Abstract: To determine the quantity of free amino acids, the D-and L-forms separately, is an important task in modern nutritional studies. The aim of our present work was to develop rapid, routine methods for fast determination of the different forms of free amino acids. We utilized two enzymes (L-amino acid oxidase, D-amino acid oxidase) with broad specificity. In our home-made reactors, the enzymes were immobilized in a thin-layer Plexi-cell on natural protein membrane. The enzyme-cell was built into a FIA system and the hydrogen peroxide generated during the enzymatic reaction was determined by an amperometric detector. The electrode potential was fixed at + 100 mV. The parameters for the biochemical and electrochemical reactions were optimized in each case. The optimal pH value for measuring L-and D-amino acids was found ~8.8 and 9.5, respectively. The LAO reactor could be used for more than 900 measurements, while the DAO reactor for about 1000 measurements. The working concentration range was between 0.1-3 and 0.2-3 mM, respectively. The same standard solution (L-and D-Methionine, 1 mM) was injected 25 times sequentially and the standard deviations were 2 and 2.7%, respectively. After determining the optimal parameters, the specificity of the immobilized enzyme preparations towards different amino acids and in samples from different stages of brewing was investigated.

Abstract: A sensor for monitoring maltose concentration (0.2-4 mM, during e.g. yeast culture and fermentation) is described and tests on optimization of its use are reported. It was based on a thin-layer reactor in which glucose oxidase and amyloglucosidase (preferred over α-glucosidase) had been immobilized on a protein membrane (e.g. pig small intestine). In a flow injection analysis system, this was followed by an amperometric measuring cell having Pt anode, Ag/AgCl reference cathode and Pt auxiliary electrode, operated with recording of the anodic current at bias +600 mV. Some interference from higher oligosaccharides, dextrins and starch occurred.

Abstract: To explore new possibilities of enzyme immobilization, we investigated bioactive layers prepared by a new procedure based on three-phase partitioning (TPP) of proteins. By this method a third phase or midlayer as a protein layer can be developed at the interface of a protein system containing two phases (organic solvent/aqueous salt solution). Proteins of meat origin partitioned together with bioselective material (e.g. an enzyme) after centrifugation resulted in excellent bioactive layers. In the newly developed sensor, glucose oxidase was immobilized in a layer, which was fixed on the surface of a platinum ring electrode. The biosensor was built in a flow injection analyzer (FIA) system, where the hydrogen peroxide generated during the enzymatic reactions was determined by an amperometric cell. The parameters for biochemical and electrochemical reactions (ion concentration and pH of buffer, flow rate) were optimized. The linear range of analysis by the newly developed sensor was from 0.5 to 10 mmol L-1 glucose. The biosensor could be used for more than 300 analysis.