Preparation of immobilized baker's-yeast glucose 6-phosphate dehydrogenase attached to modified sepharose and sephadex and a comparison of the properties of these preparations with those of the soluble enzyme

Abstract

1. Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate-NADP+ oxidoreductase, EC 1.1.1.49) from baker's yeast (Saccharomyces cerevisiae) was immobilized on CNBr-activated Sepharose 4B with retention of about 3% of enzyme activity. This uncharged preparation was stable for up to 4 months when stored in borate buffer, pH7.6, at 4 degrees C. 2. Stable enzyme preparations with negative or positive overall charge were made by adding valine or ethylenediamine to the CNBr-activated Sepharose 4B 30min after addition of the enzyme. 3. These three immobilized enzyme preparations retained 40-60% of their activity after 15 min at 50 degrees C. The soluble enzyme is inactivated by these conditions. 4. The soluble enzyme lost 45 and 100% of its activity on incubation for 3h at pH6 and 10 respectively. The three immobilized-enzyme preparations were completely stable over this entire pH range. 5. The pH optimum of the positively and negatively charged immobilized-enzyme preparations were about 8 and 9 respectively. The soluble enzyme and the uncharged immobilized enzyme had an optimum pH at about 8.5 6. Glucose 6-phosphate dehydrogenase immobilized on CNBr-activated Sephadex G-25 was unstable, as was enzyme attached to CNBr-activated Sepharose 4B to which glycine, asparitic acid, valine or ethylenediamine was added at the same time as the enzyme.

Co‐immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self‐sufficient oxidative biocatalyst

“…Regarding the immobilization on agarose based matrices, Anwar et al . immobilized a baker's yeast GDH on cyanogen bromide‐sepharose obtaining a retained activity of 3% . Persson et al .…”

“…42 Regarding the immobilization on agarose based matrices, Anwar et al immobilized a baker's yeast GDH on cyanogen bromide-sepharose obtaining a retained activity of 3%. 47 Persson et al purified a GDH from Bacillus subtilis by immobilizing it on thiopropyl-sepharose and recovered up to 65% of the initial activity. 48 However, as far as the authors know, this is the first time that GDH has been covalently co-immobilized with P450 BM3.…”

Immobilization of glucose 6-phosphate dehydrogenase in silica-based hydrogels: A comparative study

“…Some results found in the literature show that the time stability of immobilized G6PDH depends strongly on immobilization conditions. For instance Goheer et al found that the enzyme retained full activity over a period of 4 months when covalently attached to CNBr-activated Sepharose 4B; but when attached to CNBr-activated Sephadex G-25 for 2 weeks lost all of its activity on storage at room temperature and 40% of its activity on storage at 4 • C [40]. Allcock et al compared G6PDH in solution and G6PDH immobilized on polyphosphazene/alumina particles [41].…”

Mediated, Amperometric Biosensor for Glucose-6-Phosphate Monitoring Based on Entrapped Glucose-6-Phosphate Dehydrogenase, Mg2+Ions, Tetracyanoquinodimethane, and Nicotinamide Adenine Dinucleotide Phosphate in Carbon Paste

“…At higher temperature of 37°C, a sharp decrease in response was observed. Previously, Goheer et al (11) have reported on the effect of temperature on free and immobilized G6PDH. These authors found that the soluble enzyme preparations were rapidly deactivated at 37°C, retaining only 20% of the original activity after 15-min incubation.…”