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Engineering bacteria for producing L-asparaginase II and construction method and applications thereof

A technology for asparaginase and construction method, which is applied in the field of engineering bacteria expressing L-asparaginase II extracellularly, can solve problems such as less extracellular expression, and achieve less separation and purification steps, low cost and high yield Effect

Inactive Publication Date: 2010-06-23
JIANGSU POLYTECHNIC UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Through domestic and foreign patent and literature search, it is found that genetically engineered bacteria have been used to express and produce L-asparaginase, but the enzyme is located in the periplasmic space of the cell, and there are very few reports of extracellular expression, and it has not been used in industry. Production

Method used

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  • Engineering bacteria for producing L-asparaginase II and construction method and applications thereof
  • Engineering bacteria for producing L-asparaginase II and construction method and applications thereof
  • Engineering bacteria for producing L-asparaginase II and construction method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Construct L-asparaginase engineering bacterium and cultivate this engineering bacterium according to above-mentioned five-step process method, a specific embodiment is as follows:

[0056] Firstly, the known strain Escherichia coli JM109 was isolated and the chromosomal DNA was extracted. Mainly: the JM109 culture medium cultivated overnight at 37°C was collected by centrifugation, added lysozyme to a final concentration of 1 mg / mL, reacted at 0°C for 15 minutes, added RNase to a final concentration of 100 μg / mL, reacted at 37°C for 30 minutes, and added proteinase K to the final The concentration was 100 μg / mL, after 2 h at 37 °C, the protein was extracted 5 times with phenol: chloroform (1:1), the water phase was removed, 1 / 10 volume of 3 mol / L sodium acetate solution was added, and 2.5 times the volume of -20 °C pre- Cold anhydrous ethanol, after careful mixing, use a sterile toothpick to wind clockwise to take out the precipitated DNA, put it in TE buffer at 4°C to ...

Embodiment 2

[0064] In this example, the method and conditions are the same as those in Example 1, except that the final concentration of the inducer added is 5 g / L. The L-asparaginase activity in the obtained fermentation broth was 39.6U / mL.

Embodiment 3

[0066] In this example, the method and conditions are the same as those in Example 1, except that the fermentation of the engineered bacteria is carried out in a 5-liter fermenter, and the inducer is changed from IPTG to lactose.

[0067] The engineered bacteria slant was inserted into a 250mL Erlenmeyer flask containing 50mL seed medium that had been sterilized, and cultivated overnight at 37°C.

[0068] 3 L of fermentation medium (70 g / L corn steep liquor, 8 g / L glycerol, pH 7.2) was loaded into a 5 L fermenter, sterilized at 0.15 MPa for 30 min, and 30 mL of the above overnight culture solution was added. Cultivate at 37°C for 8 hours, add the inducer lactose, the final concentration is 3g / L, continue to cultivate for 12 hours, and finally the activity of L-asparaginase in the fermentation broth is 32.4U / mL.

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Abstract

The invention relates to novel engineering bacteria for producing L-asparaginaseII. An extracellular expression product of the engineering bacteria has L-asparaginaseIIprotein activity. The invention also discloses a construction method and fermentation culture conditions of the engineering bacteria, wherein recombinant plasmids pET-ASP invert colon bacillus BL21 to obtain the engineering bacteria; and the structure of the recombinant plasmids pET-ASP is shown in figure 1. The recombinant engineering bacteria are obtained by connecting the L-asparaginase II genes on expression plasmids pET22b to obtain the recombinant plasmids pET-ASP and then using the recombinant plasmids pET-ASP to invert the colon bacillus BL21. The L-asparaginase II can be produced massively by carrying out fermentation culture on the engineering bacteria. And the engineering bacteria has wide raw material source, low cost, and few separation and purification steps and high yield of the products in the fermentation liquor.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an engineering bacterium expressing L-asparaginase II extracellularly, its construction method and application. The invention uses modern biotechnology and genetic engineering methods to construct engineering bacteria expressing L-asparaginase II extracellularly, and selects a suitable medium and a culture method so that the obtained enzyme activity can reach a higher level. Background technique [0002] L-asparaginase (E.C.3.5.1.1) can catalyze the hydrolysis of L-asparagine into aspartic acid and ammonia. L-asparaginase has been obtained from different bacterial sources. [0003] In 1953, Kidd discovered that guinea pig serum had anti-tumor effects. Later, Broome confirmed that the antitumor factor of guinea pig serum was L-asparaginase in serum. L-asparaginase can disturb the metabolism of amino acids, proteins and nucleic acids in sensitive tumor cells, thereby inhibiting...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/55C12N15/70C12N9/82C12R1/19
Inventor 王利群黄达明王文兵朱劼严生虎
Owner JIANGSU POLYTECHNIC UNIVERSITY
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