Novel glutaminase, its gene and a method of producing it

Abstract

Glutaminase is purified from an Aspergillus oryzae, its partial amino acid sequence is determined, a partial sequence of glutaminase gene is obtained by PCR based on the obtained information, and DNA fragments containing glutaminase gene from Aspergillus oryzae genomic library and cDNA library, and Aspergillus nidulans genomic library by hybridization using the partial sequence as a probe.

1 - 9 . (canceled)  
     
     
         10 . An isolated DNA, which encodes a protein comprising amino acids 1-669 of SEQ ID NO: 22.  
     
     
         11 . A recombinant vector comprising the isolated DNA of  claim 10 .  
     
     
         12 . A transformed microorganism comprising the isolated DNA of  claim 10 .  
     
     
         13 . The transformed microorganism of  claim 12 , which is of a genus selected from the group consisting of  Escherichia, Aspergillus, Neurospora , and  Rhizomucor.    
     
     
         14 . The transformed microorganism of  claim 12 , which is selected from the group consisting of  Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans, Neurospora crassa , and  Rhizomucor miehei.    
     
     
         15 . A method of producing glutaminase, comprising cultivating the transformed microorganism of  claim 12  in a culture medium for a time sufficient to produce glutaminase; and collecting the glutaminase produced.  
     
     
         16 . A method of producing glutamic acid, comprising contacting glutamine with the transformed microorganism of  claim 12 .  
     
     
         17 . An isolated DNA comprising, the nucleotide sequence of SEQ ID NO: 21.  
     
     
         18 . A recombinant vector comprising the isolated DNA of  claim 17 .  
     
     
         19 . A transformed microorganism comprising the isolated DNA of  claim 17 .  
     
     
         20 . The transformed microorganism of  claim 19 , which is of a genus selected from the group consisting of  Escherichia, Aspergillus, Neurospora , and  Rhizomucor.    
     
     
         21 . The transformed microorganism of  claim 19 , which is selected from the group consisting of  Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans, Neurospora crassa , and  Rhizomucor miehei.    
     
     
         22 . A method of producing glutaminase, comprising cultivating the transformed microorganism of  claim 19  in a culture medium for a time sufficient to produce glutaminase; and collecting the glutaminase produced.  
     
     
         23 . An isolated DNA, which encodes a protein with glutaminase activity and which hybridizes under stringent conditions to SEQ ID NO: 21, wherein said stringent conditions comprise washing in 0.1×SSC and 0.1% SDS at 65° C. for 15 minutes.  
     
     
         24 . A recombinant vector comprising the isolated DNA of  claim 23 .  
     
     
         25 . A transformed microorganism comprising the isolated DNA of  claim 23 .  
     
     
         26 . The transformed microorganism of  claim 25 , which is of a genus selected from the group consisting of  Escherichia, Aspergillus, Neurospora , and  Rhizomucor.    
     
     
         27 . The transformed microorganism of  claim 25 , which is selected from the group consisting of  Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans, Neurospora crassa , and  Rhizomucor miehei.    
     
     
         28 . A method of producing glutaminase, comprising cultivating the transformed microorganism of  claim 25  in a culture medium for a time sufficient to produce glutaminase; and collecting the glutaminase produced.  
     
     
         29 . An isolated DNA comprising, the nucleotide sequence of SEQ ID NO: 25.  
     
     
         30 . A recombinant vector comprising the isolated DNA of  claim 29 .  
     
     
         31 . A transformed microorganism comprising the isolated DNA of  claim 29 .  
     
     
         32 . The transformed microorganism of  claim 31 , which is of a genus selected from the group consisting of  Escherichia, Aspergillus, Neurospora , and  Rhizomucor.    
     
     
         33 . The transformed microorganism of  claim 31 , which is selected from the group consisting of  Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans, Neurospora crassa , and  Rhizomucor miehei.    
     
     
         34 . A method of producing glutaminase, comprising cultivating the transformed microorganism of  claim 31  in a culture medium for a time sufficient to produce glutaminase; and collecting the glutaminase produced.  
     
     
         35 . An isolated DNA, which encodes a protein with glutaminase activity and which hybridizes under stringent conditions to SEQ ID NO: 25, wherein said stringent conditions comprise washing in 0.1×SSC and 0.1% SDS at 65° C. for 15 minutes.  
     
     
         36 . A recombinant vector comprising the isolated DNA of  claim 35 .  
     
     
         37 . A transformed microorganism comprising the isolated DNA of  claim 35 .  
     
     
         38 . The transformed microorganism of  claim 37 , which is of a genus selected from the group consisting of  Escherichia, Aspergillus, Neurospora , and  Rhizomucor.    
     
     
         39 . The transformed microorganism of  claim 37 , which is selected from the group consisting of  Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans, Neurospora crassa , and  Rhizomucor miehei.    
     
     
         40 . A method of producing glutaminase, comprising cultivating the transformed microorganism of  claim 37  in a culture medium for a time sufficient to produce glutaminase; and collecting the glutaminase produced.