脂肪酶的固定化及在离子液体中催化合成生物柴油的研究

【作者】 杨建军；

【导师】 马晓迅；

【作者基本信息】 西北大学 ， 生物化工， 2009， 博士

【摘要】 生物柴油是以动植物油脂为原料制造的可再生能源,可作为石油柴油的替代或部分替代燃料。生物柴油的发展不仅有利解决能源短缺问题,而且可以减少温室气体的排放量。通过对桐油的理化指标的检测分析,陕南桐油的主要理化指标为:酸值3.6601mgKOH.g^-1,过氧化值4.428mol.L^-1,磷脂含量40.634mg.Kg^-1,碘值167.2g/100g,皂化值191.6mgKOH.g^-1,不皂化值0.1502mgKOH.g^-1,挥发物含量25℃下在0.2%以内,相对密度0.9365,折射率1.5193,粘度200.4mpa.s。陕南桐油主要脂肪酸组成为:棕榈酸3.51%,亚麻酸15.61%,油酸3.58%,亚油酸1.75%,桐酸73.81%,硬脂酸1.74%。采用热重分析仪对陕南桐油基生物柴油的热解特性进行实验研究。结果表明,桐油基生物柴油热解行为与石化柴油相似,从此角度桐油适于作为生物柴油的生产原料。为了消除粗脂肪酶中杂质对固定化的影响,对Candida antarctica脂肪酶（CAL）在双水相中的分配情况进行了研究,考察了温度、PEG分子质量、NaCl浓度和（NH₄）₂SO₄浓度对分配系数(K_CAL)的影响,结果表明:温度对K_CAL影响不大;低分子质量PEG与蛋白质的疏水作用可促进CAL的分配;两相间电势差等对分配平衡有较大影响;在PEG2000/（NH₄）₂SO₄)双水相体系中,CAL最佳的萃取分离条件为:室温下,20%PEG2000、12%（NH₄）₂SO₄、1.5%NaCl,此时,K_CAL=8.16,CAL回收率Y_CAL（%）=91.6%。探讨了大孔载体吸附固定化脂肪酶的机理及其热力学性质。选取4种不同载体（NW-ZT1、NW-ZT2、NW-ZT3、NW-ZT4）,通过氮气低温吸附法对载酶载体的孔径分布情况进行分析,对吸附-交联法制备固定化脂肪酶的工艺条件进行考察。发现NW-ZT2为最适的固定化载体,其最佳固定化条件为:给酶量1400-1600ug.ml^-1,戊二醛浓度为0.05%（V/V）,缓冲液pH7.5,吸附温度30^℃,吸附时问10h。NW-ZT2载体为南极假丝酵母脂肪酶理想的吸附固定化载体。反应过程中,酶的脱落是造成固定化脂肪酶活性大大下降的一个直接原因。吸附同时用戊二醛交联,可以起一定加固脂肪酶的作用。用硅胶-PEG键合物修饰-偶联固定化脂肪酶。经红外光谱图（FT-IR）分析证明偶联最终形成了硅胶-PEG-脂肪酶固定化酶。同时对两种固定化脂肪酶在最适温度和最适反应pH方面的变化,以及它们的热稳定性和操作稳定性进行了探讨,结果表明修饰-偶联固定化脂肪酶（Limczyme）比吸附-交联法固定化脂肪酶（Liaclzyme）的各方面性能要优,两种方法固定化的脂肪酶均可有效地应用于催化反应中。由于离子液体具有不挥发、不可燃、导电性强、热容大、蒸气压小、性质稳定等优良性质,对许多无机盐和有机物有良好的溶解性。很多酶在离子液体中具有良好催化性能。通过对反应温度和反应时间的考察,确定按1:1加入氯代正丁烷与N-甲基咪唑,反应温度为75℃,反应时间为54h,此时的反应产物[BMIM]Cl得率为83.9%;第二步反应中,采用间歇微波辐射加热法合成[BMIM][BF₄],每加热15s,取出在磁力搅拌器上搅拌30s。最终确定加热反应总时间为105s,[BMIM]Cl与NaBF₄的摩尔比率为1.1:1,[BMIM][BF₄]相对于N-甲基咪唑的得率为86.3%。核磁波谱分析结果均证实且相互映证了所得产物确实为[BMIM][BF₄]。通过条件优化,得到Limczyme催化桐油利用酯交换反应制备生物柴油的最佳工艺参数:以[BMIM][BF₄]为反应介质,pH=7.5,反应温度40℃,含水量10%,固定化脂肪酶Limczyme用量5%,摇床转速应200rpm,反应时间48小时,双底物甲醇与桐油的醇油比应保持在1.0,采用分步添加的方式,提出了在此酯交换反应中甲醇添加的“酯交换反应的塔板现象”。Limczyme制备生物柴油的动力学符合双底物的乒乓Bi Bi机制的醇抑制机制。所得动力学方程为去:（?）,其中最大反应速度V_max=1.76×10^-3mol.m_<sup>-3.min^-1.g^-1,桐油的米氏常数aK_ml=0.5738mol.m^-3,甲醇的米氏常数K_m2=1.3746mol.m^-3,甲醇的抑制常数K_iMe=0.2025mol.m_-3。更多还原

【Abstract】 Biodiesel,a renewable, biodegradable, nontoxic and cleaning fuel, is made from plant oil or animal oil,is expected as a part of substitute for conventional fossil diesel. Biodiesel could have advantanges of solving energy crisis and reducing the emission of Greenhouse Gas.Respectively,tung oil from the south of Shaanxi province was assayed by chemical analysis technologe, its compositions of fatty acid was discovered with gas chromotography, and the behaviors of its thermal mass loss was investigated by thermogravimetry.As results, the main physical and chemical indexes of Tung oil were acid value 3.6601 mgKOH.g^-1, iodine value 167.2g.100g^-1,saponification number 191.6mgKOH.g^-1,paroxide value 4.428mol.L^-1.Gas chromatograpy analysis showed that the fatty acids of Tung oil consisted of hexadecanoic acid 3.51%, linolenic acid 15.61%, linoleic acid 3.58%, oleic acid 1.75%, eleostearic acid 73.81%, stearic acid 1.74%.Biodiesel from Tung oil was researched on the thermogravimetry property.The experimental results showed that the effects of different heating rates were indistinct for pyrolysis. At the same time,I found that Tung oil was the better materials for preparation biodiesel than the others.The partition behavior of Candida antarctica lipase （CAL） in aqueous two phase system（ATPS） was investigated. Respectively,temperature, molecule mass of polyethylene glycol（PEG）,the concentration of NaCl and the concentration of ammonium sulfate were studied.The results showed that in ATPS, temperature was unimportant for partition coefficient of CAL (L_CAL),while the electric potential difference between top and bottom phase was important. It was also discovered that the less the molecule mass of PEG was, the more favorable conditions for the CAL congregation into the phase because of the increased hydrophobicity were formed. Under the room temperature, the optimized extraction conditions were as follows: 20% PEG, 12% （NH₄）₂SO₄,1.5%NaCl, and K_CAL was 81.6, the recovery rate of CAL was 91.6%.To study on mechanism of Candiada antartica lipase immobilized with macroporous carriers by adsorption.For four different carriers （NW-ZT2, NW-ZT3, NW-ZT4, NW-ZT1）, their pore size distributions were investigated with the auto-absorption instrument by nitrogen low tempera -ture adsorption method. The behavior of lipase immobilized was researched by adsorption & cross-linking method, and immobilization lipase mechanism has been studied.NW-ZT2 should be the best carrier among these four carriers., and it’s optimal conditions for immobiliza -tion: the amount of lipase 1400-1600ug/ml, glutaraldehyde concentration of 0.05% （V/V）, buffer pH7.5, adsorption temperature 30℃,adsorption time 10h.We found that NW-ZT2 carrier was an ideal carrier for immobilizing Candiada antartica lipase by absorption. During the reaction processes with immobilization lipase as catalyzer, the loss of immobilization lipase activity was caused by lipase falling off the carrier. At the same time, we found it could play a role in strengthening lipase that lipase immobilized by absorption & cross-linked with NW-ZT2 and glutaraldehyde.In this paper, Candida antarctica lipase was immobilized by absorption & cross-linked method with NW-ZT2 and modified-conjugation method with silica gel-PEGThe final product silica gel-PEG-lipase（named Limczyme） was proved by analyzing IR spectrums.We have studied the optimum pH value, the optimum temperature, the thermo-stabilities and operational stabilities for two kinds of immobilized lipase.The study results show that the silica gel-PEG-lipase was better than the immobilized lipase by adsorption-cross-linked （named Liaclzyme）, and both of them were efficient for transesterification reactions.There are some advantages for ionic liquids with non-volatile, non-combustible, electrical conductivity, big heat capacity, and small vapor pressure, stabilities and good solubility for many inorganic and organic compounds. Enzymes in ionic liquids have good catalytic properties.By inspecting the reaction temperature and reaction time,（?） determined the molar ratio 1:1 for chlorophthalic add n-butane and N-methyl imidazole,and reaction temperature was 75℃,reaction time was 54h, the reaction products（FAMEs） yield was 83.9% in [BMIM]C1; For the second step reaction, using intermittent microwave irradiation heating method to prepare [BMIM][BF₄],each heating 15s, stirring 30s with the magnetic stirrer after stopping heating.Finally,the total reaction time of heating 210s, [BMIM] Cl with NaBF₄ the molar ratio of 1.1:1,the yield of [BMIM][BF₄] based on the N-methyl imidazole was 86.3%. With Magnetic Resonance Spectroscopy,the results of the analysis confirmed that the end product was indeed [BMIM][BF₄]. Optimizing reaction conditions, I obtained the best technology parameters of preparation biodiesel from Tung oil using Limczyme as catalyzer by transesterification reaction: [BMIM] [BF₄] as reaction medium, pH7.5, reaction temperature 40℃,water content of 10%, immobilized lipase Limczyme amount of 5%, shaker speed of 200rpm, reaction time of 48 hours. As double substrates, the molar ratio of methanol to Tung oil should be maintained at 1.0, used to add methanol approach step by step,named "transesterification sieve plate theory".The kinetic of Limczyme catalyzed preparation of biodiesel was consistent with the double substrates Ping-Pong Bi Bi mechanism of alcohol inhibition mechanism. Kinetic equationobtained was （?）,and the largest reactionrate V_max=1.76×10^-3mol.m^-3.min^-1.g^-1,the Michaelis constant Tung oil aK_m1=0.5738mol.m^-3, the Michaelis constant of methanol K_m2=1.3746mol.m^-3,methanol inhibition constant K_iMe= 0.2025mol.m^-3.更多还原