Neutral phenol oxidase

Abstract

The present invention relates to a neutral phenol oxidase showing small changes depending on a substrate in optimum pH for various substrates and having high enzymatic activity at pH around neutral region, a method for producing the same, and an antibody raised against the neutral phenol oxidase. According to the present invention, dyeing of fibers, coloring of hair or the like can be achieved. In addition, according to the present invention, effects on the environment, the human body, and the like can be reduced.

1 . A neutral phenol oxidase having the following properties: 
 (1) optimum pH: being from 5.0 to 7.0, and    (2) substrate specificity:    i) catalyzing a coloring reaction by an oxidation of each of N,N-dimethyl-para-phenylenediamine, ortho-aminophenol, 2,6-dimethoxyphenol, 1,3-dihydroxynaphthol, and 4-hydroxyindole at a pH of around 6.5; and    ii) catalyzing an oxidative polymerization reaction of alkali extract of lignin.    
   
   
       2 . The neutral phenol oxidase according to  claim 1 , further having the following properties (3) to (7): 
 (3) 28 kDa (calculated by SDS-PAGE),    (4) pH stability: maintaining a relative remaining activity of at least 70% under incubation conditions at a pH of 8.0 to 9.0 for 20 hours at 30° C.,    (5) optimum temperature: being from 30° to 50° C.,    (6) thermal stability:    I) maintaining a relative remaining activity of at least 80% under incubation conditions at 0° to 30° C. for 1 hour at a pH of 7.0, as compared with an activity before incubation, and    II) maintaining a relative remaining activity of at least 90% under incubation conditions at 0 to 30° C. for 1 hour at a pH of 9.0, as compared with an activity before incubation, and    (7) isoelectric point: being about 7.4;    or the following properties (3′) to (7′):    (3′) 35 kDa (calculated by SDS-PAGE),    (4′) pH stability: maintaining a relative remaining activity of at least 75% under incubation conditions at a pH of 7.0 to 10.0 for 20 hours at 30° C.,    (5′) optimum temperature: being from 30° to 60° C.,    (6′) thermal stability:    I′) maintaining a relative remaining activity of at least 90% under incubation conditions at 0° to 50° C. for 1 hour at pH 7.0, as compared with an activity before incubation, and    II′) maintaining a relative remaining activity of at least 70% under incubation conditions at 0° to 50° C. for 1 hour at a pH of 9.0, as compared with an activity before incubation, and    (7′) isoelectric point: being about 6.8;    or the following properties (3″) to (7″):    (3″) 45 kDa (calculated by SDS-PAGE),    (4″) pH stability: maintaining a relative remaining activity of at least 70% under incubation conditions at a pH of 8.0 to 10.0 for 20 hours at 30° C.,    (5″) optimum temperature: being from 30° to 60° C.    (6″) thermal stability:    I″) maintaining a relative remaining activity of at least 80% under incubation conditions at 0° to 30° C. for 1 hour at a pH of 7.0, as compared with an activity before incubation, and    II″) maintaining a relative remaining activity of at least 90% under incubation conditions at 0° to 40° C. for 1 hour at pH 9.0, as compared with an activity before incubation, and    (7″) isoelectric point: being about 6.8.    
   
   
       3 . The neutral phenol oxidase according to  claim 1  or  2 , wherein the neutral phenol oxidase is produced by a basidiomycete belonging to the genus  Flammulina.    
   
   
       4 . The neutral phenol oxidase according to  claim 1 , wherein the basidiomycete belonging to the genus  Flammulina  is a basidiomycete belonging to  Flammulina velutipes.    
   
   
       5 . The neutral phenol oxidase according to  claim 1 , wherein the basidiomycete belonging to  Flammulina velutipes  is  Flammulina velutipes  IFO 30601 strain.  
   
   
       6 . A production method of a neutral phenol oxidase, characterized in that a basidiomycete belonging to the genus  Flammulina  is cultured at a pH of 6.0 to 12.0.  
   
   
       7 . The production method according to  claim 6 , wherein the neutral phenol oxidase is the neutral phenol oxidase of  claim 1  or  2 .  
   
   
       8 . An antibody raised against the neutral phenol oxidase of any one of  claims 1  to  5   claim 1 .  
   
   
       9 . A dyeing composition comprising the neutral phenol oxidase of  claim 1 .  
   
   
       10 . The dyeing composition according to  claim 9 , further comprising a dye.  
   
   
       11 . A dyeing method, characterized in that a subject to be dyed is brought into contact with a dye in the presence of the neutral phenol oxidase of  claim 1 , thereby dyeing the subject to be dyed.