Beijing Key Lab of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China; Department of Chemistry, University of California, Berkeley, California.
Biotechnol Bioeng. 2014 Jan;111(1):50-8. doi: 10.1002/bit.25005. Epub 2013 Aug 21.
Porcine lipase has been reversibly immobilized on a monolithic polymer support containing thiol functionalities prepared within confines of a fused silica capillary and functionalized with gold nanoparticles. Use of gold nanoparticles enabled rejuvenation of the activity of the deactivated reactor simply by stripping the inactive enzyme from the nanoparticles using 2-mercaptoethanol and subsequent immobilization of fresh lipase. This flow through enzymatic reactor was then used to catalyze the hydrolysis of glyceryl tributyrate (tributyrin). The highest activity was found within a temperature range of 37-40°C. The reaction kinetics is characterized by Michaelis-Menten constant, Km = 10.9 mmol/L, and maximum reaction rate, Vmax = 5.0 mmol/L min. The maximum reaction rate for the immobilized enzyme is 1,000 times faster compared to lipase in solution. The fast reaction rate enabled to achieve 86.7% conversion of tributyrin in mere 2.5 min and an almost complete conversion in 10 min. The reactor lost only less than 10% of its activity even after continuous pumping through it a solution of substrate equaling 1,760 reactor volumes. Finally, potential application of this enzymatic reactor was demonstrated with the transesterification of triacylglycerides from kitchen oil to fatty acid methyl esters thus demonstrating the ability of the reactor to produce biodiesel.
猪脂肪酶已可逆地固定在含有巯基官能团的整体聚合物载体上，该载体是在熔融二氧化硅毛细管的限制内制备的，并通过金纳米粒子进行功能化。使用金纳米粒子，通过使用 2-巯基乙醇从纳米粒子上剥离失活的酶，并随后固定新鲜的脂肪酶，就可以简单地使失活的反应器的活性恢复。然后，这种流经式酶反应器被用于催化甘油三丁酸酯（三丁酸酯）的水解。在 37-40°C 的温度范围内发现了最高的活性。反应动力学的特点是米氏常数 Km = 10.9 mmol/L 和最大反应速率 Vmax = 5.0 mmol/L min。与溶液中的脂肪酶相比，固定化酶的最大反应速率快 1000 倍。快速的反应速率使得在仅仅 2.5 min 内就可以实现 86.7%的三丁酸酯转化率，并且在 10 min 内几乎可以完全转化。即使在连续泵送相当于 1760 个反应器体积的底物溶液通过反应器之后，其活性也仅损失了不到 10%。最后，通过将厨房油中的三酰基甘油酯转化为脂肪酸甲酯，展示了这种酶反应器的潜在应用，从而证明了该反应器生产生物柴油的能力。
