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A method for preparing asparaginase by fermentation

An asparaginase and fermentation technology, which is applied in the field of biomedicine, can solve the problems of low unit titer, unstable fermentation and amplification process, etc., and achieves great industrial application significance and value, stable process and results, and large expression amount. Effect

Active Publication Date: 2021-04-16
CHANGZHOU QIANHONG BIOPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main reasons are: ①The fermentation amplification process is unstable, and the unit titer is not high; ②The fermentation medium contains beef-derived substances, which may introduce TSE / BSE pathogenic factors

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] A method for fermenting and preparing asparaginase, comprising seed fungus slant culture, shake flask culture, seed tank culture and fermenter culture, the specific process is as follows:

[0023] (1) Incline cultivation of seed bacteria: Inoculate Escherichia coli strains (constitutively expressed E. coli) on LB solid medium plates, and cultivate them at 37°C for 18 hours. After the cultivation, single colonies were selected for strain screening, and Select a single colony with high enzyme production and carry out streak culture on LB solid slant medium for 18 hours;

[0024] The components of the LB solid medium and the LB solid slant medium are the same: peptone 10g / L, yeast powder 5g / L, sodium chloride 5g / L, agar powder 15g / L; 10g / L yeast powder, 5g / L yeast powder, 5g / L sodium chloride, and 15g / L agar powder to make LB medium, sterilize at 121°C for 30min, cool to 45°C, add a certain amount of antibiotic solution, pour a plate (such as 30ml / dish), solidified, and...

Embodiment 2

[0031] A method for fermenting and preparing asparaginase, comprising seed fungus slant culture, shake flask culture, seed tank culture and fermenter culture, the specific process is as follows:

[0032] (1) Seed fungus slant culture: inoculate constitutively expressed Escherichia coli strains;

[0033] The components and ratios of the LB solid medium and LB solid slant medium are: peptone 10g / L, yeast powder 5g / L, sodium chloride 5g / L, agar powder 15g / L;

[0034](2) Shake flask culture: inoculate a certain amount of antibiotic solution and the strains grown on the slant in (1) into the shake flask culture medium, and vibrate in a constant temperature incubator at 35°C and a speed of 200 rpm Cultivate for 6-12 hours;

[0035] (3) Seed tank culture: the seed tank adopts 200 liters of fermenter tanks, and 100 liters of seed tank culture medium is installed, and the cultured shake flask strains in (2) are inserted into the seed tank culture medium, at 35 ° C, with a rotation spe...

Embodiment 3

[0042] A method for fermenting and preparing asparaginase, comprising seed fungus slant culture, shake flask culture, seed tank culture and fermenter culture, the specific process is as follows:

[0043] (1) Seed fungus slant culture: inoculate constitutively expressed Escherichia coli strains;

[0044] The components of the LB solid medium and the LB solid slant medium are the same: peptone 10g / L, yeast powder 5g / L, sodium chloride 5g / L, agar powder 15g / L.

[0045] (2) Shake flask culture: a 500ml shake flask is used for shake flask culture, and the filling volume is 200ml. Sterilize at 121°C for 30 minutes, add a certain amount of antibiotic solution and the strains grown on the slant in (1) after cooling, and culture in a constant temperature incubator at 38°C with a rotation speed of 200 rpm for 6 hours;

[0046] (3) Seed tank cultivation: the seed tank adopts a 200-liter fermenter, and with a loading capacity of 100 liters, 100 liters of the prepared medium are added to ...

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Abstract

The invention relates to a method for preparing asparaginase by a fermentation method, comprising: (1) slant culture of seed bacteria; (2) shake flask culture: inserting the grown slant bacteria in (1) into the shake flask, Vibration culture; (3) seed tank culture: insert the cultured shake flask strains in (2) into the seed tank, and stir and cultivate under aeration conditions; (4) fermenter tank culture: culture the cultured strains in (3) The seed tank bacterial classification is moved into the fermenter, and the inoculum is 5%-10%, stirred and cultivated under aeration, then centrifuged, and the collected thalline obtains asparaginase (Esche) thallus; the fermented tank culture The components of medium, seed tank medium and shake flask medium are: corn steep steep liquor, peptone, sodium glutamate. In the present invention, the asparaginase has a large expression amount and a high titer, and there is no TSE / BSE risk factor in the culture medium, so that the applicability of the asparaginase is better, more in line with regulatory requirements, and the cost of raw materials can also be greatly reduced .

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for preparing asparaginase by a fermentation method. Background technique [0002] Asparaginase is currently applicable to the treatment of melanoma and acute lymphoblastic leukemia, and can also be used to treat acute leukemia of the monocyte type, chronic leukemia of the lymphocyte type, and non-Hodgkin's lymphoma. Asparaginases from microorganisms are relatively common, including bacteria, fungi, and ancient organisms. Among them, the asparaginase derived from Escherichia coli can provide a more stable anticancer effect, and the production cost is relatively low. In recent years, with the rapid and stable development of genetic engineering technology, there have been more and more reports on the use of constructed asparaginase genetically engineered bacteria, but they are all only in the stage of laboratory experiments, and there are no examples of standard indus...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/82C12N1/20C12R1/19
CPCC12N1/20C12N9/82C12Y305/01001
Inventor 李少龙丁财君蒋驰洲高原黄文辉
Owner CHANGZHOU QIANHONG BIOPHARMA
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