Development of Fields for Enzymatic Reaction Using Grafted Polymer Chains Immobilizing Enzyme at a High Density

对固定化酶进行了广泛的研究，因为不需要在与底物反应后回收昂贵的酶。将酶包埋在凝胶基质中，例如在卡拉胶凝胶中，是一种代表性的固定化方法，其中需要底物扩散到凝胶中以到达固定化酶的活性位点。当固有反应速率快时，总反应速率由底物向凝胶中的扩散速率决定。采用多孔膜固定化酶的对流辅助反应系统将实现更高的通量，因为底物到酶的扩散路径可以被最小化。我们的研究目的是评估一个高性能的酶反应系统，使用多孔中空纤维膜含有接枝在孔表面的聚合物链。以淀粉为底物，通过在聚合物链上引入苯基，将其接枝到多孔中空纤维膜上，结合α-淀粉酶。所得的含苯基多孔膜（Ph纤维）的内径和外径分别为0.73和1.45 mm。迫使α-淀粉酶溶液通过Ph纤维的孔径向向外渗透。固定化的α-淀粉酶的量为12 mg/g Ph纤维。在缓冲液（0.010 M Tris-HCl + 0.10 M CaCl_2，pH 7）中的20 g/L淀粉溶液的渗透期间，在高达2，200 h-1的空速下实现淀粉的定量水解<-1>;这是在0.15 MPa的操作压力下实现的，因为由于对流流动，淀粉对α-淀粉酶的扩散传质阻力可以忽略不计。虽然固定化使α-淀粉酶的活性降低到10%，但被接枝链捕获的α-淀粉酶的稳定性高于可溶性α-淀粉酶。

Extensive research has been carried out on immobilized enzymes because recovery of expensive enzymes after reactions with substrates is not required. Entrapping enzymes in a gel matrix, for example, in a caragenan gel, is a representative immobilization method where the substrate is required to diffuse into the gel to reach the active site of the immobilized enzyme. When an intrinsic reaction rate is fast, the overall reaction rate is governed by the diffusion rate of the substrate into the gel. A convection-aided reaction system using an enzyme immobilized on a porous membrane will realize a higher throughput, because the diffusional path of the substrate to the enzyme can be minimized.The objective of our study was to evaluate a high performance enzyme reaction system using a porous hollow-fiber membrane containing a polymer chain grafted on the pore surface. Here, alpha-amylase was bound by a phenyl group introduced onto the graft chain, and starch was used as a substrate.A phenyl group was introduced onto the polymer chain grafted onto a porous hollow-fiber membrane to bind alpha-amylase. The resulting phenyl-group-containing porous membrane (Ph fiber) had inner and outer diameters of 0.73 and 1.45 mm, respectively. An alpha-amylase solution was forced to permeate radially outward through the pores of the Ph fiber. The amount of alpha-amylase immobilized was 12 mg per g of the Ph fiber. Quantitative hydrolysis of starch during permeation of a 20 g/L starch solution in buffer (0.010 M Tris-HCl + 0.10 M CaCl_2, pH 7) was attained up to a space velocity of 2,200 h^<-1> ; this was achieved at an operating pressure of 0.15 MPa because of negligible diffusional mass-transfer resistance of the starch to the alpha-amylase due to convective flow. Although the activity of alpha-amylase was lowered to 10% by immobilization, the stability of alpha-amylase captured by the graft chain was higher than that of soluble alpha-amylase.