Method for improving secretory expression of L-asparaginase

An asparaginase, secretory expression technology, applied in the field of genetic engineering, can solve the problems of complex extraction process, low L-asparaginase content, low L-asparaginase yield, etc., so as to reduce production cost and yield. The effect of improving enzyme capacity and improving production efficiency

Active Publication Date: 2015-05-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Because the content of L-asparaginase in animal serum is low, and the extraction process is complicated, microorganisms have the advantages of easy cultivation and low cost, which have become the focus of scholars' research. The L-asparaginase-producing microorganisms currently studied mainly include Escherichia coli, Erwinia carotovora,...

Method used

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  • Method for improving secretory expression of L-asparaginase
  • Method for improving secretory expression of L-asparaginase
  • Method for improving secretory expression of L-asparaginase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1 asparaginase gene fusion signal peptide

[0034] Nucleotide sequence of signal peptide pelB: ATGAAATACCTGCTGCCGACCGCTGCTGCGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCC

[0035] Nucleotide sequence of signal peptide ompA: ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCCGCTCCG

[0036] Nucleotide sequence of signal peptide torT: ATGCGCGTACTGCTATTTTTACTTCTTTCCCTTTTCATGTTGCCGGCATTTTCGGCTGAT

[0037] The nucleotide sequence of the signal peptide TorA: ATGAACAATAACGATCTCTTTCAGGCATCACGTCGGCGTTTTCTGGCACAACTCGGCGGCTTAACCGTCGCCGGGATGCTGGGGCCGTCATTGTTAACGCCGCGACGTGCGACTGCGGCGCAAGCG

[0038] Nucleotide sequence of signal peptide sufI: ATGTCACTCAGTCGGCGTCAGTTCATTCAGGCATCGGGGATTGCACTTTGTGCAGGCGCTGTTCCCCTGAAGGCCAGCGCAGCCGGG

[0039] Nucleotide sequence of signal peptide DsbA: ATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTTAGCGTTTAGCGCATCGGCGGCGCAG

[0040] Nucleotide sequence of signal peptide Dmsa: ATGGAACGCAGAAGTTTTCTAAAAATGAGTGCAGCCATGGGCTGCGCAGCAACGGTCACTGGCTGT

[0...

Embodiment 2

[0049] Embodiment 2 fusion fragment is connected with carrier pET-20b (+)

[0050] The recovered product was double digested with restriction endonucleases NdeI and XhoI, and the target fragment was further recovered using a column recovery kit. The fusion fragment was ligated with the carrier pET-20b(+), and the ligation system: 4 μL of the fusion fragment, 1 μL of the vector pET20b, 5 μL of solution I, ligated overnight at 16°C. Transform the ligated recombinant plasmid pET20b-SP-ansZ into competent E.coil JM109, and use ampicillin LB plates to pick positive colonies. The plasmid was extracted after overnight culture on a shaker at 37°C and named pET20b-SP-ansZ. After the enzyme digestion was verified to be correct, the transformant was sequenced by Shanghai Sangon. Transform the plasmid with correct sequencing into Escherichia coli Rosetta (DE3) to obtain asparaginase-producing genetically engineered bacteria.

Embodiment 3

[0051] Example 3 Verification of high-yield asparaginase production strains

[0052] The plasmid with correct sequencing was transformed into E.coli Rosetta (DE3), the selected transformants were inoculated into LB liquid medium, cultured at 37°C for 12 hours, and then transferred into TB medium with an inoculum size of 3%. Bacteria grow to OD 600 When it was 1.5, IPTG was added to induce, and the culture temperature was lowered to 30°C, and cultured for 24h. Collect the fermentation supernatant and detect the enzyme activity of the fermentation supernatant, the results are as follows: figure 1 shown. The experimental results showed that compared with the starting strain, the enzyme activities were significantly improved, and the enzyme activities in the recombinant bacteria fused with signal peptides ansZ, TorA, Sufl, Tort, DsbA, Dmsa, ompA, and pelB were 1.43, 1.74, 1.40, 5.40, 0.98, 3.45, 6.89, 9.72, 31.71U / ml, the asparaginase activity of the recombinant strain R20b-pel...

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Abstract

The invention discloses a method for improving secretory expression of L-asparaginase, and belongs to the field of genetic engineering. According to the method, by fusing a pelB signal peptide at an N terminal of asparaginase, the enzyme activity of the extracellular asparaginase of a recombinant bacterium is increased by 20 times, and further through coexpression of lepB signal peptidase, the enzyme activity of the asparaginase can be increased by 1.45 times on the above basis. The enzyme-producing capacity of a modified bacterial strain is significantly improved; the method is more suitable for industrial application; by the method, the production cost can be reduced and the production efficiency can be improved.

Description

technical field [0001] The invention relates to a method for improving the secretion and expression of L-asparaginase, which belongs to the field of genetic engineering. Background technique [0002] L-asparaginase (EC3.5.1.1) is a protease with anticancer activity, which can specifically catalyze the hydrolysis of L-asparagine into aspartic acid and NH 3 . The physiological effect of L-asparaginase is mainly manifested as the inhibitory effect on some tumors, especially effective on acute leukemia and malignant lymphoma. L-asparaginase has become a very effective drug for treating leukemia and has no inhibitory effect on bone marrow cells. L-asparaginase can reduce the formation of acrylamide in food. Acrylamide is mainly produced by the Maillard reaction of reducing sugar and asparagine in food raw materials during high-temperature heating. Adding asparaginase to food can hydrolyze asparagine and reduce the formation of acrylamide from the source. [0003] Some microor...

Claims

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Application Information

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IPC IPC(8): C12N9/82C12N15/70C12N1/21
CPCC12N9/82C12Y305/01001
Inventor 刘松阮洁冯岳陈坚堵国成黎清华
Owner JIANGNAN UNIV
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