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Method for enhancing glucose oxidase secretion through transforming folding and secreting pathway of protein

A glucose oxidase and protein technology, applied in the field of genetic engineering, can solve problems such as high cost and complicated separation and extraction

Active Publication Date: 2018-03-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are still difficulties in large-scale production of highly active GOD
During the production of GOD by fermentation, a large amount of miscellaneous protein is produced, and the separation and extraction are complicated and costly

Method used

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  • Method for enhancing glucose oxidase secretion through transforming folding and secreting pathway of protein
  • Method for enhancing glucose oxidase secretion through transforming folding and secreting pathway of protein
  • Method for enhancing glucose oxidase secretion through transforming folding and secreting pathway of protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 The influence of each different module gene on the secretion of glucose oxidase

[0042] 1.1 The effect of the folding module genes in the modified endoplasmic reticulum on the exogenous expression of GOD

[0043] The protein folding rate in the endoplasmic reticulum (ER) and the secretion rate in the cytoplasm are important factors affecting the secretion of exogenous proteins in yeast cells. The resident proteins Kar2p, Cne1p and Ero1p involved in the protein folding mechanism in the ER can help the nascent unfolded peptide chain to fold into the correct conformation. In order to study the effect of the ER folding module on the secretion of exogenous GOD, the ER resident protein coding genes KAR2, CNE1 and ERO1 in P. pastoris were overexpressed in the P. pastoris engineering strain PP-GOD to enhance the transformation of the engineered strain cells. The expression levels of the corresponding functional proteins within are shown in Table 3. After co-expr...

Embodiment 2

[0062] Example 2 Effect of protein secretion pathway module optimization on exogenously expressed glucose oxidase

[0063] The co-expression of each module gene in Example 1 can improve the production of GOD in recombinant P. pastoris, which indicates that each module can be modified to secrete exogenous GOD from P. pastoris. In order to further improve the ability of recombinant P. pastoris to produce exogenous GOD, the influence of the optimized combination of each module gene on the ability to produce exogenous GOD was studied. Due to the interconnection between each functional block, simply superimposing the functional module genes in a functional block cannot make the result show an additive increase. Therefore, in this example, the genes of different functional modules are combined with each other through modular optimization and transformation, so as to further improve the production of exogenous GOD by recombinant P. pastoris. The functional genes in Example 1 are div...

Embodiment 3

[0071] Example 3 Effect of Modular Transformation on Intracellular Active Oxygen Levels of Pichia pastoris

[0072] The changes of intracellular ROS and cell growth of the recombinant strain S17 were investigated by flow cytometry. Since the use of methanol will also have an impact on the accumulation of intracellular ROS, the wild-type GS115 strain was also compared as a control strain. Such as Figure 4 As shown, after transformation, the cell survival rate of the S17 strain was significantly improved, and the cell survival rate reached 77.4% after 144 hours of fermentation, which was 16.6% higher than that of the original strain PP-GOD. Through the detection of intracellular ROS levels, it was found that due to the induction by methanol, the bacteria will also produce ROS while using methanol to increase the level of ROS in the cells, such as Figure 4 As shown in B, the number of cells with high ROS levels in the control strain GS115 increased as the induction time prolo...

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Abstract

The invention discloses a method for enhancing the glucose oxidase secretion by transforming folding and secreting pathway of a protein, and belongs to the technical field of gene engineering. KAR2, CNE1, ERO1, SSA4, SSO2, SEC53, BMH2, HRD1, UBC1, GCN4 and other genes in Pichia pastoris are respectively cloned and linked into pGAPZ series derived plasmids of a Pichia pastoris expression vector bya gene recombination technology, the obtained plasmids and GOD was co-expressed and transferred into Pichia pastoris GS115 strains, the effect of the strain is investigated, modular combination optimization is carried out, and the strains are screened and identified to obtain a strain having a higher glucose oxidase secretion ability than the original strains. The enzyme activity of the glucose oxidase expressed by the strain in a 3 L fermentation tank is 1972.9 U / mL. The method lays a good foundation for the large-scale production of the glucose oxidase.

Description

technical field [0001] The invention relates to a method for enhancing the secretion of glucose oxidase by transforming the protein folding secretion pathway, which belongs to the technical field of genetic engineering. Background technique [0002] Glucose oxidase is one of the most important tool enzymes in the biological field. Since Updike and Hicks fixed GOD on the surface of Clark oxygen electrode and applied it to blood glucose measurement in 1967, GOD has been widely used in food, feed, medicine and many other related fields. [0003] In the food industry, the presence of oxygen causes many chemical reactions that are not conducive to product quality and creates conditions for the growth of many microorganisms. At present, many countries have widely used GOD as a safe antioxidant for workers in various foods and food processing techniques. Although it has various uses, the functions of GOD mainly lie in four aspects: removing glucose, removing oxygen, forming hydro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N9/04C12R1/84
CPCC07K14/39C12N9/0006C12Y101/03004
Inventor 张娟陈坚顾磊堵国成
Owner JIANGNAN UNIV
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