Immobilization of L-glutamate oxidase and peroxidase for glutamate determination in flow injection analysis system

Streptomyces SP.N 14, isolated from soil samples, produced extracellular L-glutamate oxidase (GOD) in liquid culture. After a two-step ammonium sulfate purification and dextran G-150 chromatography, the specific activity was reached at 28.2 U/mg. The partial purified enzyme and horseradish peroxidase (HRP) were covalently coupled to alkylamine controlled pore glass (CPG) by means of glutaraldehyde. About 200-300 U/g of immobilized GOD and 300-400 U/g of immobilized HRP were obtained. The immobilized enzymes were packed Into a teflon tube ana used in flow injection analysts (FIA) for glutamate in broth. A good linear range was observed for this immobilized enzyme system at 0.1-2.0 mM, and the precision was 2.8% (n = 25). More than 80 samples were measured within an hour. One enzyme column with about 4 U of immobilized GOD and 5 U of immobilized HRP, applied for 50 assays/d, has been used for more than 50 d. The concentration of L-glutamate remaining lower than 2.0 mM, the determination of glutamate in this system was not affected by pH and temperature within the range of 6.0-7.0 and 25-35 degrees C, respectively. The system was applied to determine L-glutamate in broth samples during L-glutamate fermentation, and good correlation was achieved between results obtained with the system and with the Warburg's method.