D-aminoacylases, method for producing the same, and method for producing D-amino acids using the same

Abstract

D-aminoacylase derived from fungi is provided. The fungi capable of producing D-aminoacylase include those belonging to the genus Hypomyces, Fusarium, Auricularia, Pythium, and Menisporopsis. The fungal D-aminoacylase is useful for efficiently producing D-amino acids from N-acetyl-D-amino acids.

What is claimed is:  
     
         1 . A D-aminoacylase derived from a fungus.  
     
     
         2 . The D-aminoacylase according to  claim 1 , wherein said fungus belongs to the genus selected from the group consisting the genera Hypomyces, Fusarium, Auricularia, Pythium, and Menisporopsis.  
     
     
         3 . The D-aminoacylase according to  claim 2 , wherein said fungus belongs to the species selected from the group consisting of  Hypomyces aurantius, Hypomyces broomeanus, Hypomyces chrysospermus, Hypomyces rosellus, Hypomyces sepulcralis, Hypomyces subiculosus, Hypomyces mycophilus, Fusarium solani, Auricularia auriculajudae, Pythium aphanidermaatum,  and  Menisporopsis novaezelandiae.    
     
     
         4 . The D-aminoacylase according to  claim 3 , wherein said fungus belongs to the strain selected from the group consisting of  Hypomyces aurantius  IFO 6847,  Hypomyces broomeanus  IFO 9164,  Hypomyces rosellus  IFO 6911,  Hypomyces chrysospermus  IFO 6817,  Hypomyces sepulcralis  IFO 9102,  Hypomyces subiculosus  IFO 6892,  Hypomyces mycophilus  ATCC 76474 or IFO 6785,  Fusarium solani  IFO 9974 or IFO 9975,  Auricularia auriculajudae  IFO 5949,  Pythium aphanidermaatum  IFO 7030, and  Menisporopsis novaezelandiae  IFO 9179.  
     
     
         5 . A D-aminoacylase, which has the physico-chemical properties (a) through (f) below: 
 (a) function: the enzyme acts on N-acetyl-D-amino acids to produce corresponding D-amino acids;    (b) molecular weight: the molecular weight of the enzyme is estimated to be about 64,000 daltons by SDS-polyacrylamide gel electrophoresis, and about 56,000 daltons by gel filtration chromatography on Superdex 200 Hi-Load 6/16 (Amersham Pharmacia Biotech);    (c) substrate specificity: the enzyme acts on N-acetyl-D-tryptophan, N-acetyl-D-phenylalanine, N-acetyl-D-valine, N-acetyl-D-leucine, and N-acetyl-D-methionine, but not on N-acetyl-L-tryptophan, N-acetyl-L-phenylalanine, N-acetyl-L-valine, N-acetyl-L-leucine, or N-acetyl-L-methionine;    (d) thermostability: when heated at pH 9.5 for 30 min, the enzyme is stable at 45° C., but inactivated at higher than 60° C.;    (e) optimal temperature for activity: for the reaction at pH 7.5, the enzyme activity is optimal at about 45° C.; and    (f) stabilizer: the enzyme activity is stabilized by reducing agents, and further activated by ICH 2 CONH 2 .    
     
     
         6 . The D-aminoacylase according to  claim 5 , which is derived from the fungus belonging to the genus Hypomyces.  
     
     
         7 . The D-aminoacylase according to  claim 6 , wherein said fungus belongs to the species  Hypomyces mycophilus.    
     
     
         8 . The D-aminoacylase according to  claim 7 , wherein said fungus is  Hypomyces mycophilus  ATCC 76474 or IFO 6785 strain.  
     
     
         9 . The D-aminoacylase according to  claim 5 , comprising the amino acid sequences described in SEQ ID NOs: 1 through 5.  
     
     
         10 . A DNA encoding the D-aminoacylase according to  claim 5 .  
     
     
         11 . A method for producing D-amino acids, wherein said method comprises reacting a fungus capable of producing D-aminoacylase or D-aminoacylase derived from a fungus with N-acyl-DL-amino acid or its salt represented by the formula (I):  
       
         
           
           
               
               
           
         
       
       wherein R 1  and R 2  may be identical or different and each represents a hydrogen atom or a substituted or unsubstituted hydrocarbon group, provided that R 2  does not represent a hydrogen atom; and X is H, NH 4 , or a metal ion.  
     
     
         12 . The method for producing D-amino acids according to claim  11 , wherein said fungus belongs to a genus selected from the group consisting of the genera Hypomyces, Fusarium, Auricularia, Pythium and Menisporopsis.  
     
     
         13 . The method for producing D-amino acids according to  claim 12 , wherein said fungus belongs to the species selected from the group consisting of  Hypomyces aurantius, Hypomyces broomeanus, Hypomyces chrysospermus, Hypomyces rosellus, Hypomyces sepulcralis, Hypomyces subiculosus, Hypomyces mycophilus, Fusarium solani, Auricularia auriculajudae, Pythium aphanidermaatum,  and  Menisporopsis novaezelandiae.    
     
     
         14 . The method for producing D-amino acids according to  claim 13 , wherein said fungus is a strain selected from the group consisting of  Hypomyces aurantius  IFO 6847,  Hypomyces broomeanus  IFO 9164,  Hypomyces rosellus  IFO 6911,  Hypomyces chrysospermus  IFO 6817,  Hypomyces sepulcralis  IFO 9102,  Hypomyces subiculosus  IFO 6892,  Hypomyces mycophilus  ATCC 76474 or IFO 6785,  Fusarium solani  IFO 9974 or IFO 9975,  Auricularia auriculajudae  IFO 5949,  Pythium aphanidermaatum  IFO 7030,  Menisporopsis novaezelandiae  IFO 9179.  
     
     
         15 . The method for producing D-amino acids according to  claim 11 , wherein said D-aminoacylase has the physico-chemical properties (a) through (f) below: 
 (a) function: the enzyme acts on N-acetyl-D-amino acids to produce the corresponding D-amino acids;    (b) molecular weight: apparent molecular weight of the enzyme is estimated to be about 64,000 daltons by SDS-polyacrylamide gel electrophoresis, and about 56,000 daltons by gel filtration chromatography on Superdex 200 Hi-Load 6/16 (Amersham Pharmacia Biotech);    (c) substrate specificity: the enzyme acts on N-acetyl-D-tryptophan, N-acetyl-D-phenylalanine, N-acetyl-D-valine, N-acetyl-D-leucine and N-acetyl-D-methionine, but not on N-acetyl-L-tryptophan, N-acetyl-L-phenylalanine, N-acetyl-L-valine, N-acetyl-L-leucine and N-acetyl-L-methionine;    (d) thermostability: when heated at pH 9.5 for 30 min, the enzyme is stable at 45° C., but inactivated at higher than 60° C.;    (e) optimal temperature for activity: for the reaction at pH 7.5, the enzyme activity is optimal at about 45° C.; and    (f) stabilizer: the enzyme activity is stabilized by reducing agents, and further activated by ICH 2 CONH 2 .    
     
     
         16 . The method for producing D-amino acids according to  claim 15 , wherein said D-aminoacylase is derived from a fungus belonging to the genus Hypomyces.  
     
     
         17 . The method for preparing D-amino acids according to  claim 16 , wherein said fungus belongs to the species  Hypomyces mycophilus.    
     
     
         18 . The method for preparing D-amino acids according to  claim 17 , wherein said fungus is  Hypomyces mycophilus  ATCC 76474 or IFO 6785.  
     
     
         19 . The method for producing D-amino acids according to  claim 15 , wherein said D-aminoacylase comprises the amino acid sequences described in SEQ ID NOs: 1 through 5.  
     
     
         20 . The method for producing D-amino acids according to  claim 11 , wherein R1 and R2 in the above-described formula (I) may be identical or different and each represents a substututed or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or aralkyl group.  
     
     
         21 . The method for producing D-amino acids according to  claim 20 , wherein R 1  is an indolyl, benzyl, thiomethylethyl, isopropyl, or 2-methyl-propyl group and R 2  is a methyl, chloromethyl, phenyl, or aminomethyl group.  
     
     
         22 . A method for producing D-aminoacylase, wherein said method comprises culturing a fungus.  
     
     
         23 . The method for preparing D-aminoacylase according to  claim 22 , wherein said fungus belongs to a genus selected from the group consisting of the genera Hypomyces, Fusarium, Auricularia, Pythium, and Menisporopsis.  
     
     
         24 . The method for producing D-aminoacylase according to  claim 23 , wherein said fungus belongs to the species selected from the group consisting of  Hypomyces aurantius, Hypomyces broomeanus, Hypomyces chrysospermus, Hypomyces rosellus, Hypomyces sepulcralis, Hypomyces subiculosus, Hypomyces mycophilus, Fusarium solani, Auricularia auriculajudae, Pythium aphanidermaatum,  and  Menisporopsis novaezelandiae.    
     
     
         25 . The method for producing D-aminoacylase according to  claim 24 , wherein said fungus is a strain selected from the group consisting of  Hypomyces aurantius  IFO 6847,  Hypomyces broomeanus  IFO 9164,  Hypomyces rosellus  IFO 6911,  Hypomyces chrysospermus  IFO 6817,  Hypomyces sepulcralis  IFO 9102,  Hypomyces subiculosus  IFO 6892,  Hypomyces mycophilus  ATCC 76474 or IFO 6785,  Fusarium solani  IFO 9974 or IFO 9975,  Auricularia auriculajudae  IFO 5949,  Pythium aphanidermaatum  IFO 7030, and  Menisporopsis novaezelandiae  IFO 9179.