IMMOBILIZATION OF L-GLUTAMATE OXIDASE AND PEROXIDASE FOR GLUTAMATE DETERMINATION IN FLOW-INJECTION ANALYSIS SYSTEM

Streptomyces SP.N 14, isolated from soil samples, produced extracellul ar L-glutamate oxidase (GOD) in liquid culture. After a two-step ammon ium sulfate purification and dextran G-150 chromatography, the specifi c activity was reached at 28.2 U/mg. The partial purified enzyme and h orseradish peroxidase (HRP) were covalently coupled to alkylamine cont rolled 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. T he 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 withi n 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 t han 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, resp ectively. The system was applied to determine L-glutamate in broth sam ples during L-glutamate fermentation, and good correlation was achieve d between results obtained with the system and with the Warburg's meth od.