TREHALASE IMMOBILIZATION ON AMINOPROPYL GLASS FOR ANALYTICAL USE

Trehalase is the enzyme which hydrolyzes the disaccharide trehalose in to two alpha-D-glucose molecules. In this article, we present the immo bilization of trehalase on aminopropyl glass particles. The enzyme was extracted from Escherichia coli Mph2, a strain harboring the pTRE11 p lasmid, which contains the trehalase gene. The partially purified enzy me had a specific activity of 356 U/mg and could be used for quantifyi ng trehalose in the presence of sucrose, maltose, lactose, starch, and glycogen. Partially purified trehalase was immobilized by covalent co upling with retention of its catalytic activity. The support chosen fo r the majority of the experiments reported was aminopropyl glass, alth ough spherisorb-5NH(2) and chitin were also tested. The immobilized en zyme was assayed continuously for 40 h, at pH 6.0 and 30 degrees C, an d no release of enzyme molecules was detected during this procedure. T he best condition found for storing the enzyme-support complex was at 4 degrees C in the presence of 25 mM sodium maleate, containing 7 mM b eta-mercaptoethanol, 1 mM ethylenediaminetetraacetic acid (EDTA), and 50% glycerol. The enzyme under these conditions was stable, retaining approximately 100% of its initial activity for at least 28 days. The i mmobilized enzyme can be employed to detect trehalose molecules in mic romolar concentration. The optimum pH value found was 4.5 and the K-m app. 4.9 x 10(-3) M trehalose at pH 4.6 and 30 degrees C, with V-max o f 5.88 mu mol glucose . min.(-1), as calculated by a Lineweaver-Burk p lot. (C) 1997 John Wiley & Sons, Inc.