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Mutated glucose oxidase with increased expression quantity and encoding gene and application thereof

A glucose oxidase and expression technology, applied in the field of mutant glucose oxidase and its coding gene and application, can solve the problems of low enzyme activity, complex detection methods, low yield, etc.

Active Publication Date: 2015-10-28
GUANGDONG VTR BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Low yield, low enzyme activity, and complicated detection methods are the limitations of GOD industrialization

Method used

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  • Mutated glucose oxidase with increased expression quantity and encoding gene and application thereof
  • Mutated glucose oxidase with increased expression quantity and encoding gene and application thereof
  • Mutated glucose oxidase with increased expression quantity and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The glucose oxidase gene that the expression level of embodiment 1 improves

[0044] On the basis of the gene sequence of Genebank: FJ979866.1; two amino acid sites are mutated, specifically Y76C and Q279K, the two sites can be mutated by chemical synthesis or site-directed mutagenesis, and the resulting The GOD mutant amino acid sequence is shown in SEQ ID NO.1.

Embodiment 2

[0045] Example 2 Construction and Identification of Glucose Oxidase Genetic Engineering Bacteria

[0046] The GOD gene whose sequence is shown in Genebank: FJ979866.1 was obtained by PCR method or chemical total synthesis method. The PCR method primer sequence is as follows:

[0047] GODF:5'-CGGAATTCAGCAATGGCATCGAAGCCAGCCTC-3'

[0048] GODR: ​​5'-ATAGTTTAGCGGCCGCTCACTGCATGGAAGCATAATC-3'

[0049] PCR reaction system: Add 1 μl of Aspergillus niger GIM 3.452 (CICC 2377) DNA in sequence to a 0.2 mL PCR tube as a template; 1 μl of upstream and downstream primers; 25 μl of 2x pfu enzyme; add double distilled water to a final volume of 50 μl; Proliferation conditions: pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 30 seconds, annealing at 63°C for 30 seconds, extension at 72°C for 4 minutes (30 cycles); extension at 72°C for 10 minutes; the purified PCR product (GOD) was recovered by retention gel.

[0050] The EcoRI restriction site in the GOD gene sequence was ...

Embodiment 3

[0073] Example 3 High Expression of Glucose Oxidase Recombinant Strain

[0074] The above-mentioned single colonies of the starting bacterium P. pastoris X33-pPICZαA-GOD and the mutant recombinant bacterium P. pastoris X33-pPICZαA-GODmut were subjected to high-density fermentation culture. Configure 20L of basic salt medium, sterilize it in a 50L automatic control fermenter, and cool it to room temperature for later use. The pH value of the fermentation broth is adjusted to 5.0 with ammonia water and phosphoric acid, the dissolved oxygen is controlled to be greater than 30% by adjusting the rotating speed and air flow rate, and the fermentation temperature is 30°C. The whole fermentation process is divided into 3 stages: the first stage is the cell culture stage, the recombinant bacteria are inoculated into the fermenter according to the inoculation amount of 10%, and 4L of 50% glucose that has been sterilized is added, cultivated for 24-30h, and then It is marked by the comp...

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Abstract

The invention relates to the field of genetic engineering, in particular to mutated glucose oxidase with the increased expression quantity and an encoding gene and application thereof. The amino acid sequence of the mutated glucose oxidase is shown as SEQ ID NO.1. The site-specific mutagenesis technology is adopted for carrying out site-specific mutagenesis on a gene (Genebank:FJ979866.1) of glucose oxidase (GOD) of aspergillus niger GIM 3.452(CICC 2377) to enable mutation sites of the amino acid sequence of the mutated glucose oxidase to be Y76C and Q279K; the mutant gene is cloned and connected to a pichia pastoris expression vector pPICZalphaA, and pichia pastoris X33 is converted, and screened to obtain a glucose oxidase pichia pastoris strain P.pastoris X33-pPICZalphaA-GODmut with the increased expression quantity. Enzymatic property determination shows that the mutated glucose oxidase gene can be expressed and inherited in pichia pastoris stably and efficiently, and the enzymatic activity and the stability of glucose oxidase expressed by the strain are remarkably higher than those of an original strain, which lays a good foundation for large-scale production of glucose oxidase.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a mutant glucose oxidase with increased expression and its coding gene and application. Background technique [0002] Glucose oxidase (glucose oxidase, GOD) can specifically catalyze β-D-glucose to generate gluconic acid and hydrogen peroxide under aerobic conditions. GOD is a homodimeric molecule containing two flavin adenine dinucleotide (FAD) binding sites. Each monomer contains two completely different domains: one is non-covalently but tightly bound to part of FAD, mainly for acoustic folding; the other binds to the substrate β-D-glucose, and is supported by 4 α-helices for a trans Parallel beta-sheets. GOD is widely distributed in animals, plants and microorganisms. Due to the rapid growth and reproduction of microorganisms and wide sources, they are the main source of GOD production. The main production strains are Aspergillus niger and Penicillium. [0003] GOD has ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/81C12N1/19C12R1/84
CPCC12N9/0006C12Y101/03004
Inventor 聂金梅李阳源周银华陈丽芝刘金山李天碧
Owner GUANGDONG VTR BIO TECH
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