| The unique redox cycling properties of PQQ and the enzyme specific reactions of quinoprotein dehydrogenases are ideal for use as bioelectrocatalysts in highly selective and sensitive electrochemical-based sensor devices. Therefore, two quinoproteins, methanol dehydrogenase (MDH) from Methylobacterium extorquens AM1 and ethanol dehydrogenase (EDH) from Pseudomonas aeruginosa, were purified by chromatographic and ultrafiltration methods. MDH was obtained in an excellent overall yield with a final enzyme purity of greater than 97%. Storage at -80 °C in 20 mM phosphate buffer pH 7.0 showed only a negligible loss of enzyme activity after six months. A satisfactory purity of EDH was also obtained with similar stability.; A methanol sensor was developed by immobilizing MDH and EDH onto glassy carbon electrodes. Novel electron transfer (ET) mediators ferrocene (Fc), ferrocenecarboxylic acid (FcCOOH), N,N,N',N'-tetramethyl-1,4-phenylenediamine (TMPD), and N,N-dimethyl-p-phenylenediamine dihydrochloride (DMPD) for the enzyme electrode sensor were studied and compared with known mediators phenazine methosulfate (PMeS), and Wurster blue (WB). By using Fc as the preferred ET mediator, the sensor can detect both methanol and ethanol at muM levels with a rapid response. Also, O2 had no effect on the detection. By combining these enzymes, the detection of methanol in the presence of ethanol can be achieved by using two enzyme electrodes MDH/GC and EDH/GC together in series.; The separation of two highly negatively charged enantiomeric organic disulfates containing two chiral centers, which could not be separated by chiral HPLC, was investigated by capillary electrophoresis (CE). Different chiral selectors were studied, and the optimum separation for the enantiomers was achieved in less than 3 min with quaternary ammonium-beta-cyclodextrin (QA-beta-CD) as the selector. The method resulted in baseline resolution, excellent linearity, and highly reproducible migration times allowing facile evaluation of the enantiomeric purity of the individual isomers. The basis of the chiral discrimination between QA-beta-CD and the enantiomers was investigated by UV-visible spectroscopy, electrospray ionization mass spectrometry (ESI-MS) and 1H NMR spectroscopy. A parallel two-step complexation model was used to rationalize the NMR and the chiral discrimination observed during separation of the enantiomers. |
