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Construction of engineered strain capable of efficiently expressing phytase

A technology of engineering strains and phytase, applied in genetic engineering, bacteria, hydrolytic enzymes, etc., can solve the problems of excessive content, increased production costs, waste of phosphorus sources, etc.

Inactive Publication Date: 2014-02-05
蒋和生 +1
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Problems solved by technology

However, the digestive tract of monogastric animals (such as pigs, chickens, etc.) and the small intestine of fish lack enzymes to effectively digest phytate phosphorus (MaenzDD, Classen HL. Phytase activity in the small intestinal brush border membrane of the chicken[J ].Poult Sci, 1998, 77(4): 557-563), phytate phosphorus is directly excreted without being fully digested and absorbed, which brings many problems to the livestock and poultry breeding industry: first, it causes waste of phosphorus sources, on the one hand The phosphorus source in the feed cannot be effectively utilized. Studies have shown that: 1g of phytic acid can be completely decomposed to release 281.6mg of inorganic phosphorus, which is a very rich source of inorganic phosphorus; Additional addition of inorganic phosphorus in the feed, along with rising international phosphorus prices, feed costs continue to increase; at the same time, due to production process problems, the content of fluorine, heavy metals and other elements in the added inorganic phosphorus exceeds the standard, which is likely to cause animal poisoning
Second, high-phosphorus feces are formed to pollute the environment. 85% of the phytate phosphorus in the plant feed is excreted with the feces without being digested, causing a large amount of phosphorus to enter the water and soil, resulting in eutrophication of the environment, which seriously damages the breeding ecological environment.
However, the expression of yeast has certain requirements on the culture conditions, and the complex composition of the medium increases the production cost, and it is generally induced for 100 hours to obtain stable protein expression; the expression of insect cells requires both a strict sterile environment and an expensive medium. Low-expression recombinant protein can be obtained about 100 hours after infection, which is not conducive to industrial production

Method used

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  • Construction of engineered strain capable of efficiently expressing phytase
  • Construction of engineered strain capable of efficiently expressing phytase
  • Construction of engineered strain capable of efficiently expressing phytase

Examples

Experimental program
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Effect test

Embodiment 1

[0158] Escherichia coli Escherichia coli BL21 (DE3) (hereinafter referred to as E. coli BL21 (DE3) appA Gene acquisition and recombinant vector construction

[0159] E. coli BL21 (DE3) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0160] Prepare LB medium: peptone 10g / L, yeast extract powder 5g / L, NaCl 10g / L, 121°C, high temperature and high pressure sterilization, and store at 4°C.

[0161] Collect bacteria: the E. coli BL21 (DE3) was inoculated onto LB medium, cultured with shaking at 180-200 r / min at 37°C for 10-12 hours, and centrifuged at 10,000 rpm for 5-10 minutes at 4°C to collect bacterial precipitates and use 1-2 mL of The bacteria were washed 1-3 times with distilled water, centrifuged at 10,000 rpm for 5-10 min at 4°C, and the bacterial precipitate was collected.

[0162] Extract the target gene: use TGuide Bacterial Genomic DNA Extraction Kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract and obtain E. coli...

Embodiment 2

[0177] Acquisition of Escherichia coli Phytase Recombinant Expression Engineering Strain

[0178] Obtaining of recombinant expression strains: the sequenced correct pET30a(+)- appA Transformation of recombinant expression plasmids into E. coli In the BL21(DE3) expressing host, detected by colony PCR method, it contains pET30a(+)- appA The positive recombinant expression engineering strain W of the recombinant plasmid.

[0179] Inoculate the W strain into 5 mL of fresh LB medium (containing 100 μg / mL kanamycin) at 1-3% inoculum amount, culture at 37°C, shake at 180-200 rpm for 4-6 hours, add lactose (final concentration is 0.45- 0.75mg / mL) for purpose induced expression. After continuous induction for 12 hours, samples were taken every hour, centrifuged at 10,000g for 5-10min at 4°C, and bacterial pellets were collected. The pellet was resuspended with 200-300μL 0.25mol / L sodium acetate buffer (pH5.5) (containing lysozyme at a final concentration of 0.05-0.15mg / mL), and in...

Embodiment 3

[0182] Mutagenesis screening was performed using lactose as inducer.

[0183] M6 mother liquor: 64g Na 2 HPO 4 , 15g KH 2 PO 4 , 2.5gNaCl, 5.0gNH 4 Cl plus 1L water for sterilization.

[0184] Improved M6 medium: under sterile conditions, take 200ml of M6 mother solution, add 2mL of 1mol / L sterilized MgSO 4 , add 100 μL of sterilized 1mol / L CaCl 2 , add 20mL of sterilized 10% lactose, add sterile water to make up to 1L.

[0185] Screening of mutagenized strains: Dilute the W strain appropriately, spread it on the modified M6 medium (containing 100 μg / mL kanamycin), and cultivate overnight at 37°C. Spread a 1% agar plate containing 100 mmol / L sodium acetate buffer (pH5.5), 20 mmol / L sodium phytate and 50 mmol / L calcium chloride on the plate, and determine the enzyme activity according to the size of the plaque formed Finally, 100 positive recombinant mutagenized strains with phytase activity were obtained.

[0186] Induced expression of mutagenized strains: Induced exp...

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Abstract

The invention provides construction of an engineered strain capable of efficiently expressing phytase, relates to the technical field of biology, and specifically relates to a mutagenesis engineered strain capable of efficiently expressing escherichia coli phytase with a relatively wide adaptation scope. A recombinant expression plasmid pET30a(+)-appA is converted and enters an escherichia coli expression host, so that a recombinant expression engineered strain W is obtained, and then a new escherichia coli phytase recombinant expression engineered strain M6 is obtained through lactose mutagenesis. Compared with the engineered strain W, the engineered strain M6 has no gene mutation, but is higher in phytase activity, and the enzyme activity reaches 807.35 U / mL by employing shaking culture. Compared with phytase expressed by the engineered strain W, phytase expressed by the engineered strain M6 is improved by 17.44% in pepsin tolerance and improved by 34.77% in trypsin tolerance. The engineered strain M6 is capable of efficiently expressing phytase with the relatively wide adaptation scope; and the mutagenic phytase can be used as a feed additive, is capable of digesting and utilizing phytate in animal alimentary canal in a relatively good manner, reducing discharge of organophosphorus and promoting ecological culture of livestock and poultry industry.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an Escherichia coli phytase mutagenesis engineering strain with a wider range of high-efficiency expression and adaptability. The invention also relates to the production and application of the mutagenesis engineering strain. Background technique [0002] Phosphorus is an essential element for animal growth, exists in all cells of the animal body, and participates in almost all physiological chemical reactions. Its content is more than 1 / 4 of the total amount of minerals in animals, and it exists in the form of phosphate in animals, of which 80% exists in bones and teeth as hydroxyphosphorus, and the other 20% exists in muscles, blood and cartilage. Long-term phosphorus deficiency can lead to inhibition of animal growth tissue, soft and brittle bones, and induce rickets in growing animals and osteoporosis in adult animals, thereby affecting the growth performance of anima...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/55C12N1/21C12N15/70C12N15/01A23K1/165C12R1/19
CPCA23K20/189C12N9/16C12N15/70
Inventor 蒋和生韦佩君潘能庆杨秀荣
Owner 蒋和生
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