固定化洋葱伯克霍尔德菌脂肪酶催化合成生物柴油工艺
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摘要
以脂肪酶为催化剂制备生物柴油是一种先进的、制造工艺,已成为生物柴油新工艺的主要发展方向之一。本论文拟通过酰基受体选择、工艺参数优化和具有协同效应复合酶法催化等三种途径减轻底物对酶的负面影响,提高反应效率,以降低生产成本。本研究以所在实验室筛选得到的具优良催化性质的洋葱伯克霍尔德菌脂肪酶为催化剂,以甲醇和乙酸甲酯为酰基受体,探讨了固定化洋葱伯克霍尔德菌脂肪酶催化大豆油酯交换制备生物柴油的可行性,系统考察了酶用量、醇油比、含水量、反应温度、反应时间等因素对生物柴油转化率的影响,并采用响应面方法优化了以甲醇为酰基受体的工艺参数。主要结果如下:
(1)研究了采用乙酸甲酯为酰基受体制备生物柴油工艺。在加酶量15%、加水量4.0%、反应温度40.0℃、反应时间12.0 h、乙酸甲酯与豆油摩尔比为9.0时,甲酯得率可达80%以上。
(2)甲醇是生物柴油制备的常用酰基受体。通过单因素实验,考察了洋葱伯克霍尔德菌固定化脂肪酶催化合成生物柴油的工艺条件如反应温度、酶用量、甲醇流加次数、底物摩尔比和体系含量水对脂肪酸甲酯得率的影响,并在单因素试验的基础上,利用响应面设计优化了各主效因子,建立了甲酯得率的二次多项回归模型方程,获得了最优工艺条件:加酶量2.4%、加水量7.1%、反应温度40.4℃、反应时间10.7 h、醇油比4.5,预测甲酯得率为96.9%。为检验模型可靠性,实验实测甲酯得率为97.2%,与响应面模型预测值非常吻合。
(3)本实验室已证实具不同特异性脂肪酶之间存在协同效应,可有效降低酶的总用量,且能显著提高酶催化效率,缩短反应时间。尝试了将最常用的两种固定化脂肪酶Novozym 435、Lipozyme TLIM与固定化洋葱伯克霍尔德菌脂肪酶进行混合催化转酯反应。结果显示,复合脂肪酶能有效解决单一脂肪酶在有机溶剂体系中加酶量过高和甲酯得率低的问题,有可能发展出一条新的酶法制备生物柴油新工艺。
Enzymatic preparation of biodiesel with lipase is a green manufacturing process, which is becoming one of the main directions of new biodiesel techniques. In order to improve catalyzing effeciency and reduce production costs of biodiesel, three probable ways: the choice of new acyl receptor, optimization of process parameters, utilization of synergistic effect of combined lipases, can be employed. In this study, with methanol and methyl acetate as acyl receptor,the Burkholderia cepacia lipase isolated by ourself was applied to catalyze production of biodiesel through transesterification of soybean oil. The effects of enzyme loading, methanol/ oil molar ratio,water content, reaction temperature and reaction time on the conversion rate of biodiesel were studied in details, the parameters of biodiesel preparation with methanol as acyl receptor were optimized by using response surface methodology. The results were showed as follows:
(1) With methyl acetate as acyl receptor, under the following conditions: enzyme loading15%, water content 4.0%, reaction temperature 40.4℃,reaction time 12 h, methyl acetate /oil molar ratio 9.0, yield of methyl ester were over 80%.
(2) Methanol is usually used as acyl receptor in biodiesel preparation. In the single factorial experiments, the effects of conditions of immobilized Burkholderia cepacia lipase catalyzed production of biodiesel such as the enzyme loading, methanol/ oil molar ratio,water content, reaction temperature and reaction time on the conversion rate of biodiesel were studied. Based on the single factorial experiments, the major impact factors were optimized by response surface method, the quadric regression equation on the yield of methyl ester was established. The results showed that the optimum conditions were: enzyme loading 2.4%, water content 7.1%, reaction temperature 40.4℃, reaction time 10.7 h, methanol/ oil molar ratio 4.5,the predicted yield of methyl ester were 96.9%. In order to check up the reliability of the model, the measuring yield of methyl ester were 97.2%, which coincided with the predicted value 96.9% very well.
(3) The synergistic effect which has been proved between lipases with different characteristics can effectively reduce the total amount of enzyme loading, and significantly improve the catalyzing efficiency, so as to shorten the reaction time. The respective combination of two usual immobilized enzymes Novozym 435, Lipozyme TLIM with immobilized B. cepacia lipase from was tried. The results showed that the compound lipases could effectively solve the problems of high enzyme loading and relative lower yield of methyl ester in organic solvent system when single lipase was applied. This suggests that a new biodiesel preparation technique may be developed with the above immobilized lipases.
(1)研究了采用乙酸甲酯为酰基受体制备生物柴油工艺。在加酶量15%、加水量4.0%、反应温度40.0℃、反应时间12.0 h、乙酸甲酯与豆油摩尔比为9.0时,甲酯得率可达80%以上。
(2)甲醇是生物柴油制备的常用酰基受体。通过单因素实验,考察了洋葱伯克霍尔德菌固定化脂肪酶催化合成生物柴油的工艺条件如反应温度、酶用量、甲醇流加次数、底物摩尔比和体系含量水对脂肪酸甲酯得率的影响,并在单因素试验的基础上,利用响应面设计优化了各主效因子,建立了甲酯得率的二次多项回归模型方程,获得了最优工艺条件:加酶量2.4%、加水量7.1%、反应温度40.4℃、反应时间10.7 h、醇油比4.5,预测甲酯得率为96.9%。为检验模型可靠性,实验实测甲酯得率为97.2%,与响应面模型预测值非常吻合。
(3)本实验室已证实具不同特异性脂肪酶之间存在协同效应,可有效降低酶的总用量,且能显著提高酶催化效率,缩短反应时间。尝试了将最常用的两种固定化脂肪酶Novozym 435、Lipozyme TLIM与固定化洋葱伯克霍尔德菌脂肪酶进行混合催化转酯反应。结果显示,复合脂肪酶能有效解决单一脂肪酶在有机溶剂体系中加酶量过高和甲酯得率低的问题,有可能发展出一条新的酶法制备生物柴油新工艺。
Enzymatic preparation of biodiesel with lipase is a green manufacturing process, which is becoming one of the main directions of new biodiesel techniques. In order to improve catalyzing effeciency and reduce production costs of biodiesel, three probable ways: the choice of new acyl receptor, optimization of process parameters, utilization of synergistic effect of combined lipases, can be employed. In this study, with methanol and methyl acetate as acyl receptor,the Burkholderia cepacia lipase isolated by ourself was applied to catalyze production of biodiesel through transesterification of soybean oil. The effects of enzyme loading, methanol/ oil molar ratio,water content, reaction temperature and reaction time on the conversion rate of biodiesel were studied in details, the parameters of biodiesel preparation with methanol as acyl receptor were optimized by using response surface methodology. The results were showed as follows:
(1) With methyl acetate as acyl receptor, under the following conditions: enzyme loading15%, water content 4.0%, reaction temperature 40.4℃,reaction time 12 h, methyl acetate /oil molar ratio 9.0, yield of methyl ester were over 80%.
(2) Methanol is usually used as acyl receptor in biodiesel preparation. In the single factorial experiments, the effects of conditions of immobilized Burkholderia cepacia lipase catalyzed production of biodiesel such as the enzyme loading, methanol/ oil molar ratio,water content, reaction temperature and reaction time on the conversion rate of biodiesel were studied. Based on the single factorial experiments, the major impact factors were optimized by response surface method, the quadric regression equation on the yield of methyl ester was established. The results showed that the optimum conditions were: enzyme loading 2.4%, water content 7.1%, reaction temperature 40.4℃, reaction time 10.7 h, methanol/ oil molar ratio 4.5,the predicted yield of methyl ester were 96.9%. In order to check up the reliability of the model, the measuring yield of methyl ester were 97.2%, which coincided with the predicted value 96.9% very well.
(3) The synergistic effect which has been proved between lipases with different characteristics can effectively reduce the total amount of enzyme loading, and significantly improve the catalyzing efficiency, so as to shorten the reaction time. The respective combination of two usual immobilized enzymes Novozym 435, Lipozyme TLIM with immobilized B. cepacia lipase from was tried. The results showed that the compound lipases could effectively solve the problems of high enzyme loading and relative lower yield of methyl ester in organic solvent system when single lipase was applied. This suggests that a new biodiesel preparation technique may be developed with the above immobilized lipases.
引文
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