摘要
作为一种重要的有机精细化工产品,油酸丁酯在多个领域有着广泛的应用。目前,商品化油酸丁酯都是通过化学方法合成的,但生产过程中存在反应条件剧烈、环境污染大、产品品质低等缺点。为克服油酸丁酯传统生产方法的不足,本研究选取两种脂肪酶(黑曲霉脂肪酶和假丝酵母脂肪酶)为催化剂,考察了生物法合成油酸丁酯的可行性及影响因素,同时,考虑到游离酶在工业应用上的缺陷,还研究了脂肪酶的固定化及其在油酸丁酯合成中的应用。主要研究结果如下:
(1)游离脂肪酶催化反应表明,黑曲霉脂肪酶和假丝酵母脂肪酶都能催化合成油酸丁酯,但假丝酵母脂肪酶的催化能力明显优于黑曲霉脂肪酶。对相关影响因素的考察表明,反应溶剂对转化率影响较大,反应溶剂logP＞2时转化率较高,本研究选择的是正己烷。假丝酵母脂肪酶的最佳反应条件为:温度为37℃,摇床速度200r·min~(-1),底物正丁醇与油酸的摩尔比为1,给酶量为5000IU/25mL溶剂时,其4小时的最高转化率为59.1%。
(2)为了克服游离酶应用上的缺陷,从固定化载体选择、固定化影响因素等角度考察了两种脂肪酶的固定化效果,并分析了固定化酶的性能。结果表明,在5种不同性质的固体膜中,PS膜为最适宜的固定化载体,固定化后两种酶的负载量和酶活都较高,但固定化假丝酵母脂肪酶性能明显优于固定化黑曲霉脂肪酶。假丝酵母脂肪酶的最佳固定化条件为:吸附时间为10h,吸附温度为25℃,脂肪酶溶液pH值为6.0时,固定化脂肪酶的蛋白负载量为0.09mg·cm~(-2),固定化酶活为0.55U·cm~(-2);固定化假丝酵母脂肪酶的性能分析显示,与游离酶相比,固定化酶在高温下有更好的稳定性;重复催化水解橄榄油5次后,残余活性为39.0%;在4℃下保存10天后,残余活性仍能达到72.7%。
(3)以PS固定的假丝酵母脂肪酶催化合成油酸丁酯。结果表明,与游离酶催化相比,反应最适温度没有变化,但温度对固定化脂肪酶催化反应的影响较小,在实验温度范围内固定化脂肪酶催化反应转化率的最大变化幅度仅为游离酶的42%。这主要是由于固定化酶的热稳定性比游离酶有了较大的提高,热稳定性实验显示,固定化酶和游离酶在50℃下热处理2h后催化酯化反应,其转化率分别是原来的65.3%和29.9%。而且,固定化脂肪酶重复催化酯化反应4批后,相对反应转化率仍可为原来的65.6%,达到了较好的催化效果,使脂肪酶得以重复利用。以带无纺布支撑层的PTFE疏水膜为载体,利用过滤.交联法制备了固定化假丝酵母脂肪酶,用其催化合成油酸丁酯,反应4h后最高转化率达到82.0%,重复使用9次后转化率没有显著降低。
As an important fine chemical product,n-butyl oleate has been extensively used in many fields.However,presently the commercial n-butyl oleate was chemically synthesized,which had many disadvantages,such as violent reaction conditions,environmental pollution and poor product quality.In order to overcome the shortcoming of traditional method in synthesizing n-butyl oleate,two lipases(Candida sp.and Aspergillus niger)were used as biocatalyst to investigate their feasibility and influence factors in synthesizing n-butyl oleate.Moreover,due to shortcoming of free lipase,two lipases were immobilized to investigate their feasibility in synthesizing n-butyl oleate.Also, the immobilization conditions and reaction conditions were optimized.The main results as followed:
1.Two free lipases were used in synthesis of n-butyl oleate.The results indicated that both Candida sp.lipase and Aspergillus niger lipase can catalyze synthesis of n-butyl oleate,but Candida sp.lipase was more suitable to catalyze synthesis of n-butyl oleate than Aspergillus niger lipase. Among impact factors,polarity of reaction solvent had great effect on conversion rate,and solvents with logP＞2 were more suitable.In present study,hexane was used as reaction solvent.For Candida sp.lipase,the optimum conditions were:temperature 37℃,vibrating rate 200r·min~(-1), molar ratio of oleic acid to n-buanol 1,lipase dose 5000IU/25mL solvent, and the greatest conversion rate was 59.1%in 4h reaction.
2.In order to overcome shortcoming of free lipase in application,lipase immobilization was carried out,and effects of support characterics and impact factors were also investigated.The results indicated that polysulfone(PS)membrane was the most suitable carrier among five kinds of membrane.Compared two lipases,immobilized Candida sp.lipase had higher protein loading and lipase activity than immobilized Aspergillus niger.The optimum conditions for immobilization of Candida sp.lipase were:adsorption time 10h,adsorption temperature 25℃,lipase solution pH 6.0,and the protein loading and lipase activity were 0.09mg·cm~(-2)and 0.55U·cm~(-2),respectively.Investigation on characteristic of immobilized Candida sp.lipase showed that it was more stable than free lipase under high temperature,and it had good repeatability with remaining activity 39.0%after 5 cycles reuse.Moreover,immobilized Candida sp.lipase had good storage stability and the residual activity was 72.7%after 10 days under 4℃.
3.Candida sp.lipase immobilized on PS membrane was used to catalyze synthesis of n-butyl oleate.The results indicated that,compared to free lipase,the optimum temperature of immobilized lipase didn't shift,but its ability to endure temperature fluctuation increased.Study on thermal stability of lipase showed that relative conversion rates of free and immobilized lipase were 29.9%and 65.3%of initial activity after 2h under 50℃,respectively.In addition,relative conversion rate was 65.6%after 4 cycles reuse,which indicated that immobilization increased lipase reusability.The immobilization was futher studied.Candida sp.lipase was immobilized on PTFE membrane which has non-woven fabric as support layer through filtering and crosslinking.Then the immobilized lipase was used to catalyze synthesis of n-butyl oleate,the result shows that reaction conversion rate was up to 82.0%in 4h,and there was no significant decrease after 9 cycles reuse.
引文
[1]宋启煌.精细化工工艺学[M].北京:化学工业出版社,1995,95
[2]李继忠.油酸丁酯的合成研究[J].化学推进剂与高分子材料,2004,2(4):43-44
[3]郭永成,杨丽娜,沈健,袁兴东,焦宏宁,韩庆玮.含磺酸基的介孔分子筛SO_3H-SBA-15催化合成油酸丁酯[J].化工科技,2005,13(5):39-41
[4]吴洪特,于兵川,葛胜祥.以SnO/ZnO为催化剂合成油酸正丁酯[J].塑料助剂,2007.3:20-22
[5]Habulin M,Knez Z,Enzymatic synthesis of n-butyl oleate in a hollow fiber membrane reactor[J].Journal of Membrane Science,1991,61:315-324
[6]Ghamgui H,Chaabouni M K,Gargouri Y.1-Butyl oleate synthesis by immobilized lipase from Rhizopus oryzae:a comparative study between n-hexane and solvent-free system[J].Enzyme and Microbial Technology,2004,35:355-363
[7]刘瑛,刘俊康,高飞.微乳凝胶固定化酶催化油酸丁酯的合成[J].江南大学学报(自然科学版),2003,2(3):297-299
[8]王军.脂肪酶固定化及催化酯化反应[J].日用化学工业,1995,3:4-7
[9]陈宁.酶工程[M].中国轻工业出版社,2005,3-24
[10]Lee K T,Akoh C C.Structured lipids:synthesis and applications[J].Food Research International,1998,14(1):17-34
[11]郭铮,张根旺.脂肪酶的结构特征和化学修饰[J].中国油脂,2003,28(7):5-10
[12]Yahya A R M,Anderson W A.Ester synthesis in lipase-catalyzed reactions[J].Enzyme Microbial Technology,1998,23(7):438-450
[13]Zaks A,Russell A J.Enzymes in organic solvents:properties and applications[J].Journal of Biotechnology,1988,8(4):259-269
[14]Zaks A,Klibanov A M.Enzymatic catalysis in organic media at 100 degrees C[J].Science,1984,224(4654):1249-1251
[15]Zaks A.Industrial biocatalysis[J].Current Opinion in Chemical Biology,2001,5(2):130-136
[16]Schmid R,Partali V.Regioselective hydrolysis of diesters of(Z)- and (E)-2-methyl-butenedioic acids by PLE[J].Tetrahedron Letters,2001,42:8543-8545
[17]徐刚,戴军强,吴坚平,杨立荣.有机相中酶催化1-苯基乙胺的不对称酰胺化反应[J].化工学报,2007,58(7):1741-1745
[18]Chamouleaua F,Coulona D,Girardina M,Ghoul M.Influence of water activity and water content on sugar esters lipase-catalyzed synthesis in organic media[J].Tetrahedron,1999,55(28):8555-8566
[19]Karboune S,Safari M,Lue B M,Faustinus K Y,Kermasha S.Lipase-catalyzed biosynthesis of cinnamoylated lipids in a selected organic solvent medium[J].Journal of Biotechnology,2005,119(3):281-290
[20]Zhao H Z,Lu Z X,Bie X M,Lu F X,Liu Z M.Lipase catalyzed acidolysis of lard with capric acid in organic solvent[J].Journal of Food Engineering,2007,78(1):41-46
[21]徐岩,章克昌,王亚非.微生物脂肪酶在正庚炕中合成短链芳香酯的研究[J].生物工程学报,1998,14(2):214-218
[22]李慧清,宗敏华.非水相脂肪酶在油脂工业中的应用[J].广州食品工业科技, 2000,16(2):64-67
[23]Brenda H J,Casimir C A.Lipase-catalyzed modification of rice bran oil to incorporate capric acid[J].Journal of Agriculture and Food Chemistry,2000,48:4439-4443
[24]Garfield I L,Haarmann,Reimer G H,Holzminden,West G.Enzymatic synthesis of esters in nonaqueous systems[J].Enzyme Engineering:Proceedings of the International Enzyme Engineering Conference,1984,434:569-572
[25]沈芳,梁静娟,刘雄民.脂肪酶催化ω-羟基十五烷酸甲酯合成环十五内酯[J].应用化学,2007,24(1):63-66
[26]Haffner T,Tressl R.Biosynthesis of(R)-γ-decanolactone in the yeast sporobolomyces odorus[J].Jounal of Agriculture Food Chemistry,1996,44(5):1218-1223
[27]姜忠义,陈洪钫.游离和膜固定化脂肪酶在双液相介质中催化二肽合成的研究[J].有机化学,2002,22(12):1050-1052
[28]彭立风.有机相脂肪酶催化的有机物合成反应[J].合成化学,2000,8(4):294-299
[29]Laane C,Boeren S,Kees V.On optimizing organic solvents in multi-liquid-phase biocatalysis[J].Trends in Biotechnology,1985,3(10):251-252
[30]Theil F.Enhancement of selectivity and reactivity of lipases by additives[J].Tetrhaedron,2000,56:2905-2919
[31]Yumiko T,Masyaa K,Hiorshi K.Synthesis of Optically active novel 1,1,2-trifluoro-1-alken-3-ols thorugh liPase-catalyzed reaetion[J].Tetrhaedorn Leettrs,1999,40:2801-2802
[32]Yuri L,Khmelnitsky,Stephanie H.Salts darmatically enhance activity of enzymes suspended in ogranic solvents[J].J.Am.Chem.Soc,1994,116:2647-2648
[33]Bornscheuer U,Herar A,Kreye L.Factors affecting the lipase catalyzed transesterifications of 3-hydroxy esters in organic solvents[J].Tetrahedron:Asymmetry,1993,4:1007-1016
[34]Ahern T J,Klibanov.A M.The mechanism of irreversible enzyme inactivation at 100℃[J].Science,1985,228:1280-1284,
[35]袁勤生.现代酶学[M].华东理工大学出版社,2007(2):245
[36]Dorothy M O,Sanyal G,Volkin D B,Marcy A I,Chan H K,Ryan J A,Russell C.Middaugh.Stabilization of the FK506 binding protein by ligand binding[J].Biochemical and Biophysical Research Communications,1991,179(2):741-748
[37]Afflectk R,Xu Z F,Suzaw V.Enzymatic catalysis and dynamics in low-water environments[J].Proceedings of the National Academy Science.1992,89(3):1100-1104
[38]Hailing P J.Thermodynamic predictions for biocatalysis in nonconventional media:Theory,tests,and recommendation for experimental design and analysis[J].Enzyme and Microbial Technology,1994,16(3):178-206
[39]Berglund P,Holmquist M,Hult K.Alcohols as enantioselective inhibitors in a lipase catalyzed esterification of a chiral acyl donor[J].Biotechnology Letter,1995,17:55-60
[40]Kuo S J,Parkin K L.Acetylacylglycerol formation by lipase in microaqueous milieu:effects of acetyl group donor and environmental factors[J].Jounal of Agriculture and Food Chemistry,1995,43:1775-1783
[41]Rangheard M S,Langrand G,Triantaphylides C,Baratti J.Multicompetitive enzymetic reactions in organic media:application to the determination of lipase alcohol specificity[J].Enzyme Microbial Technology,1992,14:966-974
[42]李秀文,万婷.非水介质中酶促反应的研究进展[J].高等函授学报(自然科学版),2005,18(2):43-59
[43]吴可克,安庆大.酶促反应合成蔗糖棕榈酸酯的研究[J].中国油脂,2004,29(3):37-39
[44]Zaks A,Arkady S,Pikovsky,Kurths J,Symbolic dynamics behind the singular continuous power spectra of continuous flows,Physica D:Nonlinear Phenomena,1998,117(1-4):77-94
[45]Bornscheuer U T.Lipase-catalyzed syntheses of monoacylglycerols[J].Enzyme and Microbial Technology,1995,17(7):578-586
[46]Ferreira D S,da Fonseca M M R.Production of monoglycerides by glycerolysis of olive oil with immobilized lipases:Effect of the water activity[J].Bioprogress Engineering,1995;(12):327-337
[47]谭天伟,鹿长荣.生物化工现状及发展趋势[J].现代化工,2001,21(1):11-15
[48]Li Z Y.Acidolysis of sardine oil by lipase to concentrate eicosapentaenoic and docosahexenoic acid in glycerides[J].Journal of the American Oil Chemists' Society,1993,70:745-751
[49]姜楠,张正.生物柴油的现状与发展前景[J].世界农业,2005,(3):11-12
[50]Asano Y,Fukuda H,Shimada Y,Tnaka A,Ueda M.Biofuel production process by novel biocatalysts[J].Journal of Molecular Catalysis B:Enzymatic,2002,17:111-119
[51]谭天伟.固定化酶法生产生物柴油技术[J].中国高校科技与产业化,2007,6:77-80
[52]杨继国,林炜铁,吴军林.酶法合成生物柴油的研究进展[J].化工环保,2000,24(2):116-120
[53]汤鲁宏,张浩.非水相脂肪酶催化合成L-抗坏血酸棕榈酸酯的研究Ⅰ.生物工程学报[J].2000,16(3):363-267
[54]Bousquet M P,Willemot R M,Monsan P.Enzymatic Synthesis of Unsuatrated Fatty Acid Glucoside Esters of Demro-Cosmetic Applications[J].Biotechnology and Bioengineering,1999,63(6):730-736
[55]Humeua,Mgirardin,Rovel B.Enzymatic synthesis of aftty acid as cobrylesters[J].Journal of Molecular Catalysis B:Enzymatic,1998,5:19-23
[56]Maugard T,Tudella J,Legoy M D.Study of vitamin ester synthesis by lipase-catalyzed transesterification in organic media[J].Biotechnol Prog,2000,16(3):358-362
[57]刘涛,尹春华,谭天伟.脂肪酶催化合成维生素A酯[J].现代化工,2005,25(2):4-6
[58]卢世珩,刘光烨,江跃林,黄德英,吴衍庸.合成己酸乙酯脂肪酶产生菌的筛选及产酶条件[J].微生物学报,1994,21(1):23-25
[59]葛清秀,陈建平,黄祖新,柳荣金,孙斌.碱性脂肪酶在正庚烷中催化合成乙酸乙酯的研究[J].食品工业科技,2006,2:156-158
[60]王雨来,天然表面活性剂在日化产品中的应用[J].福建轻纺,2001,2:27-29
[61]Sodemrana O,Johanssonb U I.polyhydroxyl-based surafctants and their Physico-chemical Properties and application[J].Current Opinion in Colloid&Interafce Science,2000,4:391-401
[62]寇秀芬,徐家立.酶法合成糖及糖醇酯[J].微生物学报,2000,40(2):1193-197
[63]Klibanov A M,Cambou B.Enzymatic production of optically active compounds in biphasic aqueous-organic systems[J].Methods in Enzymology,1987,136:117-137
[64]曾庆冰,马安德,崔铭玉.酶法合成可生物降解高分子材料[J].国外医学生物医学工程分册,1997,20(6):325-329
[65]Patil D R,Dordick J S,Rethwisch D G.Chemoenzymatic synthesis of novel sucrose-containing polymers[J].Macromolecules,1991.24:3462-3463
[66]宋缪毅,马凤国,邵自强,谭惠民.合成纤维素高级脂肪酸酯的研究[J].化学世界,2001,4:216-218
[67]Sereti V,Stamatis H,Koukios E,Kolisis F N.Enzymatic acylation of cellulose acetate in organic media[J].Journal of Biotechnology,1998,66(2):219-223
[68]李树本,酶化学[M].化学工业出版社,2008,103
[69]曹国民,盛梅,高光达.脂肪酶的固定化及其性质研究[J].生物技术,1997,7(3):14-17
[70]刘勇,吾满江·艾力,夏木西卡玛尔,张永学,甘争艳,孙燕.黑曲霉脂肪酶的耦合固定化及特性[J].分子催化,2006,20(3):260-265
[71]刘汝宽,李昌珠,张玉军,肖志红.树脂固定脂肪酶技术的研究[J].河南工业大学学报(自然科学版),2007,28(3):68-71
[72]孔维,周慧,王立平,陈尊,李惟,沈家骢.重氮法固定化糖化酶活力和稳定性[J].高等学校化学学报,1994,15(5):681-684
[73]汤亚杰,吴思方,程婉农.壳聚糖固定胰蛋白酶的研究[J].食品科学,1999,1:29-31
[74]曾淑华,杨江科,徐莉,闫云君.脂肪酶固定化及其稳定性研究[J].生物技术,2006,16(4):54-57
[75]杨芳霞,董武子,苏印泉.甲壳糖固定化脂肪酶活力和稳定性的测定[J].家畜生态学报,2006,27(3):31-34
[76]陈姗姗,仇农学.阴离子交换树脂固定化果胶酶及其酶学性质的研究[J].广西农业生物科学,2007,26(4):339-343
[77]张军,徐家立.固定化假丝酵母1619催化油酸油醇酯的合成[J].生物工程学报,1995,11(4):325-331
[78]柏正武,尹传奇,吴莉.甲壳胺-硅胶复合载体的制备及其在脂肪酶固定化中的应用[J].应用化学[J],2002,19(12):1194-1196
[79]Shin J H,Marxer S M,Schoenfisch M H.Nitric Oxide-Releasing Sol-Gel Particle/Polyurethane Glucose Biosensors[J].Analytical Chemistry,2004,76(15):4543-4549
[80]Matthijs G,Schacht E.Comparative study of methodologies for obtaining β-glucosidase immobilized on dextran-modified silica[J].Enzyme and Microbial Technology,1996,19(8):601-605
[81]Hornebecq V,Antonietti M,Cardinal T,Mona T D.Stable silver nanoparticles immobilized in mesoporous silica[J].Chemistry and Materials,2003,15:1993-1999
[82]陈盛,黄智跃,刘艳如.壳聚糖固定化纤维素酶的研究[J].生物化学与生物物理进展,1996,23(3):250-254
[83]Akkus S,Cetinus,Oztop H N.Immobilization of catalase into chemically crosslinked chitosan beads[J].Enzyme and Microbial Technology,2003,23(7):889-894
[84]龚伟中,魏甲乾,周剑平等.聚丙烯酰胺固定化糖化酶特性的研究[J].分子催化,2004,18(4):291-294
[85]Benevides C C,Mateo C,Carrascosa A V,Vian A,Garcia J L,Rivas G,Alfonso C,Guisan J M,Lafuente R F.One-step purification,covalent immobilization,and additional stabilization of a thermophilic poly-his-tagged β-galactosidase from Thermus sp.strain T2 by using novel heterofunctional chelate-epoxy sepabeads[J].Biomacromolecules,2003,4(1):107-113
[86]曲红波,丛威,韦新桂.多孔醋酸纤维素球形载体固定化糖化酶的研究[J].生物化学与生物物理进展,1998,25(2):155-158
[87]El-Masry M M,Maio A D,Martino S D,Diano N,Bencivenga U,Rossi S,Grano V,Canciglia P,Portaccio M,Gaeta F S,Mita D G.Modulation of immobilized enzyme activity by altering the hydrophobicity of nylon-grafted membranes:Part 1.Isothermal conditions[J].Journal of Molecular Catalysis B:Enzymatic,2000,9:219-230
[88]Dyal A,Loos K,Noto M,Chang S W,Spagnoli C,Kurikka V.P.M.Shafi,Abrah Ulman,Cowman M,Richard A.Gross.Activity of Candida rugosa Lipase Immobilized on γ-Fe_2O_3 Magnetic Nanoparticles[J].Journal of the American Chemical Society,2003,125(7):1684-1685
[89]Liao M H,Chen D H.Immobilization of yeast alcohol dehydrogenase on magnetic nanoparticles for improving its stability[J].Biotechnology Letters,2001,23(20):1723-1727
[90]刘薇,白姝,孙彦.磁性纳米粒子的制备及脂肪酶的固定化[J].过程工程学报,2004.4:362-366
[91]Kato N,Samejima S,Takahashi F.Isomaltose synthesis in the reversed hydrolysis catalyzed by amyloglucosidase immobilized in the thermosensitive gel[J].Material Science Engineering:C,2001,17:155-160
[92]Pronk W,Boswinkel G,Riet K.Parameters influencing hydrolysis kinetics of lipase in a hydrophilic membrane bioreactor[J].Enzyme and Microbial Technology,1992,14(3):214-220
[93]Pronk W,Kerklof P J A M,Van Helden C.The hydrolysis of triglycerides by immobilized lipase in a hydrophiiic membrane reactor[J].Biotechnology and Bioengineering,1988,32(4):512-518
[94]Lye G J,Pavlou O P,Rosjidi M,Stuckey D C.Immobilization of Candida cylindracea lipase on colloidal liquid aphrons(CLAs)and development of a continuous CLA-membrane reactor[J].Biotechnology and Bioengineering,1996,51(1):69-78
[95]吴茜茜,吴克,刘斌,杨本宏,赵丽君,蔡敬民,潘仁瑞.壳聚糖固定化德氏根霉脂肪酶的研究[J].工业微生物,2003,33(4):9-12
[96]李娟,张耀庭,曾伟,罗璇,廖长春.应用考马斯亮蓝法测定总蛋白含量[J].中国生物制品学杂志,2000,13(2):118-120
[97]徐凤杰,谭天伟.生物法合成维生素C棕榈酸酯[J].生物工程学报,2005,21(6):987-992