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Super-heat-resistant glucose oxidase AtGOD as well as gene and application thereof

A technology of glucose oxidase and sugar oxidase, which is applied in the field of genetic engineering, can solve the problems of rancidity and deterioration of feed, animal harm, and reduce the nutritional value and palatability of feed, so as to reduce the cost of formula, have excellent properties, and meet the physiological characteristics of digestion. Effect

Active Publication Date: 2021-06-01
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

The multivitamins, carotene and oils and other substances added to the feed are easily oxidized by oxygen in the air, thereby reducing the nutritional value and palatability of the feed, and even leading to rancidity and deterioration of the feed, producing peroxides that are harmful to animals

Method used

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  • Super-heat-resistant glucose oxidase AtGOD as well as gene and application thereof
  • Super-heat-resistant glucose oxidase AtGOD as well as gene and application thereof
  • Super-heat-resistant glucose oxidase AtGOD as well as gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Hyperthermable Glucose Oxidase At Cloning of the GOD-encoding gene

[0038] Aspergillus terreus Aspergillus terreus Genomic DNA and total RNA. Amplification primers were designed and synthesized according to the sequence Eco RI F / not I R:

[0039] Eco RI F: SEQ ID NO: 3, not IR: SEQ ID NO:4.

[0040] PCR amplification was carried out using the above-mentioned genomic DNA and total RNA as templates, respectively. The parameters of the touchdown PCR reaction were: denaturation at 95°C for 5 min; denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 5 min, 25 cycles, and incubation at 4°C for 10 min. A fragment of about 1800 bp was obtained, which was recovered and connected to the pEazy-T3 vector and sent to BGI Sequencing Company for sequencing.

Embodiment 2

[0041] Example 2 Hyperthermable Glucose Oxidase At Construction of GOD engineering strain

[0042] The expression vector pPIC9 was double digested ( Eco RI+ not 1), simultaneously with the gene encoding superthermostable glucose oxidase At GOD double digestion ( Eco RI+ not I), the digested glucose oxidase gene fragment is connected with the expression vector pPIC9 to obtain a hyperthermogenic glucose oxidase At GOD gene recombinant plasmid pPIC9 -atgod and transform Pichia pastoris GS115 to obtain recombinant Pichia pastoris strain GS115 / atgod .

Embodiment 3

[0043] Example 3 Preparation of Ultra-thermostable Recombinant Glucose Oxidase

[0044] (1) Massive expression of glucose oxidase at shake flask level in Pichia pastoris

[0045] Transformants with good thermal stability and high enzyme activity were screened out, inoculated in 1 L Erlenmeyer flasks with 300 mL of BMGY liquid medium, shaken at 220 rpm at 30°C for 48 h; centrifuged at 4500 rpm for 5 min, discarded Then add 200 mL of BMMY liquid medium containing 0.5% methanol to the cells, induce culture at 30°C, 220 rpm for 48 h. During the induction culture period, methanol solution was added once every 24 hours to compensate for the loss of methanol, so that the methanol concentration was kept at about 0.5%; centrifuged at 12,000×g for 10 minutes, collected the supernatant fermentation liquid, detected the enzyme activity and carried out SDS-PAGE protein electrophoresis analysis .

[0046] (2) Purification of ultrathermostable recombinant glucose oxidase

[0047] Collect ...

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Abstract

The invention relates to the field of gene engineering, in particular to super-heat-resistant glucose oxidase AtGOD as well as a gene and application thereof. The residual enzyme activity of the glucose oxidase treated at 90 DEG C for 2 minutes reaches 90%, and the residual enzyme activity of the glucose oxidase treated at 100 DEG C for 2 minutes reaches 60%. The glucose oxidase disclosed by the invention has super-heat-resistant thermal stability, and can meet the application requirements of industries such as feed, food, medicine, textile and the like.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a superthermostable glucose oxidase At GOD and its genes and applications. Background technique [0002] Glucose oxidase (Glucose oxidase, E.C.1.1.3.4) is widely derived from plants and microorganisms. There are many types of microorganisms and are easy to cultivate, so microorganisms are the main source of glucose oxidase. According to literature reports, most of glucose oxidase comes from fungi, mainly Aspergillus and Penicillium. In addition to microbial sources, the enzymatic activity of glucose oxidase can be detected in the saliva secreted by honeybees, tobacco budworms, cotton bollworms, beet armyworms, and spodoptera, and in the cuticle of grasshoppers, which can protect against harmful The role of fungi and bacteria. [0003] Glucose oxidase is a flavin adenine dinucleotide (FAD)-dependent oxidase, which can specifically catalyze β-D-glucose, and has almost no ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/56C12N15/81C12N1/19A23K20/189A23L29/00C12R1/84
CPCA23K20/189A23L29/06C12N9/0006C12N15/815C12Y101/03004
Inventor 黄火清涂涛罗会颖
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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