High-throughput hydrolysis of starch during permeation across α-amylase-immobilized porous hollow-fiber membranes

Abstract

Two kinds of supporting porous membranes, ethanolamine (EA) and phenol (Ph) fibers, for immobilization of α-amylase were prepared by radiation-induced graft polymerization of an epoxy-group-containing monomer, glycidyl methacrylate, onto a porous hollow-fiber membrane, and subsequent ring-opening with EA and Ph, respectively. An α-amylase solution was forced to permeate radially outward through the pores of the EA and Ph fibers. α-Amylase was captured at a density of 0.15 and 6.6 g/L of the membrane by the graft chain containing 2-hydroxyethylamino and phenyl groups, respectively. A permeation pressure of 0.10 MPa provided a space velocity of 780 and 1500 h⁻¹ for the α-amylase-immobilized EA and Ph fibers, respectively. Quantitative hydrolysis of starch during permeation of a 20 g/L starch solution in the buffer across the α-amylase-immobilized Ph fiber was attained up to a space velocity of about 2000 h⁻¹; this was achieved because of negligible diffusional mass-transfer resistance of the starch to the α-amylase due to convective flow, whereas an enzyme reaction-controlled system was observed for the α-amylase-immobilized EA fiber.

Introduction

Membrane chromatography using functional porous membranes is a powerful technique for purifying biomolecules at a higher rate and a lower operating pressure than column chromatography using functional beads (Brandt et al., 1988; Iwata et al., 1991; Tennikova et al., 1991; Kubota et al., 1996a; Gebauer et al., 1997). A target biomolecule is transported to the functional group due to convective flow driven by transmembrane pressure. We have so far prepared various types of porous membranes containing ion-exchange groups (Tsuneda et al., 1994; Matoba et al., 1995), hydrophobic-interaction ligands (Kubota et al., 1995; Kubota et al., 1996b), and pseudo-affinity ligands (Iwata et al., 1991; Kim et al., 1991), and demonstrated that the functional porous membrane is superior to the conventional gel-bead packed bed with respect to the collection rate of proteins when the protein-ligand reaction is instantaneous (Kubota et al., 1996a).

Extensive research (Horigome et al., 1974; Ohtsuka, 1984; Yajima et al., 1986; Tarhan and Buca-Izmir, 1989; Siso et al., 1990; Yamaguchi, 1992; Ivanova and Dobreva, 1994) has been carried out on immobilized enzymes because applying this technique 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 as illustrated in Fig. 1.

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, α-amylase was bound by a 2-hydroxyethylamino or phenyl group introduced onto the graft chain as illustrated in Fig. 2, and starch was used as a substrate.