Low-temperature neutral phytase PhyH with double structure domains as well as gene and application thereof

A phytase and double-structure technology, applied in the field of genetic engineering, can solve problems such as low catalytic efficiency and difficulty in meeting production needs

Active Publication Date: 2011-11-23
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the fish growth environment and body temperature in aquaculture are low, and the pH is neutral, the application of HAP phytases is limited, so the BPP phytases with neutral pH characte...

Method used

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  • Low-temperature neutral phytase PhyH with double structure domains as well as gene and application thereof
  • Low-temperature neutral phytase PhyH with double structure domains as well as gene and application thereof
  • Low-temperature neutral phytase PhyH with double structure domains as well as gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Enzyme-producing characteristics of Bacillus sp.HJB17

[0061] Xinjiang Tianshang No. 1 glacier permafrost samples were cultured in a phytase screening medium (low phosphorus medium LPM), then diluted plates according to routine, and cultured at 4, 20, and 37°C respectively. Then the grown single colonies with transparent circles were separated and purified, and the active strains were identified. After 16S identification, the strains belonged to the genus Bacillus and were named Bacillus sp.HJB17.

[0062] The formula of low phosphorus medium LPM is as follows:

[0063] Casein peptone 1g; soybean peptone 0.45g; glucose 4g; glutamate 0.5g; sodium chloride 5g; MgCl 2 1.7mM; MgSO 4 1.4mM; KCl, 0.47mM; CaCl 2 0.3mM; Dissolve in 900mL 50mM pH 7.5 Tris-HCl, dilute to 1000mL, aliquot and sterilize for 15min before use.

[0064] The optimal growth conditions for Bacillus sp.HJB17 are 37°C, pH 7.0. It was detected that the bacteria had phytase activity at 37°C (0...

Embodiment 2

[0065] Example 2 Cloning of Bacillus sp.HJB17 Phytase Encoding Gene phyH

[0066]Genomic DNA of Bacillus sp.HJB17 was extracted using a Bacterial Genome Extraction Kit (Tiangen Company), and the specific operation room instructions. According to the conserved sequence of BPP phytase gene, degenerate primers BPP-F and BPP-R were designed and synthesized: BPP-F: 5′-GACGCAGCCGAYGAYCCNGCNITNTGG-3′ and BPP-R: 5′-CAGGSCGCANRTCIACRTTRTT-3′. PCR amplification was performed using the total DNA of Bacillus sp.HJB 17 as a template. A fragment of about 180bp was obtained, which was recovered and connected to the pEASY-T3 vector for sequencing.

[0067] According to the nucleotide sequence obtained by sequencing, three TAIL-PCR specific primers were designed upstream and downstream: the design direction was the direction of the unknown region to be amplified, the position of sp2 was designed to be inside sp1, and sp3 was located inside sp2. The distance between every two primers is not s...

Embodiment 3

[0071] Embodiment 3 Preparation of recombinant phytase

[0072] Recombination and purification of PhyH:

[0073] The signal peptide-removed phytase-encoding gene phyH and the expression vector pET22b(+) were digested simultaneously (EcoRI+XhoI), connected overnight, and transformed into TransI-competent cells to obtain phytic acid containing Bacillus sp.HJB 17BPP The recombinant plasmid pET22(+)-phyH of the enzyme gene phyH was transformed into BL21(DE3) by the sequenced correct recombinant plasmid pET22(+)-phyH.

[0074] Take the BL21(DE3) strain containing the plasmid, inoculate it in 300mL LB culture medium, shake it at 220rpm at 37℃ for about 2h, then add 1mM IPTG and 1mM CaCl 2 , placed at 30° C. and 220 rpm for induction, and after about 8 hours, the intracellular and extracellular phytase activities were measured. The activity of phytase was detected in both intracellular and extracellular, and the intracellular was higher than the extracellular. Through nickel colum...

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Abstract

The invention relates to the field of genetic engineering, in particular to a low-temperature neutral phytase PhyH with double structure domains as well as a gene and application thereof. The invention provides the neutral phytase PhyH from Bacillus sp.HJB17, and the amino acid sequence of the neutral phytase PhyH is shown as SEQ ID NO.1; and the invention provides a gene PhyH for coding the neutral phytase. The phytase provided by the invention has good pH stability and still keeps higher enzyme activity in the alkaline range; the phytase has higher relative enzyme activity in a low-temperature range; the phytase has double structure domains, an N terminal is an incomplete BPP (Butyl Pivalate Peroxide) phytase structure domain and a C terminal is a complete phytase structure domain. In short, the PhyH obtained by the invention can be used as the low-temperature neutral phytase which can be applied to cultivation of fish; the PhyH-DI structure domain can increase the catalytic efficiency of other phytases and is good for improving the production and the application of the other phytases.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a low-temperature neutral phytase PhyH with double domains and its gene and application. Background technique [0002] myo-phytic acid (IP 6 , myo-inositol hexakisphosphate), also known as phytic acid, is the most common form of inositol phosphate in nature. The molecular formula of phytic acid is C 6 h 18 o 24 P 6 , the molecular weight is 660.09. [0003] Phytic acid mainly exists in plant seeds in the form of calcium, magnesium, and potassium phytate, and also exists in nucleated red blood cells of animals. Phytase is found in enzymes secreted by soil, plants, and microorganisms to the outside of the cell. It is conceivable that phytase plays an important role in the metabolic cycle of phosphorus in the natural environment. In addition, phytic acid has a strong complexing ability for most metal ions. The complexing ability is similar to EDTA, bu...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/55C12N1/20C12R1/07
Inventor 姚斌杨培龙黄火清李中媛罗会颖石鹏君柏映国孟昆袁铁铮
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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