L-asparaginase mutant with improved enzyme activity and construction method thereof

An asparaginase and mutant technology, which is applied in the field of genetic engineering, can solve the problems of low protein expression and low L-asparaginase enzyme activity, and achieves the effect of reducing the generation of acrylamide and improving the potential of industrial application.

Inactive Publication Date: 2015-11-18
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The prominent problems of heterologous expression of L-asparaginase are low protein expression and low activity of L-asparaginase

Method used

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  • L-asparaginase mutant with improved enzyme activity and construction method thereof
  • L-asparaginase mutant with improved enzyme activity and construction method thereof
  • L-asparaginase mutant with improved enzyme activity and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Embodiment 1 Contains the construction of the recombinant vector of L-asparaginase mutant

[0015] (1) Obtaining the G107D mutant: using the nucleotide sequence shown in SEQIDNO.4 as a template, Fprimer (sequence shown in SEQIDNO.5) and Rprimer (sequence shown in SEQIDNO.6) as primers, PCR is performed to obtain The recombinant gene shown in SEQ ID NO.3.

[0016] (2) Digest the recombinant gene and pMA5 with BamHI and MluI, respectively, and ligate with T4 DNA ligase overnight at 16°C after purification. The ligation product was chemically transformed into JM109 competent cells. The transformation solution was applied to an LB plate containing kanamycin (50 mg / L), the plasmid was extracted, and the recombinant plasmid constructed was verified by double enzyme digestion, which was named pMA5-G107D. The sequencing work was completed by Shanghai Sangong.

Embodiment 2

[0017] Example 2 Production of L-asparaginase Bacillus subtilis Engineering Bacteria Construction

[0018] The recombinant plasmid pMA5-G107D obtained in Example 1 was chemically transformed into B. subtilis168 competent cells, and the specific method was as follows:

[0019] (1) The solution required for the transformation experiment is as follows (g / L):

[0020] Sp-A: (NH 4 ) 2 SO 4 4,K 2 HPO 4 28. Sodium citrate 12Sp-B: MgSO 4 ·7H 2 O0.4

[0021] 100×CAYE: Casaaminoacid20, yeast powder 100SpI medium: Sp-A49%, Sp-B49%, 50% glucose 2%, 100×CAYE2% SpII medium: SpI medium 98%, 50mmol / LCaCl 2 1%, 250mmol / LMgCl 2 1%. 115°C damp heat sterilization.

[0022] (2) Inoculate a single colony of B.Subtilis168 into 2mL SpI medium (50mL centrifuge tube), and culture overnight at 37°C and 200r / min;

[0023] (3) Take 100 μL of the culture solution into 5 mL of SpI medium, and culture at 37°C and 200 r / min until the logarithmic phase (OD600 value is about 1), about 4 to 5 hours; ...

Embodiment 3

[0025] Example 3 High expression and enzyme activity determination of recombinant bacteria pMA5-G107D / B.subtilis168L-asparaginase.

[0026] (1) The recombinant bacteria pMA5-G107D / B.subtilis168 constructed in Example 2 and the control strain pMA5-ansz / B.subtilis168 expressing unmutated enzymes were inoculated in 10 mL of LB medium containing kanamycin respectively, 37 Shake culture overnight at ℃, transfer to Bacillus subtilis fermentation medium at 4% inoculum the next day, culture at 37℃ for 24h, take the fermentation broth and centrifuge at 10000r / min for 10min at 4℃, the supernatant is crude extracellular enzyme liquid, and the supernatant of broken cells was the intracellular crude enzyme solution, which was used for the determination of enzyme activity.

[0027] (2) Bacillus subtilis fermentation medium: soybean peptone 10g / L, K 2 HPO 4 2.3g / L, KH 2 PO 4 1.7g / L, corn steep liquor 15g / L, urea 3g / L, glucose 40g / L, MgSO 4 0.75g / L, NaCl5g / L. Adjust pH6.8-7.0.

[0028]...

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Abstract

The invention discloses an L-asparaginase mutant with the improved enzyme activity and a construction method thereof, and belongs to the field of gene engineering. According to the L-asparaginase mutant, on the basis of amino acid shown in the SEQ ID NO.2, the 107th glycine is mutated into aspartic acid. The obtained mutant is expressed in bacillus subtilis, fermentation is performed in a shake flask for 24 h and then the enzyme activity is 961 U/mL; the enzyme activity of the mutant is improved by 80%, the appetency of a substrate is decreased by 50% compared with protoenzyme, the catalytic efficiency is improved by 84%, and meanwhile the specific enzyme activity is improved by 83%. According to the L-asparaginase mutant, it is shown that the 107th amino acid residue has a great influence on the enzyme catalytic action, a certain foundation is provided for research on the enzyme catalytic mechanism, and the enzyme industrial application potential is improved.

Description

technical field [0001] The invention relates to an L-asparaginase mutant with improved enzyme activity and a construction method thereof, belonging to the technical field of genetic engineering. Background technique [0002] L-asparaginase (L-asparaginaseamidohydrolase, E.C.3.5.1.1) can hydrolyze and deaminate L-asparagine to form L-aspartic acid and ammonia. L-asparaginase has anti-tumor activity and has been used in the treatment of acute lymphoblastic leukemia and Hodgkinson's disease. In recent years, studies have found that L-asparaginase can also reduce the formation of acrylamide in fried foods. The size, structure and properties of L-asparaginase vary from source to source. L-asparaginase has a wide range of sources, and L-asparaginase is found in guinea pig serum, plants and microorganisms. [0003] The prominent problems of heterologous expression of L-asparaginase are low protein expression and low activity of L-asparaginase. Therefore, site-directed mutagenesi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/82C12N15/55C12N1/21A61K38/50A61P35/02A23L1/01C12R1/125A23L5/10
CPCA23L5/10A61K38/50C12N9/82C12N15/52A61K38/00C12Y305/01001
Inventor 饶志明龙水清张显杨套伟徐美娟
Owner JIANGNAN UNIV
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