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Aspergillus niger glucose oxidase optimization gene and its expression vector and application

A glucose oxidase and expression carrier technology, applied in the field of glucose oxidase genetic engineering, can solve the problems of high production cost, low secretion expression efficiency, and inability to meet industrial large-scale production applications, etc., to reduce production costs and increase secretion expression Effect

Active Publication Date: 2022-02-25
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

[0011] At present, the highest secreted expression level of glucose oxidase gene reported in the literature in yeast is only 1972U / ml (Gu L., et al., Multivariate modular engineering of the protein secretory pathway for production of heterologous glucose oxidase in Pichia pastoris[J ].Enzyme Microb Technol,2015,68(68):33-42.); Therefore, the existing glucose oxidase gene has defects such as low secretion and expression efficiency and high production cost, which cannot meet the needs of large-scale industrial production and application, and urgently need Improve

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  • Aspergillus niger glucose oxidase optimization gene and its expression vector and application
  • Aspergillus niger glucose oxidase optimization gene and its expression vector and application
  • Aspergillus niger glucose oxidase optimization gene and its expression vector and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Optimal Design and Synthesis of Glucose Oxidase Gene

[0049] 1. Test method

[0050] 1.1 Strains and plasmids

[0051] Aspergillus niger (Aspergillus niger), preserved by the inventor's laboratory;

[0052] Escherichia coli (Escherichia coli) strain Trans1-T1 was purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

[0053] Gene fragments were synthesized by Nanjing GenScript Biotechnology Company.

[0054] 1.2 Optimum design of glucose oxidase gene

[0055] According to the sequence of the original glucose oxidase gene cloned from Aspergillus niger, the gene sequence was optimized, and the process was mainly based on the following principles:

[0056] 1) The amino acid sequence (SEQ ID NO.6) encoded by the glucose oxidase gene AGOD is not changed;

[0057] 2) Reduce the stable structural regions (such as hairpin loops) that may exist in the secondary structure of the mRNA corresponding to the gene sequence, especially reduce the free energy of the...

Embodiment 2

[0072] Example 2 Construction and Screening of Glucose Oxidase Recombinant Pichia Pastoris Strains

[0073] 1. Test method

[0074] 1.1 Strains and plasmids

[0075] Trans1-T1 Escherichia coli competent cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

[0076] The expression vector pPIC9 and Pichia pastoris recipient strain GS115 are products of Invitrogen;

[0077] The original gene sequence of glucose oxidase is obtained by PCR amplification and sequencing using the genomic DNA of Aspergillus niger strain as a template.

[0078] Plasmid pUC19-simple-AGOD with original glucose oxidase gene, plasmids pUC19-simple-AGOD-m2, pUC19-simple-AGOD-m3, pUC19-simple-AGOD-m4 and pUC19 with optimized glucose oxidase gene -simple-AGOD-m5 was synthesized by Nanjing GenScript Biotechnology Company.

[0079] 1.2 Medium and other solutions

[0080] YPD medium: glucose 20g / L, peptone 20g / L, yeast extract 10g / L, without pH adjustment, autoclaved at 115°C for 30min;

[...

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Abstract

The invention discloses a glucose oxidase optimization gene, its expression carrier and application. In the present invention, the glucose oxidase gene derived from Aspergillus niger is used without changing the amino acid sequence of the protein, including codon usage frequency, distribution of GC content in different gene regions, codon adaptation index CAI, mRNA structure optimization, and 5'mRNA freedom. Multiple strategies such as optimization and deletion of unstable sequences were optimized; Pichia pastoris expression results showed that the optimized AGOD‑m3 and AGOD‑m5 genes could increase the secretion and expression of enzyme proteins in Pichia pastoris, Among them, the secreted expression of AGOD‑m3 in Pichia pastoris was significantly increased, and the enzyme activity was also the strongest. The present invention further provides the recombinant expression vector containing the optimized gene and its application in the preparation of glucose oxidase. The invention lays the foundation for the large-scale industrial production of glucose oxidase.

Description

technical field [0001] The present invention relates to an optimized glucose oxidase gene and a recombinant expression vector and a recombinant host cell containing the Aspergillus niger glucose oxidase optimized gene, and the present invention further relates to the application of the Aspergillus niger glucose oxidase optimized gene in preparing glucose oxidase, The invention belongs to the field of glucose oxidase genetic engineering. Background technique [0002] Glucose oxidase (Glucose oxidase, referred to as GOD, EC 1.1.3.4) can specifically oxidize β-D-glucose to gluconolactone and hydrogen peroxide under aerobic conditions. GOD can remove oxygen, produce acid, sterilize, and remove glucose, so it is widely used in food, medicine, feed, chemical and other industries. [0003] In the field of medicine, GOD is mainly used as a tool enzyme in blood glucose detectors. It is used in new biosensors to make blood glucose and urine glucose determination faster and easier, wh...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N15/81C12N1/19C12R1/84
CPCC12N9/0006C12N15/815C12Y101/03004
Inventor 张宇宏张伟张艳丽
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI