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Low-temperature neutral histidine phytase APPA206, and gene and application thereof

A phytase and neutral technology, applied in the field of genetic engineering, can solve problems such as environmental pollution, soil pollution, waste of phosphorus resources, etc.

Active Publication Date: 2012-07-04
WUHAN SUNHY BIOLOGICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

40-70% of phosphorus in crops such as grains, beans, and oilseeds is in the form of phytic acid, and phosphorus in the form of phytate phosphorus is difficult to be utilized due to the lack of enzymes that can decompose phytate phosphorus in monogastric animals. The utilization rate in a typical pig diet is only 15%, and the remaining 85% of the phosphorus is excreted from the manure, which causes many problems: first, the waste of phosphorus resources, on the one hand, the phosphorus in the feed cannot be effectively used, on the other hand Then need to add a large amount of inorganic phosphorus to meet the needs of animals for phosphorus, which increases the production cost; the second, the pollution of high phosphorus ight soil to the environment, 85% phytate phosphorus in the feed is directly excreted by animals, making the surrounding soil and water heavily polluted
And up to now, there are few reports with high enzymatic activity phytase under low temperature and neutral environment

Method used

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  • Low-temperature neutral histidine phytase APPA206, and gene and application thereof
  • Low-temperature neutral histidine phytase APPA206, and gene and application thereof
  • Low-temperature neutral histidine phytase APPA206, and gene and application thereof

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Experimental program
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Effect test

Embodiment 1

[0071] Cloning of embodiment 1 Pseudomonas phytase coding gene appa206

[0072] Genomic DNA extraction of Pseudomonas sp.: take the bacterial solution cultured at 37° C. for 24 hours and centrifuge at 10,000 rpm for 10 min. Take 100 mg of bacterial cells and add 500 μL of sterile water to wash, and centrifuge to get the precipitate. The precipitate was resuspended in 500 μL extract mixture, incubated at 37°C for 60 min, and centrifuged at 10,000 rpm for 10 min to remove the precipitate. The supernatant was extracted sequentially with equal volumes of phenol and chloroform. Take the upper layer solution and add 0.6-1 times the volume of isopropanol to precipitate at room temperature for 10 minutes. Centrifuge at 12000rpm for 15min. The precipitate was washed with 70% ethanol, centrifuged slightly, dried and dissolved in 30 μL sterile water for later use.

[0073] The degenerate primers were designed and synthesized according to the conserved sequence of neutral phytase. De...

Embodiment 2

[0075] The activity analysis method of embodiment 2 phytase

[0076] The enzyme activity assay method is: dilute the enzyme solution with 0.1mol / L pH7.0 Tris-HCl buffer solution containing 0.05% BSA and 0.05% Triton X-100, take 50 μL of the diluted enzyme solution and add substrate 1.5mmol / L phytic acid Sodium 950μL (prepared with 0.1mol / L pH7.0 Tris-HCl buffer solution), react at 37°C for 15min, add 1mL10% TCA to stop the reaction, add 2mL color developing solution (10g ammonium molybdate tetrahydrate+32mL sulfuric acid+73.2g sulfuric acid Ferrous, add water to make up to 1L). For the control, after adding enzyme solution, first add TCA and mix well, then add substrate. After color development, the OD value was measured at 700 nm, and the enzyme activity was calculated.

[0077] An enzyme activity unit (U) is defined as: under certain conditions, the amount of enzyme required to release 1 nmol of inorganic phosphorus per minute is an enzyme activity unit.

Embodiment 3

[0078] The preparation of embodiment 3 recombinant phytase

[0079] According to the sequence design of the gene, the expression primers for amplifying the mature protein sequence of the phytase gene: 206-mF and 206-mR (5'-TCA GAATTC GGCCGAAACGAACCGTTACGTG-3', 206mR, 5'-CGA CTCGAG AGGGTAGCGGTACGCCGTCAGCG-3'), and introduce restriction site EcoRI at the end of primer 206mF, introduce XhoI at the end of primer 206mR, use Pseudomonas sp.206 bacterial strain genomic DNA as template, carry out the PCR amplification of phytase gene. The PCR reaction cycle conditions are: 94°C for 5min; 30 cycles of 94°C for 30s, 60°C for 30s, 72°C for 1min; 72°C for 5min. The PCR product was recovered by gel and digested with EcoR I and Xho I, and then ligated with Escherichia coli vector pET222b(+) to construct the expression vector pET22b-appa206, which was transformed into Escherichia coli BL21(DE3) by electroporation method, and was used to detect phytase activity The expression of phytase g...

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Abstract

The invention relates to the field of genetic engineering, in particular to a low-temperature neutral histidine phytase APPA206, gene thereof, a recombinant carrier comprising the gene and application. The amino acid sequence of the low-temperature neutral histidine phytase APPA206 is shown as SEQ ID NO.1, and the invention also provides the gene appa 206 which encodes the low-temperature neutral histidine phytase. The low-temperature neutral histidine phytase is obtained, has high enzymatic activity at the pH range of 4.5 to 8.5, and can resist hydrolysis of trypsin in an aquatic animal body. The characteristics are suitable for a freshwater fish feed.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a low-temperature neutral phytase APPA206 and its gene and application. Background technique [0002] Phosphorus is one of the mineral factors necessary for animal growth, reproduction and metabolism. 40-70% of phosphorus in crops such as grains, beans and oilseeds is in the form of phytic acid, and phosphorus in the form of phytate phosphorus is difficult to be utilized due to the lack of enzymes that can decompose phytate phosphorus in monogastric animals. The utilization rate in a typical pig diet is only 15%, and the remaining 85% of the phosphorus is excreted from the manure, which causes many problems: first, the waste of phosphorus resources, on the one hand, the phosphorus in the feed cannot be effectively used, on the other hand Then need to add a large amount of inorganic phosphorus to meet the needs of animals for phosphorus, which increases t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/63C12N1/19C12N1/21A23K1/165
Inventor 詹志春张菁付大伟
Owner WUHAN SUNHY BIOLOGICAL
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