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Optimized gene of recombinant glucose oxidase and expression vector and application of optimized gene

A technology of glucose oxidase and expression vector, which is applied in the field of recombinant glucose oxidase, can solve the problems affecting the efficiency of transcription, etc., and achieve the effects of improving transcription efficiency, reducing the energy barrier of transcription, and improving the free energy of mRNA secondary structure

Active Publication Date: 2013-08-28
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

During the transcription process, the mRNA secondary structure complexity and free energy of the target gene significantly affect the efficiency of transcription

Method used

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  • Optimized gene of recombinant glucose oxidase and expression vector and application of optimized gene
  • Optimized gene of recombinant glucose oxidase and expression vector and application of optimized gene
  • Optimized gene of recombinant glucose oxidase and expression vector and application of optimized gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The optimal design and synthesis of embodiment 1 glucose oxidase gene

[0045] 1.1 Strains and plasmids

[0046] Penicillium notatum (Penicillium notatum) is preserved by the inventor's laboratory;

[0047] Escherichia coli (Escherichia coli) strain TOP10 and cloning vector pSP72 were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; the whole gene fragment of god-h was synthesized by Beijing Aoke Biotechnology Company.

[0048] 1.2 Optimum design of glucose oxidase gene

[0049] The present invention first analyzes the sequence of the original glucose oxidase gene (god-w) cloned from Penicillium notatum (for the cloning of the gene, please refer to: Li Zhuofu (Master's Thesis), Cloning of the Glucose Oxidase Gene and high-efficiency expression. 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 d...

Embodiment 2

[0059] Example 2 Construction and Screening of Glucose Oxidase Recombinant Pichia Pastoris Strains

[0060] 1.1 Strains and plasmids

[0061] Top10 Escherichia coli competent cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

[0062] The expression vector pPIC9 and Pichia pastoris recipient strain GS115 are products of Invitrogen.

[0063] Pichia recombinant strain 53# with the original glucoamylase gene (god-w) (enzyme activity is 130U / ml), and Pichia recombinant strain with the first modified glucoamylase gene (god-m) The strain DG16# (enzyme activity is 614U / ml) was constructed and preserved in the inventor's laboratory; the plasmid pSP72-god-h carrying the modified glucose oxidase gene (god-h) of the present invention was artificially synthesized.

[0064] 1.2 Medium and other solutions

[0065] 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.

[0066] 10×YNB (yeas...

Embodiment 3

[0100] Embodiment 3 The application effect test of the recombinant glucose oxidase prepared by the present invention on bread

[0101] 1.1 Concentration of glucose oxidase samples

[0102] The content of the recombinant glucose oxidase expressed by Pichia pastoris in Example 2 accounts for more than 90% of the total protein in the supernatant of the fermentation broth, so the supernatant of the fermentation broth can be used for later experiments after being concentrated by ultrafiltration. First, the fermented supernatant of the recombinant yeast strain Gh168# which was transferred to god-h in Example 2 was centrifuged at 10,000 rpm for 10 min to remove the bacterium sediment, and the supernatant enzyme liquid was concentrated using a tangential flow membrane filtration system of PALL Company, first using 0.1 μm microfiltration membrane to remove residual cell debris and possible impurities in the supernatant enzyme solution, and collect the filtrate. Then the filtrate was f...

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Abstract

The invention discloses an optimized gene of recombinant glucose oxidase and an expression vector and application of the optimized gene. On the premise that the amino acid sequence of the glucose oxidase is not changed, the gene sequence of the glucose oxidase is optimized according to pichia pastoris preferred codons by comprehensively considering the influencing factors such as use frequency ofthe codons, adjustment of GC content, deletion of instable sequences, secondary mRNA structure and the like; and the nucleotide sequence of the optimized glucose oxidase gene is shown as SEQ ID NO.1.The invention further provides the expression vector and a recombinant host strain containing the optimized gene of the glucose oxidase. The optimized gene is transferred to the pichia pastoris for expression, and the test results show that: compared with the gene before optimization, the secreting expression quantity of the optimized gene in the pichia pastoris is remarkably improved. The application effect tests of the glucose oxidase show that the expressed recombinant glucose oxidase has the same using effect as a commercial enzyme preparation.

Description

technical field [0001] The present invention relates to a codon-optimized mutant gene, in particular to an optimized gene modified by modifying the glucose oxidase gene according to the preferred codon of Pichia pastoris, a recombinant expression vector and a recombinant host cell containing the optimized gene, The present invention further relates to their application in the production of glucose oxidase, which belongs to the field of recombinant glucose oxidase. 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 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N15/63C12N15/81C12N1/19C12N9/04C12R1/84
Inventor 张伟张宇宏范云六姚斌刘波
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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