hydrolysis of used newspaper. Biotechnol Bioeng. 1992, 39(1), 117-120
[78] Nakagawa M, Sakai Y, Yasui T. Itaconic Acid Fermentation of Levoglucosan. J Ferment Technol, 1984, 62(1): 201-203
[79] Oshima H, Sakata M, Harano Y. Enhancement of enzymatic hydrolysis of cellulose by surfactant. Biotechnol Bioeng. 1986, 28(11): 1727-1734
[80] M Castanon , C R Wilke. Effects of the surfactant Tween 80 on enzymatic hydrolysis of newspaper. Biotechnol Bioeng, 1981, 23(1): 1365-1372
[81] Helle S S, Duff S J B, Cooper D G.. Effect of surfactants on cellulose hydrolysis. Biotech and Bioeng, 1993, 42(5): 611-617
[82] Nurdan Eken-saracoglu, Mutlu S F, Gūnes Dilmac, et al. A comparative kinetic study of acidic hemicellulose hydrolysis in corn cob and sunflower seed hull. Bioresource Technology, 1998, 65(1): 29?33
[83] Hasan Ferdi Gercel. Production and characterization of pyrolysis liquids from sunflower-pressed bagasse. Bioresource Technology, 2002, 85(2): 113?117
[84] Sanjeev K S, Krishan L K, Gurvinder S K. Fermentation of enzymatic hydrolysate of sunflower hulls for ethanol production and its scale-up. Biomass and Bioenergy, 2004,27(4): 399?402.
[85] Müjgan T O, Nurdan E S. Fermentation of sunflower seed hull hydrolysate to ethanol by pichia stipitis. Bioresource Technology, 2008, 99(7): 2162?2169.
[86]寇灵梅,李冰,郭祀远,等.磁性固定化纤维素酶的制备及其磁响应性.食品科学, 2006, 27(12): 335-338
[87]贺华君,朱元保,钟科军,等.磁场对纤维素酶的活性及构象的影响.吉首大学学报(自然科学版), 1998, 19(1): 41-46
[88]刘佳,袁兴中,曾光明,等.表面活性剂在废木质纤维素制酒精中的应用基础研究.湖南大学,硕士学位论文, 2008, 20-64
[89] Ghose T K. Measurement of cellulase activities. Pure Appl Chem, 1987, 59(2): 257?268.
[90]王琰,杨森,李十中.糠醛渣纤维素酶解研究.环境科学, 2007, 28(9): 2129-2133
[91]秦润华,姜炜,刘宏英,等.纳米磁性四氧化三铁的制备及表征.材料导报, 2003, 9(17): 66-68
[92] Gregg D J, Saddler J N. Factors affecting cellulose hydrolysisand the potentialof enzyme recycle to enhance the efficiency of an integrated wood to ethanol process. Biotechnol Bioeng , 1996, 51(3): 75?83
[93] McMillan J D. Pretreatment of lignocellulosic biomass//Himmel M E, Baker J O, Overend R P. Enzymatic Conversionof Biomass for Fuels Production.USA:ACS Symposium Series, 1994, pp. 292?324
[94] Solange I M, Marcela Fernandes, Adriane M F M, et al. Effect of hemicellulose and lignin on enzymatic hydrolysis of cellulose from brewer’s spent grain. Enzyme and Microbial Technology, 2008, 43(2): 124?129
[95]钟文文.秸秆预处理对纤维素酶水解效果的影响.湖北农业科学, 2007, 46(6): 1006?1008
[96]莫丹,袁兴中,曾光明,等. Tween80和鼠李糖脂对稻草酶解的影响.环境科学, 2008, 29(7): 2998-3004
[97]钟科军,草祉祥,文陵飞,等.超氧化物歧化酶磁效应的研究.湖南大学学报(自然科学版), 1999, 26(5): 18-23
[98]王林格,黄勇.磁场对乙基氰乙基纤维素胆甾型液晶相的影响.纤维素科学与技术, 2000, 8(4): 7-13
[99] Sugiyama. Combined infrared and electron diffraction study of the polymorphism of native cellulose. Macromolecules, 1991, 24: 2461?2466
[100] Barbara Hinterstoisser, Lennart Salmén. Application of dynamic 2D Ftir to cellulose. Vibrational Spectroscopy, 2000, 22(1-2): 111?118
[101]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
[102] Tomme P, Warren R A J, Gilkes N R, et al.Cellulose hydrolysis by bacteria and fungi.Adv Microb Physiol, 1995, 37: 1?81
[103] Gong Liangfa, Ku Ruxiong, Xu Xin, et al. Surface tension of aqueous solutions of inorganic salts and those surfactant. Journal of Beijing Institute of Petro-chemical Technology, 2000, 8(1-2): 42?46
[104]曹林秋,杨晟,袁中一.载体固定化酶-原理、应用和设计.北京:化学工业出版社, 2007: 264-265
[105] Shchipunova Y A, Karpenkoa T Y, Bakuninab I Y, Burtsevab Y V, Zvyagintsevab T N:Anew precursor for the immobilization of enzymes inside sol-gel-derived hybrid silica nanocomposites containing, poly-saccharides.JBiochem Biophys Methods 2004, 58: 25-38
[106]失俭.生物化学实验.上海:上海科学技术出版社, 1981:64
[107]任广智,李振华,何炳林.磁性壳聚糖微球用于酶的固定化研究-Ⅱ磁性壳聚糖微球对脲酶的吸附及其酶学性质的研究.离子交换与吸附, 2000, 16(4): 304-310
[108]张惟杰.糖复合物生化研究技术.杭州:浙江大学出版社, 2003: l0-l1
[109] Badjic J D. Kostic N M. Effects of encapsulation in sol-gel silica glass on esterase activity, conformational stability, and unfolding of bovine carbonic anhydrase II.Chem Mater 1999, 11(2):3671-3679
[110] Moriyama S, Noda A, Nakanishi K, Matsuno R, Kamikubo T. Thermal stability of immobilized glucoamylase entrapped in polyacrylamide gels and bound to SP-Sephadex C-50. Agric BiolChem. 1980, 44(3): 2047-2054
[111] Munjal N, Sawhney SK. Stability and properties of mushroom tyrosinase entrapped in alginate, polyacrylamide and gelatin gels. Enzym Microb Technol. 2002, 30(5): 613-619
[112] Eggers D K, Valentine J S. Crowding and hydration effects on protein conformation:a study with sol-gel encapsulated proteins. J Mol Biol. 2001, 314(4): 911-922
[113] Liu D M, Chen I W. Encapsulation of protein molecules in transparent porous silica matrices via an aqueous colloidal sol-gel process. Acta Mater 1999, 47(1):535-4544
[114] Singh D, Goel R, Johri B N. Production of 6-aminopenicillanic acid through double entrapped Ecolincim2563. Curr Sci. 1988, 57(5):1229-1231
[115] Bajpai P, Margaritis S, Ohkawa H, Asada Y, Miyake J. Immobilization of P450 monooxygenase and chloroplast for use in light-driven bioreactors. J Biosci Bioeng 1999, 87(9): 793-797