Preparation of an immobilized two‐enzyme system, β‐amylase‐pullulanase, to an acrylic copolymer for the conversion of starch to maltose. II. Cocoupling of the enzymes and use in a packed bed column

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Abstract
β‐Amylase (EC 3.2.1.2) and pullulanase (EC 3.2.1.9) have been covalently bound in a two‐enzyme system to a crosslinked copolymer of acrylarmide‐acrylic acid by using a water‐soluble carbodiimide. The coupling yields based on the amounts of added β‐amylase and pullulanase were 40% and 38%, respectively, with residual enzymic activities of 22% and 32% of those of free enzymes. A markedly increased operational stability was observed for the immobilized two‐enzyme system compared to the free enzymes in solution. In order to find optimal operational conditions the influence of different pH values and temperatures on the conversion process was investigated. The action of the immobilized β‐amylase‐pullulanase derivative on partially hydrolyzed starch (DE 3.4–10.7) in a packed bed column was studied. Analysis of the product was performed using gas‐liquid chromatography.