Nucleic acid molecules encoding phytase and pH2.5 acid phosphatase
AssigneeRohm Enzyme Finland Oy
InventorsHelena K. M. Nevalainen, Marja T. Paloheimo, Arja S. K. Miettinen-Oinonen, Tuula K. Torkkeli, Michael Cantrell, Christopher S. Piddington, John A. Rambosek, Marja K. Turunen, Richard B. Fagerstrom, Christine S. Houston
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Abstract
The present invention provides a nucleic acid molecule encoding a phytase. The present invention also provides a nucleic acid molecule encoding a pH 2.5 acid phosphatase. Also provided are vectors, host cells, and a method of overexpressing phytate degrading enzymes.
Claims91
A purified nucleic acid molecule comprising a first genetic sequence encoding a phytase having the amino acid sequence of amino acids 1-467 of SEQ ID NO:
- 8, or a catalytically active fragment thereof.
A vector comprising a first genetic sequence encoding a phytase having the amino acid sequence of amino acids 1-467 of SEQ ID NO:
- 8, or a catalytically active fragment thereof.
A purified nucleic acid molecule comprising a first genetic sequence encoding a pH 2.5 acid phosphatase having the amino acid sequence of amino acids 1-479 of SEQ ID NO:
- 2, or a catalytically active fragment thereof.
A vector comprising a first genetic sequence encoding a pH 2.5 acid phosphatase having the amino acid sequence of amino acids 1-479 of SEQ ID NO:
- 2, or a catalytically active fragment thereof.
A host cell into which has been introduced a nucleic acid molecule comprising a genetic sequence encoding a phytate degrading enzyme selected from the group consisting of a phytase having the amino acid sequence of amino acids 1-467 of SEQ ID NO:
- 8, a catalytically active fragment of said phytase, a pH 2.5 acid phosphatase having the amino acid sequence of amino acids 1-479 of SEQ ID NO:
- 2, and a catalytically active fragment of said pH 2.5 acid phosphatase.
A method for overexpressing phytate degrading enzymes in a host cell, wherein the method comprises the steps of:
- (a) preparing a DNA construct, said DNA construct comprising a first genetic sequence selected from the group consisting of a phytase having the amino acid sequence of amino acids 1-467 of SEQ ID NO:
- 8, a catalytically active fragment of said phytase, a pH 2.5 acid phosphatase having the amino acid sequence of amino acids 1-479 of SEQ ID NO:
- 2, and a catalytically active fragment of said pH 2.5 acid phosphatase;
- (b) transforming a host cell with said DNA construct;
- and
- (c) cultivating the transformed host cell under conditions suitable for growth of said host cell and for expression of said phytate degrading enzyme.
A method of cloning a fusion of the Trichoderma reesei cbh1 promoter to the Aspergillus niger var. awamori phytase signal sequence, wherein said method comprises polymerase chain amplification of a fusion of the cbh1 promoter with phytase signal sequence using a 5' primer having SEQ ID NO:
- 9 and a 3' primer having SEQ ID NO:
- 10.
A method of cloning a fusion of DNA encoding the Trichoderma reesei CBHI signal sequence to the Aspergillus niger var. awamori mature phytase coding sequence, wherein said method comprises polymerase chain amplification of a sequence encoding a fusion of the DNA encoding said CBHI signal sequence and said mature phytase coding sequence with a 5' primer having SEQ ID NO:
- 11 and a 3' primer having SEQ ID NO:
- 12.
A method of cloning a fusion of the Trichoderma reesei cbh1 promoter to the Aspergillus niger var. awamori pH 2.5 acid phosphatase signal sequence, wherein said method comprises polymerase chain amplification of a sequence encoding a fusion of said cbh1 promoter and said signal sequence with a 5' primer having SEQ ID NO:
- 15 and a 3' primer having SEQ ID NO:
- 14.
A method of cloning a fusion of DNA encoding the Trichoderma reesei CBHI signal sequence to the Aspergillus niger var. awamori coding sequence of the mature pH 2.5 acid phosphatase, wherein said method comprises polymerase chain amplification of a sequence encoding a fusion of the DNA encoding said CBHI signal sequence and said mature pH 2.5 acid phosphatase coding sequence with a 5' primer having SEQ ID NO:
- 13 and a 3' primer having SEQ ID NO:
- 14.
A method of detecting a nucleic acid molecule encoding a phytase, said method comprising
- (a) hybridizing DNA suspected of encoding said phytase to DNA having the sequence of SEQ ID NO:
- 7;
- and
- (b) detecting the formation of the resulting double-stranded nucleic acid molecule.
A method of detecting a nucleic acid molecule encoding a pH 2.5 acid phosphatase, said method comprising
- (a) hybridizing DNA suspected of encoding said phytase to DNA having the sequence of SEQ ID NO:
- 1;
- and
- (b) detecting the formation of the resulting double-stranded nucleic acid molecule.
Dependencies are inferred from the claim text.