Method of Analyzing L-Tryptophan in Biological Samples, and Kit Used Therein

Abstract

Disclosed is a method for quantifying L-tryptophan involving a step for mixing a specimen, L-tryptophan oxidase, and water, a step for allowing the obtained reaction solution to stand a predetermined period of time in the presence of oxygen, and a step for measuring the reaction product resulting from action of enzymes present in the reaction solution after allowing to stand. The L-tryptophan oxidase has a given amino acid sequence and has oxidase activity that generates hydrogen peroxide and ammonia by acting on the L-tryptophan in the presence of oxygen and water. The oxidase activity of the L-tryptophan oxidase on the L-phenylalanine is in the range of 0-3% of the oxidase activity thereof on the L-tryptophan, and the L-tryptophan oxidase does not have oxidase activity on protein-constituting amino acids other than L-tryptophan and L-phenylalanine. Also disclosed are a kit used to quantify the L-tryptophan containing L-tryptophan oxidase, and an enzyme sensor using the L-tryptophan oxidase. This method, kit and enzyme sensor use an L-tryptophan-specific enzyme, so are capable of quantifying L-tryptophan even in the presence of other amino acids.

1 . A kit for analyzing L-tryptophan comprising L-tryptophan oxidase,
 wherein the L-tryptophan oxidase comprises an amino acid selected from the group consisting of:   (a) the amino acid sequence of SEQ ID NO: 1 or 2,   (b) a variant amino acid sequence comprising SEQ ID NO: 1 or 2, but which has 1 to 50 amino acid deletions, substitutions, and/or additions, and   (c) an amino acid sequence having 90% or greater homology with the amino acid sequence of SEQ ID NO: 1 or 2, and   wherein said L-tryptophan oxidase comprises:   (i) oxidase activity on L-tryptophan in the presence of oxygen and water to produce hydrogen peroxide and ammonia,   (ii) oxidase activity on L-phenylalanine falling within a range of 0 to 3% of the oxidase activity on L-tryptophan, and   (iii) no oxidase activity on the protein-constituting amino acids other than L-tryptophan and L-phenylalanine.   
     
     
         2 . The kit according to  claim 1 , wherein the oxidase activity on L-phenylalanine falls within a range of 0 to 1% of the oxidase activity on L-tryptophan. 
     
     
         3 . The kit according to  claim 1 , wherein the L-tryptophan oxidase is in a mixture with a stabilizing agent. 
     
     
         4 . The kit according to  claim 3 , wherein the stabilizing agent is selected from the group consisting of glycerol, sucrose, sorbitol, trehalose, and combinations thereof. 
     
     
         5 . The kit according to  claim 1 , further comprising a component selected from the group consisting of: a reaction buffer, a reagent for detecting hydrogen peroxide, an ammonia-detecting reagent, an indole pyruvic acid-detecting reagent, and combinations thereof. 
     
     
         6 . A composition for analyzing L-tryptophan comprising L-tryptophan oxidase,
 wherein the L-tryptophan oxidase comprises an amino acid sequence selected from the group consisting of:   (a) the amino acid sequence of SEQ ID NO: 1 or 2,   (b) a variant amino acid sequence comprising SEQ ID NO: 1 or 2 but which has 1 to 50 amino acid deletions, substitutions, and/or additions, and   (c) an amino acid sequence having 90% or greater homology with the amino acid sequence of SEQ ID NO: 1 or 2, and   wherein said L-tryptophan oxidase comprises   (i) oxidase activity on L-tryptophan in the presence of oxygen and water to produce hydrogen peroxide and ammonia,   (ii) oxidase activity on L-phenylalanine falling within a range of 0 to 3% of the oxidase activity on L-tryptophan, and   (iii) no oxidase activity on the protein-constituting amino acids other than L-tryptophan and L-phenylalanine.   
     
     
         7 . The composition according to  claim 6 , wherein the oxidase activity on L-phenylalanine falls within a range of 0 to 1% of the oxidase activity on L-tryptophan. 
     
     
         8 . The composition according to  claim 6 , further comprising a stabilizing agent. 
     
     
         9 . The composition according to  claim 8 , wherein the stabilizing agent is selected from the group consisting of glycerol, sucrose, sorbitol, trehalose, and combinations thereof. 
     
     
         10 . A sensor for detecting or quantifying L-tryptophan comprising L-tryptophan oxidase and an electrode,
 wherein the electrode detects hydrogen peroxide, and   the L-tryptophan oxidase comprises an amino acid sequence selected from the group consisting of:   (a) the amino acid sequence of SEQ ID NO: 1 or 2,   (b) a variant amino acid sequence comprising SEQ ID NO: 1 or 2 but which has 1 to 50 amino acid deletions, substitutions, and/or additions, and   (c) an amino acid sequence having 90% or greater homology with the amino acid sequence of SEQ ID NO: 1 or 2, and   wherein said L-tryptophan oxidase comprises:   (i) oxidase activity on L-tryptophan in the presence of oxygen and water to produce hydrogen peroxide and ammonia,   (ii) oxidase activity on L-phenylalanine falling within a range of 0 to 3% of the oxidase activity on L-tryptophan, and   (iii) no oxidase activity on the protein-constituting amino acids other than L-tryptophan and L-phenylalanine.   
     
     
         11 . The sensor according to  claim 10 , wherein the oxidase activity on L-phenylalanine falls within a range of 0 to 1% of the oxidase activity on L-tryptophan. 
     
     
         12 . The sensor according to  claim 10 , wherein the L-tryptophan oxidase is mixed with a stabilizing agent. 
     
     
         13 . The sensor according to  claim 12 , wherein the stabilizing agent is at least one member selected from the group consisting of glycerol, sucrose, sorbitol, trehalose, and combinations thereof. 
     
     
         14 . The sensor of  claim 10 , wherein the electrode is an enzymatic hydrogen peroxide electrode or a membrane hydrogen peroxide electrode.