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Catalase mutant with improved enzyme activity and heat stability

A catalase and thermal stability technology, applied in the field of bioengineering, can solve problems such as temperature sensitivity, and achieve the effect of improving yield and enzymatic properties

Inactive Publication Date: 2013-12-18
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the catalase also has certain defects, especially its sensitivity to temperature, which makes it have certain limitations in industrial applications.

Method used

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  • Catalase mutant with improved enzyme activity and heat stability
  • Catalase mutant with improved enzyme activity and heat stability
  • Catalase mutant with improved enzyme activity and heat stability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The construction of embodiment 1 mutant expression plasmid and the acquisition of recombinant Bacillus subtilis

[0029] 1. Construction of mutant expression vector

[0030] Using Bacillus subtilis pSTOP1622-katA plasmid (a recombinant Bacillus subtilis with high catalase production and its construction method and application, application number: 201110328753.1; Plasmid system for the intracellular production and purification of affinity-tagged proteins in Bacillus megaterium, Biedendieck R, Yang Y, Deckwer WD, Malten M, Jahn D) were used as templates to amplify plasmids containing mutant genes in vitro by PCR.

[0031] The primers used for site-directed mutagenesis were:

[0032] katAKY primer1: 5'-ACCCGCGCGGATTTGCTGTTTTATTTTTATACTGAAGAAGGAAA-3'

[0033] katAKY primer2: 5'-TTTCCTTCTTCAGTATAAAAATAAACAGCAAATCCGCGCGGGT-3'

[0034] katAKV primer1: 5'-CCCGCGCGGATTTGCTGTTGTATTTTATACTGAAGAAGG-3'

[0035] katAKV primer2: 5'-CCTTCTTCAGTATAAAATACAACAGCAAATCCGCGCGGG-3'

[0036...

Embodiment 2

[0049] Expression of embodiment 2 mutant CAT

[0050] Activation medium: 6% (w / w) wort, pH 7.0-7.5.

[0051] Fermentation medium (g / L): Glucose 10, NaNO 3 5. MgSO 4 ·7H 2 O0.5, Na 2 HPO 4 9.52, KH 2 PO 4 0.6, FeSO 4 ·7H 2 O0.0025, pH natural; xylose induction concentration was 0.5% (w / w).

[0052] Pick the CAT-producing recombinant Bacillus subtilis screened from the LB plate, and inoculate 1-2 loops under sterile conditions in 25mL activation medium with tetracycline at a final concentration of 20ug / mL, at 37°C. Under 200rpm shaker for 16h, the seed solution was obtained; 5% of the inoculum was transferred into a 250mL Erlenmeyer flask containing 25mL of fermentation medium. The fermentation conditions were 37° C., 200 rpm. After 56 hours of fermentation, samples were taken every 4 hours, and the original strain of Bacillus subtilis (B.subtilis WSHDZ-01 / pSTOP1622-katA) was used as a control.

Embodiment 3

[0053] Enzymatic properties of CAT before and after embodiment 3 mutation

[0054] According to the method described in Example 2, the original Bacillus subtilis (B.subtilis WSHDZ-01 / pSTOP1622-katA, a recombinant Bacillus subtilis with high catalase production and its construction method and application, application number: 201110328753.1) and other mutant strains were fermented, and the extracellular enzyme activity was measured, and the results were as attached figure 1 shown, by attaching figure 1 It can be seen that the enzyme activity of the strain after the mutation is significantly improved compared with that before the mutation, and the highest enzyme activity of katAKV is 2.41 times that before the mutation. The expression levels of the catalase mutants were 2.23, 2.41, 1.46, and 1.38 times that of those before the mutation, respectively.

[0055] Through detection, it is found that the enzyme before mutation is relatively stable at 30°C-50°C, but it is relatively e...

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Abstract

The invention discloses a catalase mutant with improved enzyme activity and heat stability, and belongs to the field of biological engineering. Catalase derived from bacillus subtilis (B. subtilis WSHDZ-01) is subjected to site-specific mutagenesis, so that a lysine of an amino acid sequence coded by the catalase is respectively mutated into tyrosine Y, valine V, methionine M or isoleucine I at the site 114. The expression quantity of the catalase mutant is respectively 2.23, 2.41, 1.46 and 1.38 times of the expression quantity before mutation, and the half-life period of the catalase after mutation is respectively 2.17, 1.5, 2 and 1.67 times of the half-life period before mutation in the terms of thermal tolerance. The catalase mutant is suitable for industrialization production requirements and has wide application prospect.

Description

technical field [0001] The invention relates to a catalase mutant with improved enzyme activity and thermal stability, belonging to the technical field of bioengineering. Background technique [0002] Catalase (catalase) is referred to as CAT, and its system name is: H 2 o 2 oxidoreductase to H 2 o 2 As a specific substrate, it is finally degraded into water and oxygen by catalyzing the transfer of a pair of electrons. Catalase is widely used in textile, food, medicine, clinical and other industries. In the food processing industry, catalase can keep food fresh and act as an antioxidant to eliminate active oxygen and free radicals in beer and beverages. Together with glucose oxidase, it is used as an oxygen remover, milk sterilization and cheese raw milk sterilization; it is often used in instrument disinfection in the pharmaceutical industry; in clinical analysis, catalase is effective in studying free radical metabolic imbalance, anti-aging and tumor pathogenesis It ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/08C12N15/53C12N15/75C12N1/21C12R1/125C12R1/11
Inventor 陈坚堵国成康振曹汶龙
Owner JIANGNAN UNIV
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