title: Stabilization of a raw-starch-digesting amylase by multipoint covalent attachment on glutaraldehyde-activated amberlite beads.
abstract: Raw-starch-digesting enzyme (RSDA) was immobilized on Amberlite beads by conjugation of glutaraldehyde/ polyglutaraldehyde (PG)-activated beads or by crosslinking. The effect of immobilization on enzyme stability and catalytic efficiency was evaluated. Immobilization conditions greatly influenced the immobilization efficiency. Optimum pH values shifted from pH 5 to 6 for spontaneous crosslinking and sequential crosslinking, to pH 6-8 for RSDA covalently attached on polyglutaraldehyde-activated Amberlite beads, and to pH 7 for RSDA on glutaraldehyde-activated Amberlite. RSDA on glutaraldehyde-activated Amberlite beads had no loss of activity after 2 h storage at pH 9; enzyme on PG-activated beads lost 9%, whereas soluble enzyme lost 65% of its initial activity. Soluble enzyme lost 50% initial activity after 3 h incubation at 60 degrees C, whereas glutaraldehyde-activated derivative lost only 7.7% initial activity. RSDA derivatives retained over 90% activity after 10 batch reuse at 40 degrees C. The apparent Km of the enzyme reduced from 0.35 mg/ml to 0.32 mg/ml for RSDA on glutaraldehyde-activated RSDA but increased to 0.42 mg/ml for the PG-activated RSDA derivative. Covalent immobilization on glutaraldehyde Amberlite beads was most stable and promises to address the instability and contamination issues that impede the industrial use of RSDAs. Moreover, the cheap, porous, and non-toxic nature of Amberlite, ease of immobilization, and high yield make it more interesting for the immobilization of this enzyme.
authors.affiliation: Microbiology Department, Faculty of Biological Sciences, University of Nigeria, Nsukka, Nigeria. tochukwu.nwagu@unn.edu.ng
authors.forename: Tochukwu N
authors.initials: TN
authors.lastname: Nwagu
authors.forename: Bartho N
authors.initials: BN
authors.lastname: Okolo
authors.forename: Hideki
authors.initials: H
authors.lastname: Aoyagi
mesh_terms: D000681:Amylases / Q000737:chemistry* / Q000378:metabolism; D001230:Aspergillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D055162:Biocatalysis; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D012117:Resins, Synthetic / Q000737:chemistry; D013213:Starch / Q000737:chemistry* / Q000378:metabolism; D013696:Temperature
chemical_list: D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D012117:Resins, Synthetic; D013213:Starch; C046797:amberlite; D000681:Amylases