L-glutamate is an important amino acid widely used as a food additive because of its taste enhancing property. In neurochemistry, it is a major excitatory neurotransmitter of the central nervous system and the enteric nervous system. L-glutamate can be measured by chromatographic methods which are complicated, time-consuming and require extensive sample pretreatment. The enzymatic method is chosen to overcome the problems mentioned above. Glutamate dehydrogenase (GDH) and glutamate decarboxylase (GDC) have been employed for the determination of L-glutamate. However, the GDC and GDH have some drawbacks due to poor substrate specificity and the requirement for expensive coenzyme such as NAD⁺. L-glutamate oxidase (GLOD) is used instead due to the relatively high substrate specificity comparing to GDH and GDC and no requirement for additional coenzyme.

L-glutamate oxidase is an enzyme that specifically catalyzes the oxidative deamination of L-glutamate in the presence of water and oxygen with the formation of a-ketoglutarate, ammonia and hydrogen peroxide. The hydrogen peroxide formed in this reaction can easily be detected by the chromogenic peroxidase reaction or amperometric method. Therefore, L-glutamate oxidase holds excellent potential for use as the principle component in the determination of L-glutamate.

In this study, we conducted a screening for glutamate oxidase-producing microorganisms from natural sources and investigating the physical and biochemical characteristics of the GLOD after the purification steps.

The extracellular GLOD was produced in wheat bran medium containing 2.0% wheat bran, 0.5% NaCl and 0.5% MSG. The maximum GLOD production was obtained from the cultivation of Streptomycessp. 18G in wheat bran medium at 30ºC for 60 hrs with shaking at 200 rpm. The enzyme subjected to purification steps including precipitation with ammonium sulfate, ion-exchange chromatography and gel filtration chromatography, respectively. The overall purification was 990 fold with a yield of 16.65%. The purified enzyme showed a single band in SDS-PAGE and had a specific activity of 152.35 U mg^-1.

The relative molecular weight (M_r) of the native enzyme was estimated to be approximately 120,000 by Superdex 200 HR 10/30 gel filtration chromatography. The subunit structure of the enzyme was analyzed by SDS-PAGE in 3 different polyacrylamide separating gel concentrations, i.e. 10%, 12% and 14% using a 4% stacking gel. Molecular weight of the enzyme subunits calculated from the three regression equations at different gel concentrations were 59,816, 63,252 and 60,044, respectively. Therefore, the molecular weight of the GLOD subunit was estimated to be 61,000. Since the native enzyme was approximately twice the size of the enzyme subunit, the results suggested that the enzyme consisted of two identical subunits.

The enzyme showed maximum activity in the pH range from 7.0 to 7.4. The enzyme was more stable in alkaline pH than in acidic pH. Moreover, the enzyme showed maximum activity at 37ºC under standard assay conditions. The enzyme was relatively stable from 30 to 55ºC. At 65ºC, the enzyme showed approximately 50% of the original activity. The enzyme was completely inactivated at 75ºC.

The activity of GLOD on various amino acids was investigated. L-glutamate was almost exclusively oxidized by the enzyme. In addition to L-glutamate, D-glutamate and L-aspartate were oxidized but with relative activities of 0.79% and 0.53%, respectively. The activities on other amino acids tested were undetectable.

Based on the results of this study, the GLOD from Streptomyces sp. 18G may have a potential for development of analytical systems for the specific determination of L-glutamate such as biosensors or kits for clinical diagnosis, bioprocess monitoring and food quality control. Additional studies are needed to obtain deeper insight into catalytic and physiochemical properties of the enzyme. Besides, molecular biology and bioprocess control may help promoting the production and stabilization of the enzyme.Note:

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