Authenticity and reconstitution of immobilized enzymes: characterization and denaturation/renaturation of glucoamylase II.
Glucoamylase II (GA II) immobilized to Eupergit C and CIZ as a porous and nonporous matrix shows enzymatic characteristics indistinguishable from those of the free enzyme, except for reduced specific activity. Since this decrease is equally observed for both matrices, it has to be ascribed to nonproductive fixation of the enzyme or steric hindrance rather that perturbations caused by "inner diffusion" effects. Authenticity refers to the optimum pH for catalytic activity, Michaelis constants for starch and maltoheptaose, as well as identical stability toward temperature, pH, and guanidinium chloride (GdmCl). On the basis of these data, the two-state mechanism observed for the equilibrium transitions of the free enzyme may be assumed to hold also for the immobilized enzyme. Renaturation after preceding denaturation in 6.4 and 7 M GdmCl leads to widely differing yields depending on the conditions. Shifting the denaturant concentration stepwise back to nondenaturing GdmCl concentrations leads to a broad range of "hysteresis" accompanied by aggregation. Rapid dilution of the free and immobilized enzymes at pH greater than 6 and sufficiently low protein concentration leads to reactivation yields of 80 and 45%, respectively. For the free enzyme, reconstitution at lower pH is determined by the kinetic competition of folding and aggregation. In the case of the immobilized enzyme, "entangling" of the matrix with the unfolded polypeptide chain competes with renaturation.
Gottschalk
N
N
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Federal Republic of Germany.
Jaenicke
R
R
D001234:Aspergillus niger / Q000201:enzymology*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011489:Protein Denaturation; D013696:Temperature
D004800:Enzymes, Immobilized; D005087:Glucan 1,4-alpha-Glucosidase