title: Trehalase immobilization on aminopropyl glass for analytical use.
abstract: Trehalase is the enzyme which hydrolyzes the disaccharide trehalose into two alpha-D-glucose molecules. In this article, we present the immobilization of trehalase on aminopropyl glass particles. The enzyme was extracted from Escherichia coli Mph2, a strain harboring the pTRE11 plasmid, which contains the trehalase gene. The partially purified enzyme had a specific activity of 356 U/mg and could be used for quantifying trehalose in the presence of sucrose, maltose, lactose, starch, and glycogen. Partially purified trehalase was immobilized by covalent coupling with retention of its catalytic activity. The support chosen for the majority of the experiments reported was aminopropyl glass, although spherisorb-5NH(2) and chitin were also tested. The immobilized enzyme was assayed continuously for 40 h, at pH 6.0 and 30 degrees C, and no release of enzyme molecules was detected during this procedure. The 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 beta-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 immobilized enzyme can be employed to detect trehalose molecules in micromolar 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) of 5.88 micromol glucose x min x(-1), as calculated by a Lineweaver-Burk plot.
authors.affiliation: Departamento de Bioquímica, Instituto de Química, Centro de Tecnologia, Universidade Federal do Rio de Janeiro, 21941-900 Rio de Janeiro, Brazil.
authors.forename: N
authors.initials: N
authors.lastname: Bachinski
authors.forename: A S
authors.initials: AS
authors.lastname: Martins
authors.forename: V M
authors.initials: VM
authors.lastname: Paschoalin
authors.forename: A D
authors.initials: AD
authors.lastname: Panek
authors.forename: C L
authors.initials: CL
authors.lastname: Paiva