Evaluation of a thermophile enzyme for a carbon paste amperometric biosensor: L-glutamate dehydrogenase

An amperometric L-glutamate sensor was prepared by the immobilization of thermophilic L-glutamate dehydrogenase (L-glutamate: NADP+ oxido-reductase, deaminating and transaminating, EC 1.4.1.4.) in a carbon paste working electrode with a polyethylenimine Toluidine Blue O redox polymer mediator and lactitol/DEAE dextran stabilizing system. The response of the sensor was both pH and temperature dependent. Decreases in Kmapp (mM) and increases in Vmax (μA) were observed with increasing temperature, whilst increasing pH resulted in increases in both Kmapp and Vmax parameters. The measurements were based on the amperometric detection of the product of the enzymatic reaction, NADPH, via mediated reoxidation to NADP+ at Eapp = 100 mV versus Ag/AgCl. The major characteristics of this method are its simplicity and reproducibility, although at higher temperatures (333 K and above) the properties of the carbon paste matrix itself limited the physical stability of the system.