酶法制备脂肪酸甲酯的研究
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摘要
脂肪酸甲酯是重要的化工中间体,具有广泛的应用领域,如可用来合成多种表面活性剂;用作洗涤剂以及其他许多化学制品的原料;用作高级润滑油的添加剂、机械加工的切削油、冷却液等。目前,脂肪酸甲酯最主要的作用就是作为生物柴油进行开发,将脂肪酸或脂肪转化为脂肪酸甲酯用作生物柴油可为潲水油等提供有效利用途径,因而更具有重要的食品安全意义。脂肪酸甲酯一般通过动植物油脂和甲醇的酯交换反应获得,根据催化剂性质,可将脂肪酸甲酯的制备方法分为酸催化法、碱催化法和酶催化法等。酶催化具有能耗低、产品回收简单、无污染等优点,是最具潜力的制备方法之一。本文从降低生产成本、提高反应效率着手,以菜籽油为原料,以凯泰脂肪酶为催化剂,研究了脂肪酸甲酯的酶法制备工艺,研究内容及结论如下:
对游离酶催化酯交换反应的条件进行了研究。首先探讨了反应体系和甲醇添加方式,确定了反应体系为乳化体系,甲醇分三次加入反应体系中。其次以甲酯得率为指标,通过单因素实验研究了水分含量、醇油摩尔比、反应温度、反应时间、酶用量等因素对酯交换反应的影响,并在单因素实验的基础上选择了水分含量、反应温度和醇油摩尔比等影响较显著的三个因素,采用响应面实验设计和分析方法,进一步对酶催化酯交换反应条件进行了优化。结果表明,游离酶催化酯交换反应的最适条件为:反应时间16h、酶用量400IU/g,水分含量21.5%、反应温度41℃、醇油摩尔比4.75:1,在此工艺条件下,甲酯得率为84.02%。
研究了凯泰脂肪酶的固定化。以酶的活力回收率为指标,首先对大孔树脂D101、大孔树脂AB-8、大孔树脂X-5、硅藻土、壳聚糖、海藻酸钠等固定化载体材料进行了筛选,结果表明,海藻酸钠是最合适的固定化载体。然后通过单因素实验和正交实验,对海藻酸钠固定化脂肪酶的条件进行了优化,结果表明,海藻酸钠固定化脂肪酶的最佳工艺条件为:海藻酸钠浓度为1.25%,CaCl2浓度为12%,脂肪酶用量为200IU/g,注射器针头外径0. 65mm。此条件下固定化脂肪酶时,酶活力回收率为74.36%。
在乳化反应体系中,对固定化脂肪酶催化菜籽油与甲醇的酯交换反应条件进行了优化。利用响应面设计和分析方法对酯交换反应条件进行优化的结果表明,固定化酶催化酯交换反应的最佳工艺条件为:水分含量34%、反应温度44℃、醇油摩尔比7.6:1、反应时间20h、固定化酶用量400IU/g。在此工艺条件下甲酯得率达到84.89%。固定化酶连续使用5次之后,甲酯得率仍能达到68.35%。GC-MS对所得样品的分析结果表明,脂肪酸甲酯主要有棕榈酸甲酯、硬脂酸甲酯、亚油酸甲酯、油酸甲酯、芥酸甲酯,其中油酸甲酯含量最高。
通过碱炼工艺对所得脂肪酸甲酯粗品进行初步提纯。考察了加碱量和碱炼的初始温度两个因素对产品酸价的影响。结果表明当加碱量为0.070g/g油、初始温度60℃的条件下,产品酸价为0.45mgKOH/g油,达到了生物柴油.标准对酸价的规定。
比较游离脂肪酶与固定化脂肪酶催化酯交换反应,固定化酶对甲醇具有更好的耐受性;在酶活性相当的条件下,固定化酶能达到与游离酶相同的催化效率;固定化酶能反复利用,与游离酶相比,降低了生产成本。
Fatty acid methyl ester is a significant chemical engineering intermediate, which had extensive application area, such as: synthesized multiple surface active agents; served for detergents and other kinds of chemicals; served for the additive of high lubricating oil;、cutting oil、cutting solution and so on. At the present time, the major application of FAME is used as biodiesel to develop. To produce FAME from fatty acid and fat could imply the useful loads for cooking waste oil, so it had the significance of food security. Generally, we used transesterification reaction of oils with methanol to obtain the FAME, based on the characteristic of the catalyst, the production methods of FAME could separate as acid catalysis、base catalysis and enzyme catalysis. Enzyme catalysis was one of the best potential methods, which was equipped with many merits such as low energy consumption、simple recycle、free from contamination and so on. In order to lower the cost of production and improve the efficiency of reaction, the rapeseed oil was used as raw material, the kaitai lipase was catalyst, this article researched on the technological of enzyme catalysis to prepare FAME, the research contents as follows:
The condition of free lipase catlysis transesterification was studied. Firstly, chosen the optimum reaction system and the methods of appendding methanol, the reaction system was emulsified system, the methanol was appended in the reaction system by three times. Secondly, to use the yield of FAME as index, analyzed the effect of water quantity, molar ration of methanol to oil, temperature, time、lipase quantity on transesterification by monofactorial tests. On the basis of the monofactorial tests, chosen three more marked factors, water quantity、temperature t、the molar ration of methanol to oil ,then, the response surface methodology was adopt to optimize the transesterification r eaction conditions. The results showed that, the optimized conditions of the reaction was: the reaction time was 16 hours、the lipase quantity was 400IU/g, the water quantity was 21.5%、the temperature is 41℃、the molar ration of methanol to oil is 4.75:1. Under these optimal conditions, the yield of FAME reached 84.02%.
The immobilized condition of kaitai lipase was studied. Firstly, to use the activity recovery rate of the immobilized enzyme as index, chosen the carrier material, such as: macroporous resin D101、macroporous resin AB-8、macroporous resin X-5、diatomite、chitosan、sodium alginate.the results showed that the sodium alginate was the optimal carrier material.secondly,designing monofactorial and orthogonal tests to optimize the conditions of the immobilized lipase.The results showed that, the optimal condition was: the consistency of sodium alginate is 1.00%, the consistency of calcium chloride is 12%, the dosage of the lipase is 200IU/g, the outside diameter of the syringe needle is 0. 61mm, Under these conditions the activity recovery rate which was 74.36%.
Optimized the immobilized lipase catalyze transesterification reaction of rapeseed oil with methanol which was in emulsified reaction system. The response surface methodology was used to optimize the transesterification reaction conditions, the results showed that, the optimized conditions of the reaction was: the amount of water was 34%, the reaction temperature wais 44℃, molar ratio of methanol and oil was 7.6:1, the reaction time was 20 hours、the quantity of immobilized lipase was 400IU/g.Under these optimal conditions, the methyl ester yield reached 84.89%. When the immobilized enzyme was used five times, the methyl ester ratio could reach 68.35%. This sample was analyzed by GC-MS technique, the results showed that the main component of FAME from rapeseed oil was methyl palmitale、methyl stearate、methyl linoleate、methyl oleate、methyl erucicate and the Content of methyl oleate was the highest.
To purify the crude product of FAME, alkali was apendded, inspecting the effect of the dosage of alkali and the initial temperature on acid value. The results showed that when the dosage of alkali is 0.070g/g、the initial temperature is 60℃, the acid value is 0.45mgKOH/g, which reached the standard of acid value of biodesel.
Ccomparing the free lipase with immobilized lipase catalysis transesterification: The immobilized lipase put up better survivability than free lipase; When the dosage of the enzyme activity was nearly; the immobilized lipase could reach the same catalytic efficiency; Compared with free lipase, the immobilized lipase could recycle which reduced the output cost.
对游离酶催化酯交换反应的条件进行了研究。首先探讨了反应体系和甲醇添加方式,确定了反应体系为乳化体系,甲醇分三次加入反应体系中。其次以甲酯得率为指标,通过单因素实验研究了水分含量、醇油摩尔比、反应温度、反应时间、酶用量等因素对酯交换反应的影响,并在单因素实验的基础上选择了水分含量、反应温度和醇油摩尔比等影响较显著的三个因素,采用响应面实验设计和分析方法,进一步对酶催化酯交换反应条件进行了优化。结果表明,游离酶催化酯交换反应的最适条件为:反应时间16h、酶用量400IU/g,水分含量21.5%、反应温度41℃、醇油摩尔比4.75:1,在此工艺条件下,甲酯得率为84.02%。
研究了凯泰脂肪酶的固定化。以酶的活力回收率为指标,首先对大孔树脂D101、大孔树脂AB-8、大孔树脂X-5、硅藻土、壳聚糖、海藻酸钠等固定化载体材料进行了筛选,结果表明,海藻酸钠是最合适的固定化载体。然后通过单因素实验和正交实验,对海藻酸钠固定化脂肪酶的条件进行了优化,结果表明,海藻酸钠固定化脂肪酶的最佳工艺条件为:海藻酸钠浓度为1.25%,CaCl2浓度为12%,脂肪酶用量为200IU/g,注射器针头外径0. 65mm。此条件下固定化脂肪酶时,酶活力回收率为74.36%。
在乳化反应体系中,对固定化脂肪酶催化菜籽油与甲醇的酯交换反应条件进行了优化。利用响应面设计和分析方法对酯交换反应条件进行优化的结果表明,固定化酶催化酯交换反应的最佳工艺条件为:水分含量34%、反应温度44℃、醇油摩尔比7.6:1、反应时间20h、固定化酶用量400IU/g。在此工艺条件下甲酯得率达到84.89%。固定化酶连续使用5次之后,甲酯得率仍能达到68.35%。GC-MS对所得样品的分析结果表明,脂肪酸甲酯主要有棕榈酸甲酯、硬脂酸甲酯、亚油酸甲酯、油酸甲酯、芥酸甲酯,其中油酸甲酯含量最高。
通过碱炼工艺对所得脂肪酸甲酯粗品进行初步提纯。考察了加碱量和碱炼的初始温度两个因素对产品酸价的影响。结果表明当加碱量为0.070g/g油、初始温度60℃的条件下,产品酸价为0.45mgKOH/g油,达到了生物柴油.标准对酸价的规定。
比较游离脂肪酶与固定化脂肪酶催化酯交换反应,固定化酶对甲醇具有更好的耐受性;在酶活性相当的条件下,固定化酶能达到与游离酶相同的催化效率;固定化酶能反复利用,与游离酶相比,降低了生产成本。
Fatty acid methyl ester is a significant chemical engineering intermediate, which had extensive application area, such as: synthesized multiple surface active agents; served for detergents and other kinds of chemicals; served for the additive of high lubricating oil;、cutting oil、cutting solution and so on. At the present time, the major application of FAME is used as biodiesel to develop. To produce FAME from fatty acid and fat could imply the useful loads for cooking waste oil, so it had the significance of food security. Generally, we used transesterification reaction of oils with methanol to obtain the FAME, based on the characteristic of the catalyst, the production methods of FAME could separate as acid catalysis、base catalysis and enzyme catalysis. Enzyme catalysis was one of the best potential methods, which was equipped with many merits such as low energy consumption、simple recycle、free from contamination and so on. In order to lower the cost of production and improve the efficiency of reaction, the rapeseed oil was used as raw material, the kaitai lipase was catalyst, this article researched on the technological of enzyme catalysis to prepare FAME, the research contents as follows:
The condition of free lipase catlysis transesterification was studied. Firstly, chosen the optimum reaction system and the methods of appendding methanol, the reaction system was emulsified system, the methanol was appended in the reaction system by three times. Secondly, to use the yield of FAME as index, analyzed the effect of water quantity, molar ration of methanol to oil, temperature, time、lipase quantity on transesterification by monofactorial tests. On the basis of the monofactorial tests, chosen three more marked factors, water quantity、temperature t、the molar ration of methanol to oil ,then, the response surface methodology was adopt to optimize the transesterification r eaction conditions. The results showed that, the optimized conditions of the reaction was: the reaction time was 16 hours、the lipase quantity was 400IU/g, the water quantity was 21.5%、the temperature is 41℃、the molar ration of methanol to oil is 4.75:1. Under these optimal conditions, the yield of FAME reached 84.02%.
The immobilized condition of kaitai lipase was studied. Firstly, to use the activity recovery rate of the immobilized enzyme as index, chosen the carrier material, such as: macroporous resin D101、macroporous resin AB-8、macroporous resin X-5、diatomite、chitosan、sodium alginate.the results showed that the sodium alginate was the optimal carrier material.secondly,designing monofactorial and orthogonal tests to optimize the conditions of the immobilized lipase.The results showed that, the optimal condition was: the consistency of sodium alginate is 1.00%, the consistency of calcium chloride is 12%, the dosage of the lipase is 200IU/g, the outside diameter of the syringe needle is 0. 61mm, Under these conditions the activity recovery rate which was 74.36%.
Optimized the immobilized lipase catalyze transesterification reaction of rapeseed oil with methanol which was in emulsified reaction system. The response surface methodology was used to optimize the transesterification reaction conditions, the results showed that, the optimized conditions of the reaction was: the amount of water was 34%, the reaction temperature wais 44℃, molar ratio of methanol and oil was 7.6:1, the reaction time was 20 hours、the quantity of immobilized lipase was 400IU/g.Under these optimal conditions, the methyl ester yield reached 84.89%. When the immobilized enzyme was used five times, the methyl ester ratio could reach 68.35%. This sample was analyzed by GC-MS technique, the results showed that the main component of FAME from rapeseed oil was methyl palmitale、methyl stearate、methyl linoleate、methyl oleate、methyl erucicate and the Content of methyl oleate was the highest.
To purify the crude product of FAME, alkali was apendded, inspecting the effect of the dosage of alkali and the initial temperature on acid value. The results showed that when the dosage of alkali is 0.070g/g、the initial temperature is 60℃, the acid value is 0.45mgKOH/g, which reached the standard of acid value of biodesel.
Ccomparing the free lipase with immobilized lipase catalysis transesterification: The immobilized lipase put up better survivability than free lipase; When the dosage of the enzyme activity was nearly; the immobilized lipase could reach the same catalytic efficiency; Compared with free lipase, the immobilized lipase could recycle which reduced the output cost.
引文
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