Genetic-engineering L-asparaginase amidohydrolase modified through site-specific mutagenesis

An asparaginase and mutant technology, applied in the field of genetic engineering, can solve the problems of low L-asparaginase enzyme activity and low protein expression, and achieve the effect of improving industrial application potential

Inactive Publication Date: 2015-11-18
JIANGNAN UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 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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  • Genetic-engineering L-asparaginase amidohydrolase modified through site-specific mutagenesis
  • Genetic-engineering L-asparaginase amidohydrolase modified through site-specific mutagenesis
  • Genetic-engineering L-asparaginase amidohydrolase modified through site-specific mutagenesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1 Contains the construction of the recombinant vector of L-asparaginase mutant

[0019] (1) Obtaining of the S166A mutant: using the nucleotide sequence shown in SEQIDN0.4 as a template, Fprimer (sequence shown in SEQIDN0.5), and Rprimer (sequence shown in SEQIDN0.6) as primers, PCR is obtained The recombinant gene shown in SEQIDN0.3.

[0020] (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-S166A. The sequencing work was completed by Shanghai Sangong.

Embodiment 2

[0021] Example 2 Production of L-asparaginase Bacillus subtilis Engineering Bacteria Construction

[0022] The recombinant plasmid pMA5-S166A obtained in Example 1 was chemically transformed into B. subtilis168 competent cells, and the specific method was as follows:

[0023] (1) The solution required for the transformation experiment is as follows (g / L):

[0024] Sp-A: (NH 4 ) 2 SO 4 4,K 2 HPO 4 28. Sodium citrate 12Sp-B: MgSO 4 ·7H 2 O0.4

[0025] 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.

[0026] (2) Inoculate a single colony of B.Subtilis168 into 2mL SpI medium (50mL centrifuge tube), and culture overnight at 37°C and 200r / min;

[0027] (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

[0029] Example 3: High expression and enzyme activity determination of recombinant bacteria pMA5-S166A / B.subtilis168L-asparaginase.

[0030] (1) The recombinant strain pMA5-S166A / B.subtilis168 constructed in Example 2 and the original strain pMA5-ansz / B.subtilis168 were respectively inoculated in 10 mL of LB medium containing kanamycin, cultured with shaking at 37°C overnight, and Transfer to the fermentation medium of Bacillus subtilis at 4% daily inoculum, culture at 37°C for 24 hours, take the fermentation broth at 4°C, and centrifuge at 10,000r / min for 10min, the supernatant is extracellular crude enzyme liquid, and the supernatant of broken cells The solution is the intracellular crude enzyme solution, which is used for the determination of enzyme activity.

[0031] (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.

[003...

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Abstract

The invention discloses an activity-improved L-asparaginase amidohydrolase mutant and a construction method thereof, and belongs to the field of genetic engineering. The mutant is characterized in that the 166th serine is mutated into alanine on the basis of the amino acid shown in SEQ ID No.2. The mutant is expressed in bacillus subtilis, the activity is 657.1 U / ml after flask fermentation is carried out for 24 h, the mutation activity is improved by 23%, the substrate affinity is reduced by 20% compared with original amidohydrolase, the catalytic efficiency is improved by 8.4%, and meanwhile the specific activity is improved by 25%. The mutant and the construction method show that the 166th serine residue has large influences on the catalytic action of the amidohydrolase, a certain foundation is provided for researching the catalytic mechanism of the amidohydrolase, and the industrial application potential of the amidohydrolase 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 invention adopts the single-point mutation technology and based on the homology modeling method, optimizes the molecular structure of L-asparaginase of Bacillus su...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/82C12N15/75C12N1/21C12R1/125
CPCC12N9/82C12Y305/01001
Inventor 饶志明龙水清张显杨套伟徐美娟
Owner JIANGNAN UNIV
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