Novel phenol oxidizing enzymes

Abstract

Disclosed herein are novel phenol oxidizing enzymes naturally-produced by strains of the species Stachybotrys which possess a pH optima in the alkaline range and which are useful in modifying the color associated with dyes and colored compounds, as well as in anti-dye transfer applications. Also disclosed herein are biologically-pure cultures of strains of the genus Stachybotrys, designated herein Stachybotrys parvispora MUCL 38996 and Stachybotrys chartarum MUCL 38898, which are capable of naturally-producing the novel phenol oxidizing enzymes. Disclosed herein is the amino acid and nucleic acid sequence for Stachybotrys phenol oxidizing enzymes as well as expression vectors and host cells comprising the nucleic acid. Disclosed herein are methods for producing the phenol oxidizing enzyme as well as methods for constructing expression hosts.

We claim:  
     
         1 . A phenol oxidizing enzyme obtainable from Stachybotrys and having at least 80% identity to the phenol oxidizing enzyme having the amino acid sequence as disclosed in SEQ ID NO:2.  
     
     
         2 . The phenol oxidizing enzyme of  claim 1  wherein said Stachybotrys includes  S. parvispora, S. chartarum, S. kampalensis, S. theobromae, S. bisbyi, S. cylindrospora, S. dichroa, S. oenanthes  and  S. nilagerica.    
     
     
         3 . The phenol oxidizing enzyme of  claim 1  having the amino acid sequence as disclosed in SEQ ID NO:2.  
     
     
         4 . An isolated polynucleotide encoding the amino acid having the sequence as shown in SEQ ID NO:2.  
     
     
         5 . The isolated polynucleotide of  claim 4  having at least 65% identity to the nucleic acid sequence disclosed in SEQ ID NO:1 or SEQ ID NO:3.  
     
     
         6 . The isolated polynucleotide of  claim 5  having the nucleic acid sequence as disclosed in SEQ ID NO:1.  
     
     
         7 . The isolated polynucleotide of  claim 5  having the nucleic acid sequence as disclosed in SEQ ID NO:3.  
     
     
         8 . An isolated polynucleotide capable of hybridizing to the polynucleotide having the sequence as shown in SEQ ID NO:1 under conditions of high stringency.  
     
     
         9 . An expression vector comprising the polynucleotide of  claim 4 .  
     
     
         10 . An expression vector comprising the polynucleotide of  claim 5 .  
     
     
         11 . An expression vector comprising the polynucleotide of  claim 8 .  
     
     
         12 . A host cell comprising the expression vector of  claim 9 ,  claim 10 , or  claim 11 .  
     
     
         13 . The host cell of  claim 12  that is a filamentous fungus.  
     
     
         14 . The host cell of  claim 13  wherein said filamentous fungus includes Aspergillus species, Trichoderma species and Mucor species.  
     
     
         15 . The host cell of  claim 13  that is a yeast.  
     
     
         16 . The host cell of  claim 15  wherein said yeast includes Saccharomyces, Pichia, Schizosaccharomyces, Hansenula, Kluyveromyces, and Yarrowia species.  
     
     
         17 . The host cell of  claim 13  wherein said host is a bacterium.  
     
     
         18 . The host cell of  claim 17  wherein said bacterium includes Bacillus and Escherichia species.  
     
     
         19 . A method for producing a phenol oxidizing enzyme obtainable from Stachybotrys in a host cell comprising the steps of: 
 (a) obtaining a host cell comprising a polynucleotide encoding said phenol oxidizing enzyme obtainable from Stachybotrys wherein said enzyme has at least 65% identity to the amino acid sequence disclosed in SEQ ID NO:2;    (b) growing said host cell under conditions suitable for the production of said phenol oxidizing enzyme; and    (c) optionally recovering said phenol oxidizing enzyme produced.    
     
     
         20 . The method of  claim 19  wherein said phenol oxidizing enzyme is obtainable from a Stachybotrys including  S. parvispora, S. chartarum, S. kampalensis, S. theobromae, S. bisbyi, S. cylindrospora, S. dichroa, S. oenanthes  and  S. nilagerica.    
     
     
         21 . The method of  claim 19  wherein said phenol oxidizing enzyme is obtainable from S. chartarum and has the amino acid sequence as disclosed in SEQ ID NO:2.  
     
     
         22 . The method of  claim 19  wherein said polynucleotide comprises the sequence as shown in SEQ ID NO:1 or SEQ ID NO:3.  
     
     
         23 . The method of  claim 19  wherein said host cell includes filamentous fungus, yeast and bacteria.  
     
     
         24 . The method of  claim 23  wherein said yeast includes Saccharomyces, Pichia, Schizosaccharomyces, Hansenula, Kluyveromyces, and Yarrowia species.  
     
     
         25 . The method of  claim 23  wherein said filamentous fungus includes Aspergillus species, Trichoderma species and Mucor species.  
     
     
         26 . The method of  claim 25  wherein said filamentous fungus is a species of Aspergillus.  
     
     
         27 . The method of  claim 26  wherein the filamentous fungus is  Aspergillus niger  var.  awamori.    
     
     
         28 . The method of  claim 23  wherein said filamentous fungus is a species of Trichoderma.  
     
     
         29 . The method of  claim 28  wherein said Trichoderma species is  Trichoderma reseei.    
     
     
         30 . A method for producing a host cell comprising a polynucleotide encoding a phenol oxidizing enzyme obtainable from Stachybotrys and having at least 65% identity to the amino acid sequence disclosed in SEQ ID NO:2 comprising the steps of: 
 (a) obtaining a polynucleotide encoding said phenol oxidizing enzyme;    (b) introducing said polynucleotide into said host cell; and    (c) growing said host cell under conditions suitable for the production of said phenol oxidizing enzyme.    
     
     
         31 . The method of  claim 30  wherein said host cell includes filamentous fungus, yeast and bacteria.  
     
     
         32 . The method of  claim 31  wherein said filamentous fungus includes Aspergillus species, Trichoderma species and Mucor species.  
     
     
         33 . The method of  claim 32  wherein said Aspergillus species is  Aspergillus niger  var.  awamori.    
     
     
         34 . The method of  claim 32  wherein said Trichoderma species is  Trichoderma reseei.    
     
     
         35 . The method of  claim 31  wherein said yeast is a Saccharomyces species.  
     
     
         36 . The method of  claim 35  wherein said Saccharomyces species is  Saccharomyces cerevisiae.    
     
     
         37 . The method of  claim 30  wherein said polynucleotide has at least 65% identity to the nucleic acid shown in SEQ ID NO:1 or SEQ ID NO:3.  
     
     
         38 . The method of  claim 30  wherein said polynucleotide has the nucleic acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:3.