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Glucose oxidase mutant gene, expression and application thereof

A technology of glucose oxidase and mutant gene, which is applied in the field of preparation of glucose oxidase, can solve problems such as affecting the expression amount, and achieve the effect of increasing the secretion and expression amount

Inactive Publication Date: 2011-01-26
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the foreign gene contains more host non-preferred codons, especially if they appear continuously, it will seriously affect its expression in the host bacteria

Method used

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  • Glucose oxidase mutant gene, expression and application thereof
  • Glucose oxidase mutant gene, expression and application thereof
  • Glucose oxidase mutant gene, expression and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Optimal Design and Synthesis of Glucose Oxidase Gene

[0038] 1.1 Strains and plasmids

[0039] Peniciliium notatum (Peniciliium notatum), preserved by the inventor's laboratory;

[0040] Escherichia coli (Escherichia coli) strain TOP10 and cloning vector pSP72 were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; small gene fragments were synthesized by Beijing Aoke Biotechnology Company.

[0041] 1.2 Optimum design of glucose oxidase gene

[0042] First, the sequence of the original glucose oxidase gene cloned from Penicillium notatum was analyzed (for gene cloning, please refer to: Li Zhuofu (Master's Thesis), Cloning and High Efficiency Expression of Glucose Oxidase Gene. Changchun University of Science and Technology , 2008), under the premise of not changing the amino acid sequence of the protein, comprehensively considering the codon usage frequency, the adjustment of GC content, the deletion of unstable sequences and other influencing facto...

Embodiment 2

[0095] Example 2 Construction and Screening of Glucose Oxidase Recombinant Pichia Pastoris Strains

[0096] 2.1 Strains and plasmids

[0097] Top10 Escherichia coli competent cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

[0098] The expression vector pPIC9 and Pichia pastoris recipient strain GS115 are products of Invitrogen.

[0099] The plasmid pBluescriptSK-GOD with the original glucoamylase gene and the plasmid pSP72-GOD-M with the modified glucoamylase gene were constructed by the inventor's laboratory.

[0100] 2.2 Medium and other solutions

[0101] YPD medium: peptone 20g / L, yeast extract 10g / L, glucose 20g / L (solid medium contains 1.5% agar powder), sterilized at 108°C for 15min.

[0102] 10×YNB (yeast without amino acid nitrogen source): 134g YNB solid was dissolved in 1L deionized water, sterilized by filtration, and stored at 4°C;

[0103] 500×biotin: biotin 20mg / 10omL water, filter sterilized, store at 4°C.

[0104] 10×glucose: Dissol...

Embodiment 3

[0135] Embodiment 3 Glucose oxidase enzymatic property determination

[0136] 3.1 Concentration of glucose oxidase samples

[0137] The content of the glucose oxidase expressed by Pichia pastoris in the fermentation broth supernatant accounts for more than 90% of the total protein, so the fermentation broth supernatant can be used for later-stage property determination by ultrafiltration concentration ( Figure 8 ). First, centrifuge the supernatant of the fermentation broth at 10,000rpm for 10min to remove bacterial precipitates. The supernatant enzyme solution is concentrated by PALL’s tangential flow membrane filtration system. Cell debris and possible impurities are collected and the filtrate is collected. Then the filtrate was filtered through an ultrafiltration membrane with a molecular weight cut-off of 10 kDa, and the filtrate was discarded to obtain a concentrated enzyme solution for the determination of enzymatic properties.

[0138] 3.2 Determination of glucose o...

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Abstract

The invention discloses a glucose oxidase mutant gene, expression and application thereof. In the invention, 272 basic groups are changed through codon optimization and GC content change and the content of GC is reduced to 48.44% from 55.54% so as to obtain the glucose oxidase mutant gene, wherein the basic group is represented by SEQ ID NO.2. The glucose oxidase mutant gene is transferred into pichia yeast to express; the experimental result shows that the secretory expression level of the glucose oxidase mutant gene in the pichia yeast is significantly improved by comparing with the same before mutation; compared with partial research at home and abroad, the final secretory expression of the glucose oxidase mutant gene achieves high expression level, thereby building the foundation for further expansion of industrial production. The determination of enzymatic properties of the glucose oxidase mutant gene shows that the recombinant glucose oxidase protein expressed by the glucose oxidase mutant gene has good thermal stability and high enzyme activity.

Description

technical field [0001] The invention relates to a glucose oxidase gene, in particular to a glucose oxidase mutant gene after codon optimization, and also relates to the application of the glucose oxidase mutant gene in preparing glucose oxidase, which belongs to the field of glucose oxidase preparation. Background technique [0002] Glucose Oxidase (Glucose Oxidase, referred to as GOD) scientific name β-D-glucose oxidoreductase (EC1.1.3.4), it can specifically oxidize β-D-glucose into gluconic acid and hydrogen peroxide (Pluschkell S, Hellmuth K and Rinas U, Kinetics of glucose oxidase excretion by recombinant Aspergillus niger. Biotechnology and bioengineering, 1996, 51: 215-220.). GOD is widely used in food, feed, medicine and many other related fields (Bankar SB, Bule MV, Singhal RS, et al., Glucose oxidase-An overview. Biotechnol Adv, 2009). [0003] The main role of GOD in the food industry is to remove glucose, remove oxygen, form hydrogen peroxide, and form gluconic ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/63C12N15/81C12N1/15C12N1/19C12N1/21C12N5/10C12N9/04
Inventor 张伟姚斌范云六张宇宏
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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