Nguyen Le Truc, Seow Nianjia, Yang Kun-Lin
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, 117576, Singapore.
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, 117576, Singapore.
Colloids Surf B Biointerfaces. 2017 Mar 1;151:88-94. doi: 10.1016/j.colsurfb.2016.12.006. Epub 2016 Dec 8.
Hollow cross-linked enzyme aggregates of laccase (h-CLEA laccase) can be prepared by employing a millifluidic reactor carrying two coaxial laminar flows. In a confluence zone where acetonitrile and an aqueous solution of laccase meet, diffusion of acetonitrile into the aqueous solution gives rise to rapid precipitation of laccase aggregates at the water/acetonitrile interface, as is evidenced by fluorescence images. By controlling the flow rates carefully in the laminar flow regions, h-CLEA laccase around 220±10nm can be obtained, and the size of the h-CLEA laccase increases with increasing flow rates of both solutions. The h-CLEA laccase particles are distinctly different from CLEA laccase prepared in batch processes. The former only consist a crust of cross-linked enzymes (with a hollow core) whereas the latter has a highly porous structure. When the h-CLEA laccase is used as biocatalysts, their activity (0.26U/mg) is comparable to that of free enzymes at neutral pH due to the hollow structure. Moreover, the activity of h-CLEA laccase is higher than that of free laccase at high pH. For example, trypan blue (a dye molecule) can be decolorized completely in the presence of h-CLEA laccase within 270min even at pH 10.0, at which the free enzyme completely loses its activity. Because of their uniform sizes, h-CLEA laccase can be trapped in a membrane for continuous degradation of trypan blue up to 96h without losing any activity. This study shows the superiority of h-CLEA laccase compared to other types of immobilized enzymes.
漆酶的中空交联酶聚集体（h-CLEA漆酶）可以通过使用带有两个同轴层流的微流控反应器来制备。在乙腈和漆酶水溶液相遇的汇合区，乙腈扩散到水溶液中会导致漆酶聚集体在水/乙腈界面迅速沉淀，荧光图像证明了这一点。通过在层流区域小心控制流速，可以获得尺寸约为220±10nm的h-CLEA漆酶，并且h-CLEA漆酶的尺寸会随着两种溶液流速的增加而增大。h-CLEA漆酶颗粒与分批法制备的CLEA漆酶明显不同。前者仅由交联酶的外壳（具有中空核心）组成，而后者具有高度多孔的结构。当h-CLEA漆酶用作生物催化剂时，由于其空心结构，它们在中性pH下的活性（0.26U/mg）与游离酶相当。此外，h-CLEA漆酶在高pH下的活性高于游离漆酶。例如，即使在pH 10.0时，在h-CLEA漆酶存在下，锥虫蓝（一种染料分子）也能在270分钟内完全脱色，而此时游离酶完全失去活性。由于其尺寸均匀，h-CLEA漆酶可以被困在膜中，用于连续降解锥虫蓝长达96小时而不会损失任何活性。这项研究表明了h-CLEA漆酶相对于其他类型固定化酶的优越性。
