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Polypeptide having a phytase activity and nucleotide sequence coding therefor

A technology of phytase activity and phytase, applied in the field of polypeptides with phytase activity and nucleotide sequences encoding polypeptides, can solve problems such as limited characteristics

Active Publication Date: 2007-11-28
AB ENZYMES GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, naturally occurring phytase producers have a disadvantage: phytases are only produced in fixed amounts and have limited properties

Method used

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  • Polypeptide having a phytase activity and nucleotide sequence coding therefor
  • Polypeptide having a phytase activity and nucleotide sequence coding therefor
  • Polypeptide having a phytase activity and nucleotide sequence coding therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Embodiment 1: the mensuration of phytase activity

[0086] Phytase activity was detected with a detection mixture consisting of 0.5% phytic acid (about 5 mM), 200 mM sodium citrate, pH 5.0. After co-incubating at 37°C for 15 minutes, the same volume of 15% trichloroacetic acid was added to stop the reaction. Mix 100 microliters of the detection mixture with 900 microliters of water and 1 milliliter of 0.6M sulfuric acid, 2% ascorbic acid and 0.5% ammonium molybdate, react at 50°C for 20 minutes, and quantitatively measure the released phosphorus ions at 820nm . A standard solution of potassium phosphate was used as a reference.

Embodiment 2

[0087] Example 2: Construction of pKDa2 and pKDa4 plasmids

[0088] The sequence encoding the phytase from E. coli (Dassa et al. 1990, J. Bacteriol. 172: 5497-5500, accession number: M58704) utilizes codons from Trichoderma reesei (http: / / www.kazusa.or.jp / codon) generation and synthesis. All synthetic fragments were sequenced and the fragments with and without mutations were joined together to generate new phytase variants. In one of the resulting variants, the amino acid Val of the phytase gene 200 (GTG) becomes Tyr 200 (TAC). This variant is called Da2. The original polynucleotide ("Stammpolynucleotid") without mutations was called Da4. Mature E. coli phytase gene clones were amplified by PCR. The DNA sequence containing the CAG (Gln) codon at position 1 contains an open reading frame of 1,230 bp, which encodes a phytase of 410 amino acids (SEQ ID NO: 1 / 2).

[0089] The signal peptide of the A. niger phytase (SEQ ID NO: 3 / 4) was used to enable secretion of the E. col...

Embodiment 3

[0101] Example 3: Transformation of Trichoderma reesei with pKDa2 and pKDa4 to obtain single copy transformants

[0102] Trichoderma reesei RH 3780d was transformed with linearized expression cassettes isolated from plasmids pKDa2 and pKDa4, respectively. The technique for transforming and maintaining Trichoderma reesei refers to the technique published by Penttil et al. (1987, Gene 61: 155-164). Transformants were screened and purified twice by single spore isolation. Those transformants with the highest secretion efficiency were selected from all transformants. These transformants containing DNA from plasmid pKDa2 were designated RH 31068 and RH 31069, and those containing DNA from plasmid pKDa4 were designated RH 31071-31075 and were used for further characterization.

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Abstract

The invention relates to a recombinant DNA molecule coding for a polypeptide having a phytase activity after expression in a procaryotic or eucaryotic host cell. Said recombinant DNA molecule comprises a DNA sequence selected from a) DNA sequences that have been obtained by variations of the mature wild type E.coli phytase sequence, at least one amino acid being mutated between positions 189 and 211 and / or between positions 137 and 152 in comparison to the wild type sequence; b) DNA sequences having a homology of between 70 % and 100 % in relation to the sequences according to a); and c) DNA sequences related to the sequences according to a) and b) as a result of the degeneracy of the genetic code. During the expression of the recombinant DNA molecule in an adapted host cell, said recombinant DNA molecule is associated with an increased activity of the thus coded protein in the culture supernatant. The invention also relates to thus coded proteins.

Description

technical field [0001] The present invention relates to recombinant DNA molecules encoding polypeptides having phytase activity and the polypeptides thus encoded. In particular, the present invention relates to a recombinant DNA molecule encoding a polypeptide having phytase activity, wherein said DNA sequence is obtained by mutation of a mature Escherichia coli wild-type phytase, compared with the wild-type sequence, where the specified amino acid position occurs grooming. Furthermore, the present invention relates to the expression of recombinant phytases and their use in food and feed technology. Background of the Invention [0002] Phytic acid or inositol-1,2,3,4,5,6-dihydrogen hexaphosphate (abbreviated as phytic acid) is the main source of inositol in plant seeds and is the main storage form of phosphoric acid. About 70% of the phosphoric acid content in legume seeds is in the form of mixed potassium, magnesium and calcium salts of phytic acid. The seeds of cereals ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/63C12N15/55
CPCC12N9/16Y02P60/87
Inventor K·Q·源B·温特尔
Owner AB ENZYMES GMBH
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