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Glucose oxidase mutant and encoding gene and application thereof

A glucose oxidase and coding gene technology, applied in glucose oxidase mutants and its coding genes and application fields, can solve the problems of low expression, high production cost, hindering industrial production and application, etc., and achieve broad application prospects, Excellent thermal stability, high enzyme activity effect

Active Publication Date: 2017-09-22
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the heterologous expression of GOD has been realized by means of genetic engineering, the problem of high production cost due to the low expression level has not been effectively solved, which hinders the large-scale industrial production and application of GOD

Method used

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  • Glucose oxidase mutant and encoding gene and application thereof
  • Glucose oxidase mutant and encoding gene and application thereof
  • Glucose oxidase mutant and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1, Site-directed Mutation of Glucose Oxidase Encoding Gene

[0070] The homology modeling of glucose oxidase GODA was carried out, and the mutation site was designed as the 82nd glutamic acid was mutated to cysteine. The mutation site was introduced by Over-lap PCR method, and it was sequenced and verified to obtain the mutant gene GODB. Primers used in Over-lap PCR are shown in Table 1:

[0071] Table 1. Mutant GODB-specific primers

[0072]

[0073] Sequencing results show that the above-mentioned Over-lap PCR amplified nucleic acid fragments are: the 5' end contains the restriction site EcoRI, the 3' end contains the restriction site Not I, and the middle part has the sequence ID of SEQ ID №: 1 in the sequence table. Nucleic acid fragment showing nucleotide sequence. Among them, the nucleic acid fragment with the nucleotide sequence shown in SEQ ID No.: 1 in the sequence listing has a total of 1746 bp, and the coding region is 1743 bp long. Shown by ac...

Embodiment 2

[0074] The preparation of embodiment 2 glucose oxidase mutant GODB

[0075] (1) Preparation of recombinant plasmid pPIC9-GODB

[0076] Carry out double enzyme digestion (Eco RI+Not I) of expression vector pPIC9; Carry out double enzyme digestion (Eco RI+Not I) of the nucleic acid fragment prepared in the above-mentioned embodiment 1 at the same time; The above-mentioned two nucleic acid fragments that cut are connected , to obtain a recombinant plasmid containing the mutant gene GODB.

[0077] The recombinant plasmid obtained above was sent for sequencing to verify the correctness of the sequence. The recombinant plasmid in which the sequence of the foreign gene inserted in the obtained plasmid is the nucleotide sequence shown in SEQ ID No. 1 is named pPIC9-GODB.

[0078] (2) Preparation of recombinant bacteria GS115 / GODB

[0079] The recombinant plasmid pPIC9-GODB was transformed into Pichia pastoris GS115 cells to obtain recombinant yeast strain GS115 / GODB. The plasmid o...

Embodiment 3

[0082] Embodiment 3 The property analysis comparison of glucose oxidase mutant GODB and wild type

[0083] (1) Enzyme activity analysis and comparison

[0084] GOD enzyme activity was determined by ultraviolet spectrophotometer. The specific method is as follows: Carry out the enzymatic reaction under the given conditions. The enzymatic reaction system is: 200 μL reaction system, including 50 μL appropriate diluted enzyme solution, 20 μL 10 mM ABTS solution, 20 μL 50 U / mL HRP solution, 90 μL hydrogen phosphate Disodium-citrate Buffer, and 20μL 1M glucose solution. Measure the growth curve of optical density with time within 3min at a wavelength of 420nm, record it every 30s, and obtain the enzyme activity according to the slope of the straight line. One enzyme activity unit (U) is defined as the amount of enzyme required to generate 1 μmol of oxidized ABTS per unit time under given conditions.

[0085] After the glucose oxidase mutant GODB prepared in the above-mentioned Ex...

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Abstract

The invention discloses a GODB mutant and an encoding gene and application thereof. The GODB mutant derives from GODA of Aspergillus niger, a female parent, and is obtained through point mutation Glu82Cys. The GODB mutant has the advantages that mutant enzyme activity is increased from 229.6 U / mg of a wild type to 352.1 U / mg, an increase of 53.3%, a half-life period at 60 DEG C is increased to 119 minutes from 51 minutes of the wild type, an increase of 133%, and accordingly the GODB mutant can meet the requirements of the fields such as food, medicine, feed and the textile industry and is promising in application prospect.

Description

technical field [0001] The invention belongs to the field of genetic engineering and genetic engineering, and specifically relates to a glucose oxidase mutant, its coding gene and its application. Background technique [0002] Glucose oxidase (glucose oxidase, GOD) is also known as β-d-glucose oxidoreductase, EC number is 1.1.3.4, can specifically act on the hydroxyl group of β-d-glucose, making it dehydrogenated and oxidized to carboxyl Or aldehyde group, generating gluconolactone and hydrogen peroxide. Because GOD has the functions of removing glucose, deoxidizing, and sterilizing, it is widely used in food, medicine, feed, textile and other industries. For example, in the food industry, GOD can effectively remove glucose to inhibit food browning, and the product hydrogen peroxide is a bactericide, which can prolong the shelf life of food; in the pharmaceutical industry, using the substrate specificity of GOD can Applied to the quantitative determination of glucose in hu...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/81C12N1/19C12R1/84
CPCC12N9/0006C12N15/815C12Y101/03004
Inventor 姚斌罗会颖涂涛刘伟娜黄火清柏映国王苑苏小运王亚茹孟昆
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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