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A kind of glucose oxidase mutant and its carrier and application

A technology of glucose oxidase and mutants, applied in the field of glucose oxidase mutants and their carriers and applications, can solve the problems of high production cost, limited industrial application, low specific activity of GOD, etc., and achieve the effect of high relative enzyme activity

Active Publication Date: 2021-05-28
GUANGDONG VTR BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Wild-type Aspergillus niger has low GOD specific activity and high production cost, which limits its industrial application

Method used

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  • A kind of glucose oxidase mutant and its carrier and application
  • A kind of glucose oxidase mutant and its carrier and application
  • A kind of glucose oxidase mutant and its carrier and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, optimization and vector construction of Aspergillus niger (Aspergillus niger) glucose oxidase (GOD) gene

[0037] The glucose oxidase (GOD) gene (Genebank: FJ979866.1) of Aspergillus niger Aspergillus niger GIM 3.452 (CICC 2377), its nucleotide sequence is shown in SEQ ID NO: 2, and its amino acid sequence is shown in SEQ ID NO: 1 Show.

[0038]Through a large number of previous studies, it was found that the expression level of Aspergillus niger expression engineering bacteria was low, the operation was complicated, and it was difficult to perform high-throughput screening of mutations. At the same time, it was found that the glucose oxidase expressed in the Escherichia coli expression system had no activity and could not be used for mutation screening. Pichia pastoris is a mature exogenous protein expression platform for eukaryotic microorganisms, which has the following characteristics: rapid growth and cheap culture; simple and easy to operate, conven...

Embodiment 2

[0039] Embodiment 2, glucose oxidase gene site-directed mutation

[0040] Using the above pGAPzαA-GOX as a template, introduce site-directed mutation and transform Pichia pastoris X33

[0041] According to the three-dimensional structure information of glucose oxidase, after a lot of research, it is believed that the 34th, 51st, 60th, 90th, 106th, 243rd, 420th, and 561st amino acids in the amino acid sequence have important effects on the enzymatic activity of glucose oxidase, and saturation mutations are carried out on the above sites , Pick the yeast recombinant transformants obtained by the site saturation mutation one by one to a 24-well plate, 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 ...

Embodiment 3

[0049] Embodiment 3, glucose oxidase gene combination mutation

[0050] Effective mutation T34S, V; S51G, K, P; P60A, G, H; Q90G, A, R; V106Y, C, I; Q243E, H, V; , A are combined separately. Finally, six combined mutations with significantly improved enzyme activity were obtained through experiments and named GOX-M1, GOX-M2, GOX-M3, GOX-M4, GOX-M5, and GOX-M6, respectively.

[0051] The mutation sites included in GOX-M1 are: T34S, S51K, P60A, Q90A, V106Y, Q243E, M561S, its amino acid sequence is shown in SEQ ID NO: 4, and its corresponding nucleotide sequence is shown in SEQ ID NO: 10 Show.

[0052] The mutation sites included in GOX-M2 are: T34V, S51P, P60A, Q90R, V106Y, Q243H, M561T, its amino acid sequence is shown in SEQ ID NO: 5, and its corresponding nucleotide sequence is shown in SEQ ID NO: 11 Show.

[0053] The mutation sites included in GOX-M3 are: T34V, S51P, P60A, Q90R, V106I, Q243E, M561S, its amino acid sequence is shown in SEQ ID NO: 6, and its corresponding...

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Abstract

The invention discloses a glucose oxidase mutant, the glucose oxidase comprises the amino acid sequence of the glucose oxidase of Aspergillus niger GIM 3.452 (CICC 2377), and is included in the 34th, 51st, 60th, 90th, 106th, Mutations at amino acid positions 243 and 561, wherein the amino acid sequence of glucose oxidase from Aspergillus niger GIM 3.452 (CICC 2377) is shown in SED ID NO:1. The glucose oxidase mutant uses Pichia pastoris as a protein expression platform, which can significantly increase the enzyme activity, and can also maintain a relatively high relative enzyme activity under acidic conditions.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, more specifically, to a glucose oxidase mutant, its carrier 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. Glucose oxidase is widely distributed in nature, has the characteristics of high reaction efficiency and strong catalytic specificity, and is widely used in the food industry and feed addition. Glucose oxidase is a dehydrogenation enzyme preparation that uses β-D-glucose as a substrate. I...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/81C12N1/19C12P19/02A23K20/189A23L29/00C12R1/84
CPCA23K20/189A23L29/06C12N9/0006C12N2800/22C12P19/02C12Y101/03004
Inventor 李阳源黄江何小梅陈丽芝刘金山黄佳乐陈琼银冯国华周银华梁雪霞
Owner GUANGDONG VTR BIO TECH
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