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High heat resisting phytase yeast engineering bacteria and constructing method thereof

A technology of yeast engineering and construction method, applied in the field of bioengineering, can solve the problems of application, poor thermal stability, difficult pellets, etc., and achieve the effect of wide application prospects

Inactive Publication Date: 2013-07-17
SHANXI UNIV
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AI Technical Summary

Problems solved by technology

The phytase appA derived from Escherichia coli currently used in production practice has good pH characteristics and the highest specific activity reported so far, but its thermal stability is poor, and its activity will be lost after granulation at high temperature More than 70%, it is difficult to apply in the main pellets (Ding Qiang et al., China Agricultural Science and Technology Herald, 2010, 12(3); 27~33)

Method used

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  • High heat resisting phytase yeast engineering bacteria and constructing method thereof
  • High heat resisting phytase yeast engineering bacteria and constructing method thereof
  • High heat resisting phytase yeast engineering bacteria and constructing method thereof

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Embodiment 1

[0038] 1. Acquisition of phytase gene Appa

[0039] According to the AppA sequence (DQ513832) reported in GenBank, a biotechnology company was commissioned to synthesize the phytase gene Appa and clone it into the T-Easy vector to obtain the recombinant plasmid T-Easy-Appa.

[0040] 2. Analysis of Appa sequence glycosylation sites

[0041] When the protein contains the consensus sequence Asn-X-Ser / Thr (N-X-S / T) (X represents any amino acid), Pichia pastoris can glycosylate the amide nitrogen on the aspartyl residue to produce N- Glycosylation. According to relevant literature reports, glycosylation has an important impact on the thermal stability of the enzyme. Sequence analysis shows that the phytase encoded by the Appa gene has three glycosylation sites, namely: N139, N204, and N317 sites. The analysis found that there are two potential sites for introducing glycosylation through modification of the gene, namely: Q259, Q350, asparagine N codons: AAT, AAC, and AAC is a pref...

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Abstract

The invention relates to high heat resisting phytase yeast engineering bacteria and a constructing method thereof. Reported phytase AppA gene is analyzed by a biological information method to find a potential thermal stable associated locus. Site-specific mutagenesis is performed to the key locus of the gene by a site-specific mutagenesis technology to obtain a mutant gene Appa-M2. The mutant gene is constructed to a yeast shuttle vector pPIC9 and converted to a pichia pastoris GS115 so as to screen heat-resisting phytase strains with efficient expression. The heat resisting performance of phytase expressed by recombinant yeast engineering bacteria screened is good. The residual enzyme activity of the yeast engineering bacteria insulated for 10 minutes at 85 DEGC can reach 60%. The yeast engineering bacteria has wide application prospect in feed and food industry.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a high specific activity and high heat resistance phytase yeast engineering bacteria and a construction method thereof. Background technique [0002] Phytase is a general term for a class of enzymes that catalyze the hydrolysis of phytic acid and its salts into inositol and phosphate (salt), and belongs to phosphate monoester hydrolase. Phytase has a special spatial structure, which can sequentially separate phosphorus in phytic acid molecules, degrade phytic acid (salt) into inositol and inorganic phosphorus, and release other nutrients combined with phytic acid (salt). Phytic acid is one of the main anti-nutritional factors in plant feed, which has chelating effect on inorganic ions and proteins, thus affecting the absorption of nutrients by animals. As the main component of animal feed, plant feed contains a considerable amount of phosphorus, of which more than 50% is in the for...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N1/19C12N15/81C12R1/84
Inventor 梁爱华姚明泽王伟柴宝峰付月君张志云申泉
Owner SHANXI UNIV
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