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Medium for fermenting and producing alkaline phosphatase and preparation method of alkaline phosphatase

A fermentation culture and phosphatase technology, applied in the biological field, can solve the problems of high-density growth of bacteria, high-level expression of recombinant proteins, engineering bacteria cannot be truly applied to industrial production, and large-scale production is not easy, so as to prevent the accumulation of acid production , optimize the inducer and induction conditions, and improve the effect of viability

Active Publication Date: 2022-06-21
WUHAN HANHAI NEW ENZYMES BIOLOGICAL TECH CO LTD
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  • Summary
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  • Description
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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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  • Medium for fermenting and producing alkaline phosphatase and preparation method of alkaline phosphatase
  • Medium for fermenting and producing alkaline phosphatase and preparation method of alkaline phosphatase
  • Medium for fermenting and producing alkaline phosphatase and preparation method of alkaline phosphatase

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preparation example Construction

[0034] An embodiment of the present study also provides a method for preparing alkaline phosphatase, including the following steps: using the above-mentioned medium for fermenting alkaline phosphatase to ferment and culture recombinant Escherichia coli to obtain alkaline phosphatase. Among them, the recombinant Escherichia coli carries the 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 self-constructed recombinant Escherichia coli carrying an alkaline phosphatase gene fragment.

[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.

[0037] Wherein, the sequ...

Embodiment 1

[0066] The fermentation method of the recombinant Escherichia coli producing alkaline phosphatase provided in this embodiment comprises the following steps:

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

[0068] Step 2: A 5L fermentor with a 3L fermentation medium was added, the corresponding antibiotics at a final concentration of 50 μg / mL were added, 50% ammonia water was added, and the pH was adjusted to 7.00±0.05. 5% of the inoculum was inserted into the seed liqu...

Embodiment 2

[0072] The fermentation method of the recombinant Escherichia coli producing alkaline phosphatase provided in this embodiment comprises the following steps:

[0073] Step 1: Same as Example 1.

[0074] Step 2: A 5L fermentor with a 3L fermentation medium was added, the corresponding antibiotics at a final concentration of 50 μg / mL were added, 50% ammonia water was added, and the pH was adjusted to 7.00±0.05. 5% of the inoculum was inserted into the seed liquid cultured in step 1, and fermented at 37° C., 200 rpm, and aeration ratio of 1.5 vvm. The whole fermentation process 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: 15g / L glucose, 20g / L peptone, 10g / L yeast powder, 7g / L NaCl, 7.5g / L (NH3) 4 ) 2 SO 4 , 4g / L of KH 2 PO 4 , 6g / L of K 2 HPO 4 , 4.5mg / L MnCl 2 ·4H 2 O, 0.5mg / L Na 2 MoO 4 ·2H 2 O, 5mg / L FeCl 3 ·6H 2 O, 10 mM MgSO 4 ·7H 2 O, 0.5mM ZnCl 2 , th...

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Abstract

The invention relates to a culture medium for producing alkaline phosphatase by fermentation and a preparation method of alkaline phosphatase. The above-mentioned medium for fermenting and producing alkaline phosphatase includes: 5g / L-15g / L of glucose, 10g / L-20g / L of peptone, 3g / L-10g / L of yeast powder, 2g / L- 7g / L NaCl, 2.5g / L-7.5g / L (NH 4 ) 2 SO 4 , 3g / L-4g / L of KH 2 PO 4 , K of 5g / L‑6g / L 2 HPO 4 , 0.3mg / L‑4.5mg / L of MnCl 2 4H 2 O, 0.1mg / L-0.5mg / L of Na 2 MoO 4 2H 2 O, 1mg / L-5mg / L FeCl 3 ·6H 2 O, 3mM-10mM Mg 2+ , 0.05mM‑0.5mM Zn 2+ . The above-mentioned culture medium for fermenting and producing alkaline phosphatase can increase the unit enzyme activity and expression level of alkaline phosphatase.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a medium for fermenting alkaline phosphatase and a preparation method of alkaline phosphatase. Background technique [0002] 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 improve 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 important applications in clinical localization diagnosis, etiological analysis, tumor diagnosis and treatment. [0003] At present, Escherichia coli alkaline phosphatase is the most studied, and commercial alkaline phosphatase includes Escherichia coli phosphatase, bovine small intestine alkaline phosphatase, and shrimp al...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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