Immobilization of enzymes onto modified polyacrylonitrile membranes: Application of the acyl azide method

Abstract

Chemical reactions toward acyl azide activated polyacrylonitrile (PAN) and conditions for membrane surface modifications are described. Ultrafiltration (UF) membranes were prepared from PAN homopolymer and copolymer with methyl acrylate. Besides hydrazide formation and nitrosation, a new method to introduce acyl azide groups into carboxyl modified PAN, using azido transfer with diphenyl phosphoryl azide, was developed. Chemical conversions were characterized, especially with Fourier transform infrared spectroscopy. The heterogeneous modifications are not chemically selective due to side reactions and/or incomplete conversion. The pore structure is altered predominately via modified polymer swelling causing changed UF fluxes and selectivities. However, for the modification via PAN reaction with hydroxyl amine, acid hydrolysis, and azido transfer, the initial membrane separations performance is qualitatively preserved. Using the acyl azide ḿethod, amylo-glucosidase (AG) (EC 3.2.1.3) was immobilized onto the modified PAN UF membranes, enabling hydrolysis of starch or maltose to glucose. Enzyme activity was assayed depending on previous chemical modification (azide content) and immobilization (pH) conditions as well as hydrolysis parameters (substrate, conversion during diffusion or UF). The best results (up to 600 mU/cm2 at 40°C and pH 5.0) were obtained after modification of PAN membranes via carboxyl creation and azido transfer. AG convalently bound to PAN is not influenced much in its catalytic properties (Km = 3.48 and 3.1 mmol/L for free and bound AG, respectively, with maltose at 40°C and pH 5.0). Under UF conditions, AG effective activity can be improved by the convective flow through the membrane. UF selectivity for the polymer starch determines effective substrate concentrations in the membrane, thus affecting observed activities and product purities in the filtrate. © 1996 John Wiley & Sons, Inc.