Neutral phytase CP53 from rumen and gene and application thereof

A phytase and neutral technology, applied in the field of genetic engineering, can solve problems such as no neutral phytase

Active Publication Date: 2012-03-07
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

[0004] CP phytase is a type of phytase isolated from the rumen environment. At present, there are relatively few phytases whose properties have been tested. The optimum pH value of this type of phytase is generally between 4.0-5.0. See neutral phytase report

Method used

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  • Neutral phytase CP53 from rumen and gene and application thereof
  • Neutral phytase CP53 from rumen and gene and application thereof
  • Neutral phytase CP53 from rumen and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Extraction of Rumen Environment Metagenome

[0053] The metagenomic DNA of goat and cattle rumen fluid microorganisms were extracted by direct lysis method. The specific steps are as follows: 1. Take 50 mL of rumen juice, filter with gauze to remove undigested grass roots and large particles, and wash some microorganisms adsorbed in the grass roots with an appropriate amount of sterilized phosphate buffer; 2. Transfer the supernatant to multiple new In a 50mL centrifuge tube, add twice the volume of preheated lysis buffer, bathe in 70°C water for 2 hours, and shake gently every 10 minutes to mix; 3. After taking it out, cool to room temperature, centrifuge at 5,000rpm for 10 minutes, discard the precipitate, and repeat centrifugation Once; 4. Add 0.7 times the volume of isopropanol to the supernatant, mix gently, place on ice for 30 minutes, centrifuge at 10,000rpm for 30 minutes, wash the precipitate with 70% ethanol once, vacuum dry for 10 minutes, dissolve ...

Embodiment 2

[0054] Example 2 Cloning of phytase-encoding gene cp53

[0055] According to the conservation of CP phytase gene ([V / M / I]-D-L-R-[Q / K / E]-E-[S / T]-H and W-L-H-F-H-C-[X]-[A / E]-G -[X]-G-R-T-T) sequence design and synthesis of degenerate primers P1, P2

[0056] P1: 5'-GTGGACCTRCGRSARGARWCICA-3';

[0057] P2: 5'-GTCCGACCATTGCCTGCYTCRCARTGRAMRTGIADCCA-3').

[0058] PCR amplification was performed using purified rumen fluid metagenomic DNA as a template. Drop-down PCR reaction parameters are: denaturation at 94°C for 5min; denaturation at 94°C for 30sec, annealing at 58-52°C for 30sec, extension at 72°C for 1min, 10 cycles (1°C drop for each cycle), denaturation at 94°C for 30sec, annealing at 52°C for 30sec , 72°C for 1min, 30 cycles, 72°C for 10min. A fragment of about 400bp was obtained, which was recovered and connected with the pEASY-T3 vector and sent to Sanbo Biotechnology Co., Ltd. for sequencing.

[0059] According to the 400bp nucleotide sequence obtained by sequencing, ...

Embodiment 3

[0063] Example 3 Preparation of recombinant phytase.

[0064] The expression vector pET22b(+) was double digested (NcoRI+XhoI), and the gene cp53 encoding phytase was double digested (NcoRI+XhoI) to cut out the gene fragment encoding mature phytase and the expression vector pET22b( +) connection to obtain recombinant plasmid pET22b-cp53 containing phytase gene cp53 and transform Escherichia coli BL21 (DE3) to obtain recombinant Escherichia coli strain BL21 cp53.

[0065] The BL21 strain containing the recombinant plasmid was inoculated in 3 mL of LB (100 μg / mL ampicillin) culture solution, and cultured at 37° C. overnight. Inoculate 1% of the bacteria in 20mL LB (with 100μg / mL ampicillin), shake and culture at 37°C for about 2-3h (OD 600 After reaching 0.6-0.8), the inducer IPTG with a final concentration of 0.6mmol / L was added, and the culture was shaken at 180rpm at 20°C for 12h. Centrifuge at 12000rpm for 5min, and collect the medium supernatant.

[0066] After being ind...

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Abstract

The invention relates to the field of gene engineering, in particular to neutral phytase CP53 from a rumen and gene and application thereof. The invention provides the phytase CP53 from a metagenome DNA of the rumen, wherein the amino acid sequence of the phytase CP53 is expressed as SEQ ID NO.1; and the invention provides a genome coding gene cp53 for coding the phytase. The phytase has the following properties that: the optimal pH is 6.5, the optimal temperature is 50 DEG C, and the phytase has good stability when the pH is between 2.5 and 8.5 and high catalytic activity under neutral pH condition. The phytase serving as a novel enzyme preparation can be widely applied to ruminant and aquatic feed industry and the like.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a neutral phytase CP53 derived from the rumen and its gene and application. Background technique [0002] Ruminants can use phytate phosphorus in feed more efficiently than monogastric animals, because there may be some microorganisms that can degrade phytic acid in the rumen of animals. Yanke et al. (1998) detected phytase activity in many anaerobic bacteria isolated from the stomach, such as Selenomonas ruminantium, Prevotella ruminicola, Megasphaera elsdenii, Mitsuokella multiacidus, Mitsuokella jalaludinii, etc., among which S.ruminantium had the highest phytase activity (Yanke et al. 1998, 144:1565-1573). Different from other phytases, this phytase has its own typical sequence (conserved active site) HCXXGXXR[T / S]. [0003] In the spatial structure, it is divided into two structural domains: the large domain and the small domain. Large domains are...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/63C12N15/70C12N1/21A23K1/165C12R1/19A23K20/189
Inventor 姚斌黄火清石鹏君罗会颖杨培龙袁铁铮王亚茹柏映国孟昆史秀云
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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