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Culture medium for producing alkaline phosphatase through fermentation and preparation method of alkaline phosphatase

A phosphatase and culture medium technology, applied in the biological field, can solve the problems of limited high-level expression of recombinant proteins in high-density growth of bacterial cells, unsuitable industrial production of engineering bacteria, and difficulty in large-scale production, so as to prevent the accumulation of acid production. , optimize the inducer and induction conditions, improve the effect of vitality

Active Publication Date: 2021-11-09
WUHAN HANHAI NEW ENZYMES BIOLOGICAL TECH CO LTD
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  • Claims
  • Application Information

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Problems solved by technology

However, it is still prevalent that large-scale production is not easy and the enzyme yield is low; in the fermentation industry, engineering bacteria are often limited by the high-density growth of bacteria and the high-level expression of recombinant proteins due to expression vectors, expression hosts, and expression conditions, resulting in Engineering bacteria cannot really be applied in industrial production

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  • Culture medium for producing alkaline phosphatase through fermentation and preparation method of alkaline phosphatase
  • Culture medium for producing alkaline phosphatase through fermentation and preparation method of alkaline phosphatase
  • Culture medium for producing alkaline phosphatase through fermentation and preparation method of alkaline phosphatase

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preparation example Construction

[0034] One embodiment of this study also provides a method for preparing alkaline phosphatase, which includes the following steps: using the above-mentioned medium for fermenting and producing alkaline phosphatase to ferment and cultivate recombinant Escherichia coli to obtain alkaline phosphatase. Wherein, the recombinant Escherichia coli carries an alkaline phosphatase gene fragment.

[0035] It should be noted that the recombinant Escherichia coli can be a commercially available recombinant Escherichia coli carrying an alkaline phosphatase gene fragment, or a recombinant Escherichia coli carrying an alkaline phosphatase gene fragment independently constructed.

[0036] Specifically, the recombinant Escherichia coli uses Escherichia coli as a host, is constructed according to conventional methods in the art, and expresses alkaline phosphatase derived from bovine small intestine. The amino acid sequence encoded by the alkaline phosphatase gene is shown in SEQ ID No.1.

[003...

Embodiment 1

[0066] The fermentation method of the recombinant escherichia coli producing alkaline phosphatase provided by the present embodiment comprises the following steps:

[0067] Step 1: Inoculate the recombinant Escherichia coli -80°C glycerol-preserved strain carrying the alkaline phosphatase gene into a stoppered test tube containing 10 mL of LB medium with a volume concentration of 0.1%, and add the corresponding antibiotics, shake Cultivate overnight in bed at 37°C and 200rpm, and inoculate the first-grade seed solution with a volume concentration of 0.05% into a 500mL Erlenmeyer flask containing 150mL of LB medium, with a final concentration of 50μg / mL of the corresponding antibiotic, shaker at 37°C, 200rpm Cultivate for 6h.

[0068] Step 2: In a 5L fermenter with 3L fermentation medium, add the corresponding antibiotic at a final concentration of 50 μg / mL, add 50% ammonia water, and adjust the pH to 7.00±0.05. Insert the seed solution cultivated in step 1 according to the in...

Embodiment 2

[0072] The fermentation method of the recombinant escherichia coli producing alkaline phosphatase provided by the present embodiment comprises the following steps:

[0073] Step 1: Same as Example 1.

[0074] Step 2: In a 5L fermenter with 3L fermentation medium, add the corresponding antibiotic at a final concentration of 50 μg / mL, add 50% ammonia water, and adjust the pH to 7.00±0.05. Insert the seed liquid cultivated in step 1 according to the inoculation amount of 5%, and ferment under the conditions of 37° C., 200 rpm, and an aeration ratio of 1.5 vvm. The whole process of fermentation passes through 50% ammonia water and 10% phosphoric acid solution, and the pH is automatically controlled to be 7.00±0.05. Fermentation medium (3L) is: the glucose of 15g / L, the peptone of 20g / L, the yeast powder of 10g / L, the NaCl of 7g / L, the (NH 4 ) 2 SO 4 , 4g / L of KH 2 PO 4 , 6g / L of K 2 HPO 4 , 4.5mg / L of MnCl 2 4H 2 O, 0.5 mg / L Na 2 MoO 4 2H 2 O, 5mg / L FeCl 3 ·6H 2 O, ...

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Abstract

The invention relates to a culture medium for producing alkaline phosphatase through fermentation and a preparation method of the alkaline phosphatase. The culture medium for producing the alkaline phosphatase through fermentation comprises 5-15g / L of glucose, 10-20g / L of peptone, 3-10g / L of yeast powder, 2-7g / L of NaCl, 2.5-7.5g / L of (NH4)2SO4, 3-4g / L of KH2PO4, 5-6g / L of K2HPO4, 0.3-4.5mg / L of MnCl2.4H2O, 0.1-0.5mg / L of Na2MoO4.2H2O, 1-5mg / L of FeCl3.6H2O, 3-10mM of Mg < 2+ > and 0.05-0.5mM of Zn < 2+ >. The culture medium for producing the alkaline phosphatase through fermentation can improve the unit enzyme activity and expression quantity of the alkaline phosphatase.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a medium for fermenting and producing alkaline phosphatase and a preparation method for alkaline phosphatase. Background technique [0002] Alkaline phosphatase (alkaline phosphatase) can remove the phosphate group at the 5' end of DNA and RNA. In molecular cloning, it is often used to prevent the self-ligation of the vector and increase the insertion rate of the target fragment. Alkaline phosphatase can also prepare DNA templates for 5' end labeling. In addition, alkaline phosphatase can catalyze the hydrolysis of phosphate compounds, and is widely used in immunolabeling and chemiluminescence; it has very important applications in clinical positioning diagnosis, etiology analysis, and tumor diagnosis and treatment. [0003] At present, Escherichia coli alkaline phosphatase is the most researched, and the commercialized alkaline phosphatase includes Escherichia coli phosphatase, bov...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/16C12N15/55C12R1/19
CPCC12N9/16C12N1/20C12Y301/03001
Inventor 王梁姜涛徐灿宫安罗漫杰
Owner WUHAN HANHAI NEW ENZYMES BIOLOGICAL TECH CO LTD
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