A novel l‐glutamate oxidase from Streptomyces endus

Abstract

A new flavoenzyme using molecular oxygen to oxidize L-glutamic acid has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis, from the culture medium of Streptomyces endus. Hydrogen peroxide, 2-oxoglutaric acid and ammonia are formed as products. Among 25 amino acids tested including D-glutamic acid, L-glutamine and L-aspartic acid, only L-glutamic acid is converted. The molecular mass of the enzyme was estimated to be about 90 kDa by gel chromatography and 50 kDa by SDS/PAGE. The subunit contains 1 molecule noncovalently bound FAD. The absorption spectrum shows maxima at 273,355 and 457 nm and the isoelectric point is at pH 6.2.The K,,, value for L-glutamic acid in air-saturated phosphate pH 7.0 was estimated to be 1.1 mM, the K,,, for oxygen was calculated to be 1.86 mM at saturating concentration of L-glutamic acid. The enzymic reaction is inhibited by Ag' and Hg2+ ions. The enzyme described here distinctly differs from two microbial L-glutamate oxidases purified hitherto, with regard to extremely high substrate specificity and to the subunit structure. . Even if the biological importance of the more specific L-amino acid oxidases is still unknown, they can serve as valuable analytical tools for the determination of L-amino acids. In particular, the specific determination of Lglutanlate would provide new possibilities for the estimation of serum glutamate oxaloacetate transaminase and glutamate pyruvate transaminase in the clinical laboratory and is also of interest for evaluating the quality of foods.The hitherto described L-glutamate oxidases from Streptomycetes catalyze the oxidation of L-glutamic acid to 2-oxoglutaric acid, simultaneously generating NH3 and H 2 0 2 . However, the enzyme from S. violuscens oxidizes Lglutamine and L-histidine [13] in addition to L-glutamate and the enzyme from S. X-119-6 oxidizes L-aspartic acid [14] to some extent.In this paper we report on the isolation, purification and characterization of a novel L-glutamate oxidase from the culture filtrate of Streptomyces endus, which is entirely specific for the conversion of L-glutamic acid.

L-amino acid oxidases: properties and molecular mechanisms of action

“…LAAO широко представлены в природе: они обнаружены у многих видов змей [17][18][19][20][21], насекомых [22], некоторых видов бактерий [23][24][25][26][27][28][29], грибов [30][31][32][33], водорослей [34,35], моллюсков и рыб [36][37][38][39][40][41]. У млекопитающих LAAO выделены из печени, почек, мозга [42,43], секрета молочных желёз (у мышей) [44], и из полиморфноядерных лейкоцитов [45].…”

Isolation, purification, and characterization of L-glutamate oxidase from Streptomyces sp. 18G

“…18G was grown in wheat bran precultivation medium described by Böhmer et al 1989 with some modifications. The medium contained 2.0% wheat bran, 0.5% sodium chloride and 0.5% monosodium glutamate.…”

“…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. GLOD 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 (Kusakabe et al 1983;Böhmer et al 1989;Fukunaga et al 1998). The hydrogen peroxide formed in this reaction can easily be detected by the chromogenic peroxidase reaction or amperometric method (Böhmer et al 1989;Villarta et al 1991;Almeida and Mulchandani, 1993;Zilkha et al 1995;Niwa et al 1997;Chang et al 2003).…”

“…GLOD 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 (Kusakabe et al 1983;Böhmer et al 1989;Fukunaga et al 1998). The hydrogen peroxide formed in this reaction can easily be detected by the chromogenic peroxidase reaction or amperometric method (Böhmer et al 1989;Villarta et al 1991;Almeida and Mulchandani, 1993;Zilkha et al 1995;Niwa et al 1997;Chang et al 2003). Therefore, Lglutamate oxidase holds excellent potential for use as the principle component in the determination of L-glutamate (Chen and Su, 1991;White et al 1994;Ye et al 1995;Matsumoto et al 1998;Udomsopagit et al 1998;Valero and Garcia-Carmona, 1998;Yao et al 1998), although the presently available GLOD still has several disadvantages such as broad substrate specificity of the enzyme from some microorganisms (Kamei et al 1983) and high cost (Kusakabe et al 1983).…”

Amperometric Flow-Injection Biosensor System for the Simultaneous Determination of Urea and Creatinine

“…Native urease and CRDI were assayed using an NADH/ GLDH coupled method. 11 The activity of native GLDH was determined by an o-dianisidine/peroxidase method similar to the 4-aminoantipyrine/ peroxidase assay described by Bohmer et al 12 Continuous flow through method was used for the assay of immobilized enzymes. First, the enzyme preparation (dry weight, 10 -20 mg) was packed into a glass tube (i.d.…”