固定化重组脂肪酶催化木本植物油脂制备生物柴油研究
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摘要
本论文对木本植物油脂的提取与精制,脂肪酶的固定化,固定化酶催化酯交换反应制备生物柴油的工艺条件及生物柴油产品性能等方面进行了系统的研究。主要研究结果如下:
1.利用溶剂浸提法提取麻疯树籽和黄连木籽油,对毛油进行精制(脱胶和脱色),两种树籽的提油率分别达到:52.4%(种仁)和35.0%(净籽),并对所得油品理化性质进行了分析。
2.以本课题组的摇瓶发酵优化条件为指导,探讨了培养基种类、诱导时间、甲醇浓度等因素对发酵罐产酶工艺的影响。以无机盐培养基FM21发酵毕赤酵母菌,经过甘油补料和甲醇补料两个阶段,发酵培养96小时后得到的脂肪酶的酶活达到90U/mL,目的蛋白含量为0.92mg/m1。
3.筛选了一系列的无机和有机载体固定R. oryzae脂肪酶,发现阴离子树脂D301效果最佳。其最佳固定化条件为:1.预吸附条件:吸附时间10h (吸附温度为4℃)或吸附时间4h(吸附温度为28℃),给酶量600U/g。2.交联固定条件:给酶量600U/g,交联剂浓度0.5%,交联时间20min,转速110rpm,固定化脂肪酶的酶活为160U/g.
4.探讨了固定化脂肪酶催化食用大豆油脂制备生物柴油的工艺条件,并且对固定化酶的重复性进行了探讨。具体的工艺条件如下:醇油比:5:1、反应温度:37℃、反应时间:48h、水含量:60%(基于油重),所得产物中脂肪酸甲酯含量为92%,该固定化脂肪酶可重复使用7次,无明显酶活下降。
5.探讨了固定化酶催化木本油脂(麻风树和黄连木籽油)制备生物柴油的工艺条件。最佳工艺条件:醇油比:4.7:1、反应温度:37℃、反应时间:48h、水含量:40%(麻疯树籽油),20%(黄连木籽油)。在此条件下,脂肪酸甲酯的含量分别达到94%和92%。该固定化脂肪酶重复使用5次无明显酶活下降。
6.自制生物柴油产品主要性能指标(密度、运动粘度、热值、闪点、十六烷值)符合现行的美国和我国生物柴油标准要求,可作为柴油发动机燃料使用。
In this dissertation, the extraction and refining of woody plant oils, preparation of immobilized lipase for catalyzing transesterification of the oils, optimization of transesterification conditions, and properties of biodiesel products were systematically investigated. The main results are as follows:
1. The extraction yield of crude oil from the kernel of Jatropha curcas L. seed and the seed of Pistacia chinensis were 60% and 35.0%, respectively. The extracted oils were degummed and discolored prior to use as the raw material for the transesterification reactions.
2. Conditions related to cell growth and lipase expression sueh as the cultural medium variety, fermentation time and methanol concentration were investigated in a 10L fermentor based on the fermention conditions of shaker flasks. The effect of fed-batch with glycerol on the production and growth of engineering strain and with methanol as the inducers of lipase were compared. The lipase activity reached at 90U/ml after induction for 96h with increasing the velocity and concentration of methanol.
3. The immobilization of R. royzae lipase was conducted with different supports. It was found that the anion resin D301 is a proper matrix for immobilization of R. royzae. The optimal conditions for the preparation of immobilized lipase were as follows: absorption time 4h at 28℃or 10h at 4℃, enzyme amount 1200U/g resin, cross-lingking concentration 0.5% and cross-lingking time 20 min at 110 rpm. The optimal activity of the immobilized lipase is 160U/g under these conditons.
4. The transesterification conditions of refined soybean oil catalyzed by the immobilized lipase were discussed. The proper conditions were as follows: enzyme amout 24U/g oil, oil/alcohol molar ratio 1:4.8, water content 60% based on oil mass, temperature 37°C, and reaction time 48h. The maximum methyl esters (ME) yield could reach 92% under the conditions. The immobilized lipase could be repeatedly used for 7 times in the production of biodiesel without apparent loss of lipase activity.
5. The optimal alcoholysis conditions for woody plant oils were as follows: enzyme amont 24U/g oil, oil/alcohol molar ratio 1:4.7, water content (on water mass) 40% for Jatropha curcas oil and 20% for Pistacia chinensis seed oil, temperature 37°C, and reaction time 48h. The maximum methyl esters (MEs) yield could reach 94% and 92%, respectivly. The immobilized lipase could be repeatedly used for 5 times in the production of biodiesel without apparent loss of lipases activity.
6. The main properties of the prepared biodiesel, such as density, kinematic viscosity, heat value, flash point and cetane number, are well consistant with the current American and Chinese biodiesel standards.
1.利用溶剂浸提法提取麻疯树籽和黄连木籽油,对毛油进行精制(脱胶和脱色),两种树籽的提油率分别达到:52.4%(种仁)和35.0%(净籽),并对所得油品理化性质进行了分析。
2.以本课题组的摇瓶发酵优化条件为指导,探讨了培养基种类、诱导时间、甲醇浓度等因素对发酵罐产酶工艺的影响。以无机盐培养基FM21发酵毕赤酵母菌,经过甘油补料和甲醇补料两个阶段,发酵培养96小时后得到的脂肪酶的酶活达到90U/mL,目的蛋白含量为0.92mg/m1。
3.筛选了一系列的无机和有机载体固定R. oryzae脂肪酶,发现阴离子树脂D301效果最佳。其最佳固定化条件为:1.预吸附条件:吸附时间10h (吸附温度为4℃)或吸附时间4h(吸附温度为28℃),给酶量600U/g。2.交联固定条件:给酶量600U/g,交联剂浓度0.5%,交联时间20min,转速110rpm,固定化脂肪酶的酶活为160U/g.
4.探讨了固定化脂肪酶催化食用大豆油脂制备生物柴油的工艺条件,并且对固定化酶的重复性进行了探讨。具体的工艺条件如下:醇油比:5:1、反应温度:37℃、反应时间:48h、水含量:60%(基于油重),所得产物中脂肪酸甲酯含量为92%,该固定化脂肪酶可重复使用7次,无明显酶活下降。
5.探讨了固定化酶催化木本油脂(麻风树和黄连木籽油)制备生物柴油的工艺条件。最佳工艺条件:醇油比:4.7:1、反应温度:37℃、反应时间:48h、水含量:40%(麻疯树籽油),20%(黄连木籽油)。在此条件下,脂肪酸甲酯的含量分别达到94%和92%。该固定化脂肪酶重复使用5次无明显酶活下降。
6.自制生物柴油产品主要性能指标(密度、运动粘度、热值、闪点、十六烷值)符合现行的美国和我国生物柴油标准要求,可作为柴油发动机燃料使用。
In this dissertation, the extraction and refining of woody plant oils, preparation of immobilized lipase for catalyzing transesterification of the oils, optimization of transesterification conditions, and properties of biodiesel products were systematically investigated. The main results are as follows:
1. The extraction yield of crude oil from the kernel of Jatropha curcas L. seed and the seed of Pistacia chinensis were 60% and 35.0%, respectively. The extracted oils were degummed and discolored prior to use as the raw material for the transesterification reactions.
2. Conditions related to cell growth and lipase expression sueh as the cultural medium variety, fermentation time and methanol concentration were investigated in a 10L fermentor based on the fermention conditions of shaker flasks. The effect of fed-batch with glycerol on the production and growth of engineering strain and with methanol as the inducers of lipase were compared. The lipase activity reached at 90U/ml after induction for 96h with increasing the velocity and concentration of methanol.
3. The immobilization of R. royzae lipase was conducted with different supports. It was found that the anion resin D301 is a proper matrix for immobilization of R. royzae. The optimal conditions for the preparation of immobilized lipase were as follows: absorption time 4h at 28℃or 10h at 4℃, enzyme amount 1200U/g resin, cross-lingking concentration 0.5% and cross-lingking time 20 min at 110 rpm. The optimal activity of the immobilized lipase is 160U/g under these conditons.
4. The transesterification conditions of refined soybean oil catalyzed by the immobilized lipase were discussed. The proper conditions were as follows: enzyme amout 24U/g oil, oil/alcohol molar ratio 1:4.8, water content 60% based on oil mass, temperature 37°C, and reaction time 48h. The maximum methyl esters (ME) yield could reach 92% under the conditions. The immobilized lipase could be repeatedly used for 7 times in the production of biodiesel without apparent loss of lipase activity.
5. The optimal alcoholysis conditions for woody plant oils were as follows: enzyme amont 24U/g oil, oil/alcohol molar ratio 1:4.7, water content (on water mass) 40% for Jatropha curcas oil and 20% for Pistacia chinensis seed oil, temperature 37°C, and reaction time 48h. The maximum methyl esters (MEs) yield could reach 94% and 92%, respectivly. The immobilized lipase could be repeatedly used for 5 times in the production of biodiesel without apparent loss of lipases activity.
6. The main properties of the prepared biodiesel, such as density, kinematic viscosity, heat value, flash point and cetane number, are well consistant with the current American and Chinese biodiesel standards.
引文
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