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Asparaginase mutant with enhanced enzyme activity

An asparaginase and asparagine technology, applied in the field of enzyme engineering, can solve the problems of complex extraction process, low L-asparaginase content, low L-asparaginase yield, etc. The effect of improving enzyme production capacity and improving production efficiency

Active Publication Date: 2015-02-25
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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, Erwinia chrysanthemi, etc., but the yield of L-asparaginase in wild strains is low. In recent years, genetic engineering technology has been used to clone the L-asparaginase gene into Escherichia coli to obtain high-efficiency expression of L-asparaginase. The use of engineering bacteria to produce L-asparaginase has become an important source

Method used

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  • Asparaginase mutant with enhanced enzyme activity
  • Asparaginase mutant with enhanced enzyme activity
  • Asparaginase mutant with enhanced enzyme activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 High-efficiency secretion of asparaginase strain construction

[0021] Using NdeI and BamHI as restriction endonuclease sites, carry out double enzyme digestion on the plasmid pMA0911-wapA-SP-ansZ (fragment) and pET22b(+) plasmid (vector), use the gel recovery kit to digest The plasmid was purified and recovered, and the concentration of the recovered product was checked by electrophoresis. The recovered target gene (wapA-SP-ansZ) was ligated with the carrier pET22b(+), the connection system: 4 μL of the target gene (wapA-SP-ansZ), 1 μL of the vector (pET22b(+)), 5 μL of solutionI, overnight at 16°C . Transform the ligated recombinant plasmid pET22b-wapA-SP / ansZ into competent E.coil JM109, transform it into an ampicillin LB plate, and pick positive colonies. Such as figure 1 . The plasmid was extracted after overnight culture on a shaker at 37°C and named pET22b-wapA-SP / ansZ. After the enzyme digestion was verified to be correct, the transformants were se...

Embodiment 2

[0023] Example 2 Verification of high secretion capacity asparaginase production strain

[0024] The plasmid sequenced correctly in Example 1 was transformed into Escherichia coli E. coli rosetta. Selected transformants were inoculated into LB liquid medium, cultured at 37°C for 12 hours, and then transferred into TB medium with an inoculation amount 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. The fermentation supernatant was collected, and the enzyme activity of the fermentation supernatant was detected, and the results showed that asparaginase was secreted extracellularly. The enzyme activity is 2.68U / ml.

Embodiment 3

[0025] Embodiment 3 Obtaining of high activity and thermostable mutant strain

[0026] Using the site-directed mutagenesis kit (TaKaRa), design 3 pairs of primers (as shown in Table 1), and use the constructed pET22b-wapA-SP / ansZ as a template to carry out PCR, and the asparagine at position 133 inside the asparaginase molecule They were mutated into tyrosine and tryptophan, respectively, and valine at position 143 was mutated into isoleucine, named N133Y, N133W, and V143I, respectively. The PCR reaction conditions were: 95° C. for 5 min, 34 cycles (95° C. for 5 min, 60° C. for 30 s, 72° C. for 5 min and 40 s), and 72° C. for 10 min. PCR amplification system: template 1 μL, upstream and downstream primers 1 μL, dNTP Mix 4 μL, 5×primeSTAR Buffer 10 μL, sterilized double distilled water 32.5 μL, primeSTAR DNA polymerase 0.5 μL. The gel recovery kit was used to purify and recover the PCR product, and the concentration of the recovered product was checked by electrophoresis. The...

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Abstract

The invention discloses an asparaginase mutant with enhanced enzyme activity, and belongs to the field of enzyme engineering. According to the asparaginase mutant, hydrophilic amino acid asparaginate and valine which are contained in an asparaginase molecule are mutated into tyrosine, tryptophan and isoleucine which have high hydrophobicity in a site-specific mutagenesis mode, so that the hydrophobicity inside the asparaginase molecule is changed, the enzyme activity, which is expressed by a strain, of asparaginase is remarkably improved, and the enzyme activity of the asparaginase is enhanced by 2.57 times. The improved strain is remarkably enhanced in enzyme producing capacity, more suitable for industrial application, reduced in production cost and improved in production efficiency.

Description

technical field [0001] The invention relates to an asparaginase mutant with improved enzyme activity, which belongs to the field of enzyme 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. [0003] 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. [0004] Some microorganisms, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/82C12N1/21C12R1/19
CPCC12N9/82C12Y305/01001
Inventor 刘松冯岳陈坚堵国成陈双全王广圣陈璇
Owner JIANGNAN UNIV
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