Properties and analytical application of PQQ-dependent glycerol dehydrogenase from Gluconobacter sp. 33

Abstract

PQQ-dependent glycerol dehydrogenase (GlyDH) was purified from Gluconobacter sp. 33. Activation of this enzyme by Ca ++ and Mg ++ ions and PQQ was analysed. The purified GlyDH displayed an optimum activity at pH 7.0–7.5 and was the most stable at pH 8.5–9.5. It was determined that the rate of glycerol oxidation had a maximum at 33–37 °C. However, a rapid enzyme inactivation began at the temperatures higher than 35 °C. The calculated K M values for glycerol, d-sorbitol, and d-mannitol were 0.83, 1.0, and 2.4 mM, respectively. Several different designs of enzymatic electrodes were constructed and involved into glycerol biosensor design. Investigation of substrate selectivity of immobilised (cross-linked by glutaraldehyde) GlyDH shows, that immobilisation procedure decreases the activity of the membranes of Gluconobacter sp. 33 cells (mGlyDH) for glucose, sorbitol, mannitol, and ethanol, but increases the activity of mGlyDH for methanol and dulcitol. Soluble phenazine methosulphate (PMS) and insoluble 4-ferrocenylphenol (FP), N-(4-hydroxybenzylidene)-4-ferrocenylaniline (HBFA) were applied for construction of GlyDH-based biosensors. Inactivation of GlyDH by heavy metal ions was investigated and it was observed that biosensors based on membranes of Gluconobacter sp. 33 cells (mGlyDH) and purified PQQ-dependent glycerol dehydrogenase (pGlyDH) were sensitive to Pb ++, Cd ++, Co ++, Cu ++, Ni ++, and Hg ++. Possibility to apply GlyDH-based biosensors for detection of glycerol and triglycerides in real wine samples was shown.