L-glutamate oxidase

Abstract

The present invention provides a novel L-glutamate oxidase, a gene encoding the enzyme, and a method for producing the enzyme. By use of a gene encoding the enzyme, L-glutamate oxidase can be readily prepared at low costs through a recombinant DNA technique. The novel L-glutamate oxidase has the following physicochemical properties: (A) action: catalyzing the following reaction: L-glutamic acid+O 2 +H 2 O→α-ketoglutaric acid+H 2 O 2 +NH 3 ; (B) substrate specificity: being specific to L-glutamic acid; (C) molecular weight and subunit structure: molecular weight as determined through SDS-polyacrylamide gel electrophoresis of 70,000±6,000, molecular weight as determined through gel filtration of 140,000±10,000, and being a dimer formed of the same subunits having a molecular weight of 70,000±6,000; (D) optimum pH: around pH 6.0 to 8.5; (E) heat stability: being stable up to 60° C. at a pH of 7.4 for 30 minutes; and (F) coenzyme: flavin adenine dinucleotide (FAD).

1 . (canceled)  
     
     
         2 . An L-glutamate oxidase consisting of an amino acid sequence that is at least 90% homologous to SEQ ID NO: 1, wherein said L-glutamate oxidase has the following physicochemical properties: 
 (A) action: catalyzing the following reaction:      L-glutamic acid+O 2 +H 2 O→α-ketoglutaric acid+H 2 O 2 +NH 3 ;    (B) substrate specificity: being specific to L-glutamic acid; and    (C) coenzyme: flavin adenine dinucleotide (FAD).    
     
     
         3 .- 10 . (canceled)  
     
     
         11 . An L-glutamate oxidase which is produced through a method comprising: 
 transforming a host microorganism with an expression vector comprising an isolated polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1;    culturing the resultant transformant to thereby produce L-glutamate oxidase;    isolating the L-glutamate oxidase from the cultured product; and    purifying the L-glutamate oxidase;    wherein said L-glutamate oxidase has the following physicochemical properties:    (A) action: catalyzing the following reaction:      L-glutamic acid+O 2 +H 2 O→α-ketoglutaric acid+H 2 O 2 +NH 3 ;    (B) substrate specificity: being specific to L-glutamic acid.    
     
     
         12 . The L-glutamate oxidase of  claim 11 , wherein said host microorganism is  E. coli.    
     
     
         13 . The L-glutamate oxidase of  claim 11 , wherein said isolated polynucleotide has the nucleotide sequence of SEQ ID NO: 2.  
     
     
         14 . The L-glutamate oxidase of  claim 11 , wherein said polynucleotide is isolated from a microorganism belonging to  Streptomyces.    
     
     
         15 . The L-glutamate oxidase of  claim 2 , wherein said L-glutamate oxidase has the sequence of SEQ ID NO: 1.  
     
     
         16 . The L-glutamate oxidase of  claim 2 , wherein said L-glutamate oxidase is obtained from a microorganism belonging to  Streptomyces.    
     
     
         17 . An L-glutamate oxidase which is produced through a method comprising: 
 transforming a host microorganism with an expression vector comprising an isolated polynucleotide that hybridizes to the polynucleotide of SEQ ID NO: 2 or to the complement thereof under stringent conditions and which encodes a protein having an L-glutamate oxidase activity, wherein said stringent conditions comprise hybridization at 60° C. in a solution comprising 5×SSC, 0.1% w/v N-lauroylsarcosine sodium salt, 0.02% w/v SDS, and 0.5% w/v blocking reagent;    culturing the resultant transformant, to thereby produce L-glutamate oxidase;    isolating the L-glutamate oxidase from the cultured product; and    purifying the L-glutamate oxidase,    wherein said L-glutamate oxidase has the following physicochemical properties:    (A) action: catalyzing the following reaction:      L-glutamic acid+O 2 +H 2 O→α-ketoglutaric acid+H 2 O 2 +NH 3 ;    (B) substrate specificity: being specific to L-glutamic acid.    
     
     
         18 . The L-glutamate oxidase of  claim 17 , wherein said host microorganism is  E. coli.    
     
     
         19 . The L-glutamate oxidase of  claim 17 , wherein said polynucleotide is isolated from a microorganism belonging to  Streptomyces.