Covalent coupling of pullulanase to an acrylic copolymer using a water soluble carbodi-imide

Pullulanase (EC 3.2.1.9) prepared from a culture of Acrobacter aerogeneshas been covalently bound to an inert crosslinked copolymer of aerylamide-acrylic acid by using a water-soluble carbodi-imide. The binding yield based on the amount of added pullulanase was 34%. The residual enzymic activity was 43%, of that of free enzyme. Coupling in the presence of the substrate pullulan gave a 5-fold increase in activity over that obtained when substrate was lacking. The effect of different carbodi-imide concentrations on the coupling has been investigated. The isoelectric point of the pullulanase preparation (3.5–4.0) was determined using isoelectric, focusing, in order to find optimal pH conditions for the coupling procedure. The immobilized pullulanase in a packed bed column was used to debranch amylopeetin to low molecular weight amylose.