【作者】 杨婧；

【导师】 张卫东；

【作者基本信息】 北京化工大学 ， 化学工程， 2008， 硕士

【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。更多还原

【Abstract】 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^-2and 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.更多还原