洋葱假单胞菌脂肪酶的固定化及其催化合成生物柴油的工艺研究
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摘要
脂肪酶的固定化技术有助于提高酶的稳定性和维持酶的催化活力,使酶能够在工程化条件下重复使用,从而降低了生物工业行业的生产成本。本文研究了以四甲氧基硅烷(TMOS)和甲基三甲氧基硅烷(MTMS)为前驱体的溶胶-凝胶法(sol-gel)固定洋葱假单胞菌(Pseudomonas cepacia)脂肪酶,优化了固定化条件。结果表明,当TMOS为2 mmol和MTMS为10 mmol时,最优固定化条件是:水与硅烷前驱体摩尔比为10,PEG400为120μL,给酶量为200 mg。在此条件下固定化酶的蛋白质回收率为93.7 %,pNPP水解比活力为游离酶的1.54倍。
在此基础上,考察了固定化酶与游离酶的酶学性质差异。结果表明,80℃以下固定化酶能保持80%以上的酶活,而游离酶在50℃以上活力急剧降低,到80℃时残余酶活约为10%;固定化酶在50%的甲醇中处理48 h能保持85%的酶活,在90%的乙醇中处理48 h能保持31%的酶活,而游离酶残余酶活只有69%和0;固定化酶的pH稳定性与游离酶相比也有一定程度的提高。测定的酶反应动力学参数为:固定化酶的Vmax为12.3mmol/min·mg,Km值为4.04mmol/L,游离酶的Vmax为8.0mmol/min·mg,Km值为13.64mmol/L,该结果表明固定化酶比游离酶有更好的底物亲和性和催化活力。
随后比较了来源于日本天野酶公司的七种游离脂肪酶酯交换效率的差异,筛选出酯交换效率最高的Lipase PS,采用溶胶-凝胶法固定化后用于催化大豆油制备生物柴油;系统研究了固定化Lipase PS催化大豆油与甲醇酯交换反应合成生物柴油工艺中的各种影响因子(包括酶用量、醇油比、含水量、反应温度、反应时间、溶剂等)对酯交换效率的影响。结果表明,通过酯交换反应,催化4.5g大豆油合成生物柴油的最佳反应工艺参数为:固定化酶646mg,醇油比4:1,含水量6%,反应温度40℃。此条件下反应72 h后,甲酯的最高得率为96.33%,高出游离酶16.3个百分点,且固定化酶重复使用10次后仍能保持60%的酶活。
Lipase immobilization technology is widely used because it can improve lipase stability, maintain its activity, which renders lipase be reused in biochemical processing for several hundred times thus reduce production cost. In this paper lipase from Pseudomonas cepacia Burkholder was entrapped in hybrid organic-inorganic sol-gel powder prepared by base-catalyzed polymerization of tetramethoxysilane (TMOS) and methyltreimethoxysila (MTMS). When TMOS 2 mmol and MTMS 10 mmol, optimum preparation conditions were water/silane molar ratio 10, enzyme supply 200 mg, and PEG400 120μL. The percentage of protein immobilization was 93.7% and the resulting lipase specific activity of hydrolyzing pNPP was 1.54 times higher than that of the free lipase.
The characteristics of immobilized lipase were studied. The results showed that immobilized lipase remained over 80% of its activity when temperature was below 80℃while the residual activity of free one was decreased rapidly when temperature was over 50℃, and only 10% was remained when temperature reached 80℃. After incubating 48 h in 50% methanol solvent, the residual activities of immobilized lipase and free one were 85% and 69% respectively, and after incubating 48 h in 90% ethanol solvent, the residual activities of immobilized lipase and free one were 31% and 0 respectively. The kinetic parameters of Vmax and Km obtained using Lineweaver-Burk plot method were 12.3mmol/min·mg and 4.04mmol/L for immobilized lipase, 8.0mmol/min·mg and 13.64mmol/L for free lipase, respectively.
Seven different free lipases from AMANO ENZYME INC.(JAPAN) were tested in transesterification experiments. Transesterification capacity of Lipase PS from P. cepacia ranked the highest and the lipase was selected as the experimental enzyme to be immobilized in sol-gel matrix, and then the immobilized lipase was used to catalyze the transesterification reactions of soybean oil with methanol to produce biodiesel. The factors such as effects of water content, methanol/oil molar ratio, enzyme loading, temperature, organic solvents and time course in the reaction were studied. The results showed that the optimal conditions for converting 4.5g soybean oil were: temperature 40℃, 4:1methanol/oil molar ratio, 6% water content and 646mg immobilized lipase, under which conditions, methyl esters formation of 96.33% was obtained after 72 h, which was 16.3% higher than that of the free one. After continuously used for 10 times, 60% residual activity of immobilized lipase was remained. These results indicated the stability and activity of the lipase when immobilized were highly improved.
在此基础上,考察了固定化酶与游离酶的酶学性质差异。结果表明,80℃以下固定化酶能保持80%以上的酶活,而游离酶在50℃以上活力急剧降低,到80℃时残余酶活约为10%;固定化酶在50%的甲醇中处理48 h能保持85%的酶活,在90%的乙醇中处理48 h能保持31%的酶活,而游离酶残余酶活只有69%和0;固定化酶的pH稳定性与游离酶相比也有一定程度的提高。测定的酶反应动力学参数为:固定化酶的Vmax为12.3mmol/min·mg,Km值为4.04mmol/L,游离酶的Vmax为8.0mmol/min·mg,Km值为13.64mmol/L,该结果表明固定化酶比游离酶有更好的底物亲和性和催化活力。
随后比较了来源于日本天野酶公司的七种游离脂肪酶酯交换效率的差异,筛选出酯交换效率最高的Lipase PS,采用溶胶-凝胶法固定化后用于催化大豆油制备生物柴油;系统研究了固定化Lipase PS催化大豆油与甲醇酯交换反应合成生物柴油工艺中的各种影响因子(包括酶用量、醇油比、含水量、反应温度、反应时间、溶剂等)对酯交换效率的影响。结果表明,通过酯交换反应,催化4.5g大豆油合成生物柴油的最佳反应工艺参数为:固定化酶646mg,醇油比4:1,含水量6%,反应温度40℃。此条件下反应72 h后,甲酯的最高得率为96.33%,高出游离酶16.3个百分点,且固定化酶重复使用10次后仍能保持60%的酶活。
Lipase immobilization technology is widely used because it can improve lipase stability, maintain its activity, which renders lipase be reused in biochemical processing for several hundred times thus reduce production cost. In this paper lipase from Pseudomonas cepacia Burkholder was entrapped in hybrid organic-inorganic sol-gel powder prepared by base-catalyzed polymerization of tetramethoxysilane (TMOS) and methyltreimethoxysila (MTMS). When TMOS 2 mmol and MTMS 10 mmol, optimum preparation conditions were water/silane molar ratio 10, enzyme supply 200 mg, and PEG400 120μL. The percentage of protein immobilization was 93.7% and the resulting lipase specific activity of hydrolyzing pNPP was 1.54 times higher than that of the free lipase.
The characteristics of immobilized lipase were studied. The results showed that immobilized lipase remained over 80% of its activity when temperature was below 80℃while the residual activity of free one was decreased rapidly when temperature was over 50℃, and only 10% was remained when temperature reached 80℃. After incubating 48 h in 50% methanol solvent, the residual activities of immobilized lipase and free one were 85% and 69% respectively, and after incubating 48 h in 90% ethanol solvent, the residual activities of immobilized lipase and free one were 31% and 0 respectively. The kinetic parameters of Vmax and Km obtained using Lineweaver-Burk plot method were 12.3mmol/min·mg and 4.04mmol/L for immobilized lipase, 8.0mmol/min·mg and 13.64mmol/L for free lipase, respectively.
Seven different free lipases from AMANO ENZYME INC.(JAPAN) were tested in transesterification experiments. Transesterification capacity of Lipase PS from P. cepacia ranked the highest and the lipase was selected as the experimental enzyme to be immobilized in sol-gel matrix, and then the immobilized lipase was used to catalyze the transesterification reactions of soybean oil with methanol to produce biodiesel. The factors such as effects of water content, methanol/oil molar ratio, enzyme loading, temperature, organic solvents and time course in the reaction were studied. The results showed that the optimal conditions for converting 4.5g soybean oil were: temperature 40℃, 4:1methanol/oil molar ratio, 6% water content and 646mg immobilized lipase, under which conditions, methyl esters formation of 96.33% was obtained after 72 h, which was 16.3% higher than that of the free one. After continuously used for 10 times, 60% residual activity of immobilized lipase was remained. These results indicated the stability and activity of the lipase when immobilized were highly improved.
引文
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[3]魏铁麒,沙长青.微生物脂肪酶及其应用[J].生物技术, 1997, 7(4): 8-10
[4]李燕,潘运国,运毅.微生物脂肪酶催化及其性质研究进展[J].粮食与油脂, 2005, 10: 15-7
[5] Hsu A F, Kerby J, Thomas A F, et al. Optimization of alkyl ester production from grease using a phyllosilicate sol-gel immobilized lipase[J]. Biotechnology Letters, 2003, 25: 1713-1716
[6] Hsu A F, Kerby J, Thomas A F, et al. Transesterification activity of lipase immobilized in a phyllosilicate sol-gel matrix[J]. Biotechnology Letters, 2004, 26: 917-921
[7] Noureddini H, Gao X, Philkana R S. Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil[J]. Bioresource Technology, 2005, 96: 769-777.
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