Phytase enzymes, nucleic acids encoding phytase enzymes and vectors and host cells incorporating same

Abstract

A novel DNA is provided which encodes an enzyme having phytase activity isolated from Penicillium. Also provided for is a method of isolating DNA encoding an enzyme having phytase activity from organisms which possess such DNA, transformation of the DNA into a suitable host organism, expression of the transformed DNA and the use of the expressed phytase protein in feed as a supplement.

1 . An isolated polynucleotide derived from a fungal source of the genus  Penicillium,  which polynucleotide comprises a nucleotide sequence encoding an enzyme having phytase activity.  
     
     
         2 . The polynucleotide of  claim 1 , wherein said fungal source is selected from the group consisting of  Penicillium piceum  and  Penicillium hordei.    
     
     
         3 . The polynucleotide of  claim 1 , wherein said enzyme comprises an amino acid sequence having at least 70% identity, and optionally at least 80% identity, to an amino acid sequence as disclosed in SEQ ID NO: 4.  
     
     
         4 . An isolated polynucleotide comprising a nucleotide sequence (i) having at least 55% identity, and optionally at least 65% identity, to a nucleotide sequence as disclosed in SEQ ID. NO: 1, SEQ ID NO:2, or SEQ ID NO:3, or (ii) being capable of hybridizing to a probe derived from the nucleotide sequence disclosed in SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3 under conditions of intermediate to high stringency, or (iii) being complementary to the nucleotide sequence disclosed in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.  
     
     
         5 . The polynucleotide of  claim 4 , wherein said nucleotide sequence has at least 75% identity, and optionally at least 85% identity, to the nucleotide sequence disclosed in SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3.  
     
     
         6 . An isolated polynucleotide encoding an enzyme having phytase activity, wherein the enzyme is derived from a  Penicillium  source.  
     
     
         7 . The polynucleotide of  claim 6 , wherein said enzyme includes an amino acid sequence having at least 70% identity, and optionally at least 80% identity, to an amino acid sequence as disclosed in SEQ ID NO: 4.  
     
     
         8 . The polynucleotide of  claim 6 , wherein said polynucleotide has (i) at least 55% identity, and optionally at least 65% identity, to a nucleotide sequence as disclosed in SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, or (ii) is capable of hybridizing to a probe derived from the nucleotide sequence disclosed in SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3 under conditions of medium to high stringency, or (iii) is complementary to the nucleotide sequence disclosed in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.  
     
     
         9 . An expression construct including a polynucleotide sequence (i) having at least 55% identity, and optionally at least 65% identity, to a nucleotide sequence as disclosed in SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, or (ii) being capable of hybridizing to a probe derived from the nucleotide sequence disclosed in SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3 under conditions of medium to high stringency, or (iii) being complementary to the nucleotide sequence disclosed in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.  
     
     
         10 . A vector including the expression construct of  claim 9 .  
     
     
         11 . A host cell transformed with the vector of  claim 10 .  
     
     
         12 . A probe for use in detecting nucleic acid sequences coding for an enzyme having phytase activity derived from a microbial source, comprising: a nucleotide sequence (i) having at least 55% identity, and optionally at least 65% identity, to a nucleotide sequence as disclosed in SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, or (ii) being capable of hybridizing to a polynucleotide including a sequence as disclosed in SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3 under conditions of medium to high stringency, or (iii) being complementary to the nucleotide sequence disclosed in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.  
     
     
         13 . The probe of  claim 12 , wherein said microbial source is a fungal source.  
     
     
         14 . The probe of  claim 13 , wherein said fungal source is a  Penicillium  species.  
     
     
         15 . Food or animal feed including an enzyme having phytase activity, wherein said enzyme comprises an amino acid sequence having at least 70% identity, and optionally at least 80% identity, to an amino acid sequence as disclosed in SEQ ID NO: 4.  
     
     
         16 . Food or animal feed including an enzyme having phytase activity, wherein said enzyme is derived from a fungal source selected from the group consisting of  Penicillium hordei  and  Penicillium piceum.    
     
     
         17 . An isolated phytase enzyme wherein said enzyme is obtained from a fungus selected from the group consisting of  P. piceum  and  P. hordei,  and has the following physiochemical properties: (1) Molecular weight: between about 45-55 kDa (non-glycosylated); and (2) Specificity: phytate.  
     
     
         18 . A method of producing an enzyme having phytase activity, comprising: 
 (a) providing a host cell transformed with an expression vector comprising a polynucleotide as defined in  claim 4;     (b) cultivating said transformed host cell under conditions suitable for said host cell to produce said phytase; and    (c) recovering said phytase.    
     
     
         19 . The method of  claim 18 , wherein said host cell is an  Aspergillus  species.  
     
     
         20 . A method of separating phosphorous from phytate, comprising: 
 treating said phytate with an enzyme comprising an amino acid sequence having at least 70% identity, and optionally at least 80% identity, to an amino acid sequence as disclosed in SEQ ID NO: 4.    
     
     
         21 . A method of separating phosphorous from phytate, comprising: 
 treating said phytate with an enzyme as defined in  claim 17 .    
     
     
         22 . The polynucleotide of  claim 1 , wherein said enzyme includes an amino acid sequence having at least 70% identity, and optionally at least 80% identity, to an amino acid sequence as disclosed in  FIG. 17 .  
     
     
         23 . An isolated polynucleotide including a nucleotide sequence (i) having at least 55% identity, and optionally at least 65% identity, to a nucleotide sequence as disclosed in  FIG. 17 , or (ii) being capable of hybridizing to a probe derived from the nucleotide sequence disclosed in  FIG. 17  under conditions of intermediate to high stringency, or (iii) being complementary to the nucleotide sequence disclosed in  FIG. 17 .  
     
     
         24 . The polynucleotide of  claim 23 , wherein said nucleotide sequence has at least 85% identity to the nucleotide sequence disclosed in  FIG. 17 .  
     
     
         25 . The isolated polynucleotide of  claim 6 , wherein the enzyme is derived from  Penicillium piceum  or  Penicillium hordei.    
     
     
         26 . The polynucleotide of  claim 25 , wherein said enzyme includes an amino acid sequence having at least 70% identity, and optionally at least 80% identity, to an amino acid sequence as disclosed in  FIG. 17 .  
     
     
         27 . The polynucleotide of  claim 25 , wherein said polynucleotide includes a nucleotide sequence (i) having at least 55% identity, and optionally at least 65% identity, to a nucleotide sequence as disclosed in  FIG. 17 , or (ii) capable of hybridizing to a probe derived from the nucleotide sequence disclosed in  FIG. 17  under conditions of medium to high stringency, or (iii) complementary to the nucleotide sequence disclosed in  FIG. 17 .  
     
     
         28 . An expression construct comprising a polynucleotide including a nucleotide sequence (i) having at least 55% identity, and optionally at least 65% identity, to a nucleotide sequence as disclosed in  FIG. 17 , or (ii) being capable of hybridizing to a probe derived from the nucleotide sequence disclosed in  FIG. 17  under conditions of medium to high stringency, or (iii) being complementary to the nucleotide sequence disclosed in  FIG. 17 .  
     
     
         29 . A vector including the expression construct of  claim 28 .  
     
     
         30 . A host cell transformed with the vector of  claim 29 .  
     
     
         31 . A probe for use in detecting nucleic acid sequences coding for an enzyme having phytase activity derived from a microbial source, comprising: a nucleotide sequence (i) having at least 55% identity, and optionally at least 65% identity, to a nucleotide sequence as disclosed in  FIG. 17 , or (ii) being capable of hybridizing to a polynucleotide including a sequence as disclosed in  FIG. 17  under conditions of medium to high stringency, or (iii) being complementary to the nucleotide sequence disclosed in  FIG. 17 .  
     
     
         32 . The probe of  claim 31 , wherein said microbial source is a fungal source.  
     
     
         33 . The probe of  claim 32 , wherein said fungal source is a  Penicillium  species.  
     
     
         34 . Food or animal feed including an enzyme having phytase activity, wherein said enzyme includes an amino acid sequence having at least 70% identity, and optionally at least 80% identity, to an amino acid sequence as disclosed in  FIG. 17 .  
     
     
         35 . A method of producing an enzyme having phytase activity, comprising: 
 (a) providing a host cell transformed with an expression vector comprising a polynucleotide as defined in  claim 23;     (b) cultivating said transformed host cell under conditions suitable for said host cell to produce said phytase; and    (c) recovering said phytase.    
     
     
         36 . The method of  claim 35 , wherein said host cell is an  Aspergillus  species.  
     
     
         37 . A method of separating phosphorous from phytate, comprising: 
 treating said phytate with an enzyme (i) having phytate hydrolyzing activity and (ii) including an amino acid sequence having at least 65% identity, and optionally at least 70% identity, to an amino acid sequence as disclosed in  FIG. 17 .    
     
     
         38 . An enzyme derived from a  Penicillium  species, optionally  P. piceum  or  P. hordei;  said enzyme being encoded by a nucleotide sequence capable of hybridising to a polynucleotide sequence as shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, or  FIG. 17  under conditions of intermediate to high stringency; said enzyme having one or more of the following physiochemical properties: 
 (1) Molecular weight: between about 45-60 kDa (non-glycosylated),    (2) An activity that is specific towards phytate, phytic acid or myo-inositol hexa-phosphate, and/or lower phosphate derivatives thereof;    (3) A theoretical pl of between about 7 and 7.6; optionally, about 7.3;    (4) A pH optimum within a range of about 4.5-5.5, optionally, about 5; and/or    (5) An ambient temperature optimum with a range of from about 40 to about 45 degree C.; optionally, 42-44 degree C.