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Specific activity and heat stability improved glucose oxidase mutant and encoding gene and application of glucose oxidase mutant

A technology of glucose oxidase and thermal stability, applied in the field of genetic engineering, can solve the problems of poor thermal stability, limited industrial application, low specific activity, etc., and achieve the effects of reducing production cost and improving specific activity and thermal stability.

Active Publication Date: 2018-07-06
GUANGDONG VTR BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Compared with Penicillium GOD, Aspergillus niger produced GOD has better thermal stability, but wild-type Aspergillus niger GOD has lower specific activity and poor thermal stability, which limits its industrial application

Method used

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  • Specific activity and heat stability improved glucose oxidase mutant and encoding gene and application of glucose oxidase mutant
  • Specific activity and heat stability improved glucose oxidase mutant and encoding gene and application of glucose oxidase mutant
  • Specific activity and heat stability improved glucose oxidase mutant and encoding gene and application of glucose oxidase mutant

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, optimization and gene synthesis of Aspergillus niger (Aspergillus niger) glucose oxidase (GOD) gene

[0032] The glucose oxidase gene (Genebank: FJ979866.1) of Aspergillus niger GIM 3.452 was optimized for the codon preference of Pichia pastoris, and the optimized nucleotide sequence was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. The 5' end and 3' end of the enzyme optimization gene (GOX) were introduced into EcoRI and XbaI restriction sites, and connected to the Puc57-amp vector, GOX-Puc57 was inoculated into LBA medium, and after culturing for 24 hours, the plasmid was extracted and used EcoRI and XbaI enzyme digestion, gel cutting to recover target gene fragments, product purification and recovery, were connected to expression vectors pPICzαA and pPGAPzαA, respectively, to obtain expression vectors pPICzαA-GOX and pGAPzαA-GOX.

Embodiment 2

[0033] Embodiment 2, site-directed saturation mutation

[0034] Using the above pPICzαA-GOX as a template and using the primers in the table for PCR amplification, the specific amplification reaction system is as follows:

[0035] Q5 High Fidelity Taq Enzyme MIX

23uL

Corresponding mutant primer 1 (50uM)

1uL

Corresponding mutant primer 1 (50uM)

1uL

pPICzαA-GOX (20ng)

2uL

Add water to

50uL

[0036] When the mutation site is V20Y, the corresponding mutant primer 1: 5'-cgccggtagaacctacgactacatcattgc-3'; the corresponding mutant primer 2: 5'-gcaatgatgtagtcgtaggttctaccggcg-3'. When the mutation site is T34V, the corresponding mutant primer 1: 5'-gaccggtttgaccgttgctgctagattga-3'; the corresponding mutant primer 2: 5'-tcaatctagcagcaacggtcaaaccggtc-3'. When the mutation site is D70Q, the corresponding mutant primer 1: 5'-ggatttgaatgcctacggtcagatcttcggatcttctgtcg-3'; the corresponding mutant primer 2: 5'-cgacagaagatccgaagatc...

Embodiment 3

[0039] Embodiment 3, high-throughput screening high specific activity mutant strain

[0040] Pick the recombinant yeast transformants obtained in Example 2 to a 24-well plate one by one with a toothpick, add 1 mL of medium containing BMGY to each well, culture at 30° C., 220 rpm for about 24 hours, and centrifuge to remove the supernatant. Then add 1.6mL BMMY medium respectively for induction culture. After culturing for 24 hours, the supernatant was collected by centrifugation, and 200 μL of the above supernatant was taken out to a 96-well plate, and the enzyme activity of glucose oxidase was measured. After high-throughput screening, six effective mutation sites were obtained, namely V20Y, T34V, D70Q, Q90R, V106I, and Y509E. The relative specific activities of these six mutants are shown in Table 1.

[0041] Table 1 Relative specific activity of original glucose oxidase and mutant glucose oxidase

[0042] Numbering

[0043] It can be known from Table 1 that the ...

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Abstract

The invention discloses a specific activity and heat stability improved glucose oxidase mutant and an encoding gene and the application of the glucose oxidase mutant, and relates to the field of genetic engineering. The glucose oxidase (GOD) of Aspergillus niger GIM 3.452(CICC 2377) is genetically optimized according to the codon preference of pichia pastoris; the optimized gene is subjected to site-specific mutagenesis through the site-specific mutagenesis technology, and mutation sites comprise the second site, the tenth site, the twentieth site, the thirty fourth site, the seventieth site,the ninetieth site, the one hundred and sixth site, the one hundred and tenth site, the one hundred and sixty seventh site, the three hundreds and fifth site, and the five hundreds and ninth site. Theheat resistance property and the specific activity of the obtained mutant are higher than those of the original strain, the demands of application in the fields of foods, medicines, feeds, textile industries and the like are well met, and the mutant has a quite broad application prospect.

Description

technical field [0001] The invention relates to a glucose oxidase mutant with improved specific activity and thermal stability, its coding gene and its application, and relates to the technical field of genetic engineering. 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 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] Compared with Penicillium GOD, the GOD produced by Aspergillus niger has better thermal stability, but the wild-type Aspergillus niger GOD has lower specific activity and poor thermal stability, which limits its industrial application. CN 103981159A discloses a glucose oxidase mutant and its application. By changing th...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/81C12N1/19
CPCC12N9/0006C12N15/815C12Y101/03004
Inventor 李阳源聂金梅黄江王勇王平
Owner GUANGDONG VTR BIO TECH
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