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Method for improving acid stability of glucose oxidase, mutant Q241E/R499E, gene and application

A technology of glucose oxidase and mutants, applied in the direction of oxidoreductase, microbial-based methods, biochemical equipment and methods, etc., can solve the problem of low enzyme activity, achieve improved acid stability, enhanced acid stability, improved The effect of acid stability

Active Publication Date: 2021-12-31
INST OF ANIMAL SCI CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the pH condition for the above-mentioned glucose oxidase mutant GOD-M10 to exert the maximum enzyme activity is 6.0, and the pH stability is extremely low at pH 2.5-3.0.

Method used

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  • Method for improving acid stability of glucose oxidase, mutant Q241E/R499E, gene and application
  • Method for improving acid stability of glucose oxidase, mutant Q241E/R499E, gene and application
  • Method for improving acid stability of glucose oxidase, mutant Q241E/R499E, gene and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1, recombinant strain GS115 (pPIC9 -godm10 ) preparation

[0053] (1) Amplify the nucleic acid sequence of the maternal high thermostable glucose oxidase mutant GODM10 godm10

[0054] Amplified by PCR godm10 Gene fragments, the vector pPIC9 nucleic acid fragments were obtained by double enzyme digestion, and the two were connected by a recombination kit to obtain the recombinant plasmid pPIC9- godm10 , and transform Pichia pastoris GS115 to obtain recombinant Pichia pastoris strain GS115 (pPIC9 -godm10 ). The primers used in PCR are as follows:

[0055] godm10-pPIC9-F (GOD ID No: 9, 40bp): GGTATTGAGGCTTCCTTGTTGACTGACCCAAAGGAGGTCG

[0056] godm10-pPIC9-R (GOD ID No: 10, 40bp): TTGCATGGAGGCGTAGTCAGCCAAAACAGCGTCTGCGATC

[0057] Among them, godm10-pPIC9-F and godm10-pPIC9-R are used to amplify the gene coding sequence of glucose oxidase M10; the vector pPIC9 is obtained by extracting the preserved strains after bottle culture. After the amplificati...

Embodiment 2

[0060] Embodiment 2, recombinant bacterial strain GS115 (pPIC9 -godm10-Q241E , pPIC9 -godm10-R499E , pPIC9 - godm10-Q241E / R499E ) preparation

[0061] (1) Recombinant plasmid pPIC9- godm10-Q241E , pPIC9- godm10-R499E , pPIC9- godm10-Q

[0062] 241E / R499E build

[0063] After optimization, the design of the mutation site is to mutate glutamine and arginine at positions 241 and 499 to glutamic acid respectively, introduce the mutation site through the method of point mutation kit, and perform sequencing verification on it to obtain glucose Oxidase mutant plasmid pPIC9- godm10-Q241E , pPIC9- godm10-R499E , pPIC9- godm10-Q241E / R499E . The primers used are as follows:

[0064] Q241E-F (God ID No: 10) AGACCTAACTTGGAGGTTTTGACCGGTCAATACGT

[0065] Q241E-R (God ID No: 11) ACCGGTCAAAACCTCCAAGTTAGGTCTTTGGTAGT

[0066] R499E-F (God ID No: 12) GATGCTGACTTGGAGGCTTGGGTTGAATACATTCC

[0067] R499E-R (God ID No: 13) TTCAACCCAAGCCTCCAAGTCAGCATCGTAGGCCA

[0068] Among t...

Embodiment 3

[0071] Example 3, the acquisition of high thermostable glucose oxidase M10 and Q241E, R499E, Q241E / R499E

[0072] 1. Induced expression of GODM10 and Q241E, R499E, Q241E / R499E

[0073] The obtained recombinant expression strain GS115 ( pPIC9-godm10 ) and GS115 (pPIC9- godm10-Q241E , pPIC9- godm10-R499E , pPIC9-godm10-Q241E / R499E ) were inoculated into YPD medium for seed culture, 200 rpm, 30°C for 48 h, then transferred to BMGY medium with 1% inoculum, 200 rpm, 30°C for 48 h, and collected after enriching enough bacteria Bacteria were added to BMMY medium containing 1% methanol to induce expression.

[0074] 2. Purification of GODM10 and Q241E, R499E, Q241E / R499E

[0075] Centrifuge the induced bacterial solution at 12000 rpm for 10 min, collect the supernatant and concentrate it, then dialyze it with 10 mM disodium hydrogen phosphate solution (adjust the pH to 6.5 with citric acid), and then carry out the ion exchange layer for the dialyzed enzyme For analysis, solu...

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Abstract

The invention relates to the field of genetic engineering, in particular to a method for improving acid stability of glucose oxidase, a mutant Q241E / R499E, a gene and application. A glucose oxidase mutant GODM10 with high thermal stability is subjected to site-directed mutagenesis to obtain a mutant with improved acid stability. The glucose oxidase mutant disclosed by the invention has good enzymatic properties and can be applied to the industries of foods, feeds and the like.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for improving the acid stability of glucose oxidase, mutant Q241E / R499E, gene and application. Background technique [0002] Glucose oxidase (Glucose oxidase, GOD) is an aerobic dehydrogenase that can specifically oxidize β-D-glucose into gluconic acid and hydrogen peroxide. Glucose oxidase is widely distributed in animals, plants and microorganisms, and microorganisms are its main source, and the main producing strains are Aspergillus niger and Penicillium. Glucose oxidase, as a new feed enzyme preparation to replace antibiotics, has many functions such as improving animal intestinal microecological balance and intestinal digestive environment, protecting intestinal health, improving feed utilization, promoting animal growth, and improving body immunity. It is widely used in the feed industry. [0003] Chinese patent application CN201910835766.4 provides sources fro...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/81C12N1/19C12R1/84
CPCC12N9/0006C12N15/815C12Y101/03004C12N2800/102
Inventor 涂涛黄火清闫亚茹姚斌罗会颖张伟王苑柏映国苏小运王亚茹张杰秦星王晓璐张红莲于会民
Owner INST OF ANIMAL SCI CAAS
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