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Method for secretorily expressing glucose oxidase, and recombinant bacteria and applications thereof

A technology of glucose oxidase and glucose, applied in the field of promoting the secretion and expression of glucose oxidase, which can solve the problems of GOD inactivity, inability to post-translational processing of GOD, low yield of GOD enzyme, etc.

Pending Publication Date: 2017-12-15
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] At present, GOD is produced by microbial fermentation in this field, but the production of GOD by fermentation of Aspergillus niger or Penicillium has problems such as low enzyme yield and difficulty in separating and purifying intracellular enzymes.
However, the use of Escherichia coli to produce GOD cannot post-translationally process GOD, and the synthesized GOD is inactive.
Those skilled in the art also utilize Pichia pastoris to produce GOD, but the yield and enzyme activity are not ideal enough, and the production process of GOD needs to be further optimized

Method used

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  • Method for secretorily expressing glucose oxidase, and recombinant bacteria and applications thereof
  • Method for secretorily expressing glucose oxidase, and recombinant bacteria and applications thereof
  • Method for secretorily expressing glucose oxidase, and recombinant bacteria and applications thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0125] Embodiment 1, the construction of recombinant bacterial strain

[0126] 1. Construction of recombinant bacteria G / GODM

[0127] For the secretory expression of glucose oxidase in Pichia pastoris, the inventors selected the pPIC9K vector. Using pUC57-GOD as a template, EcoRI was introduced at the 5' end of the GOD sequence by PCR, and NotI was introduced at the 3' end. After enzyme digestion, it was ligated with pPIC9K that was also digested with the same enzyme. The construction flow chart is as follows figure 1 . More specifically, using the plasmid pUC57-GOD (the GOD nucleic acid sequence is connected to pUC57) as a template, using primers GODF and GODR, the target fragment GOD (1749bp, see figure 2 ), introduce EcoRI and NotI two enzyme cutting sites at the same time, use restriction endonucleases to linearize the amplified product and pPIC9K vector respectively, then perform ligation reaction, and finally, transform into Escherichia coli DH5α competent cells by h...

Embodiment 2

[0135] Embodiment 2, the performance investigation of recombinant bacteria

[0136] Pick and verify the correct transformants G / GMP1, G / GMP2, G / GMP3, G / GMB1, G / GMB2, G / GMZ1, G / GMG3, inoculate into a test tube containing 3mL of YPG medium, and culture for about 18h Afterwards, transfer to a 250mL shake flask containing 25mL BMGY medium, enrich culture to OD 600 4 to 6, collect the bacteria, inoculate into 500mL shake flask containing 50mL BMMY medium for culture, initial OD 600 =1, add 1% methanol solution every 24h, and take samples to measure OD at the same time 600 , to investigate the effects of co-expression of PDI1, PDI2, PDI3 and other genes on the growth of recombinant bacteria and changes in enzyme activity.

[0137] 1. Cell growth

[0138] Depend on Figure 9 It can be seen that the growth trend of the recombinant bacteria G / GMP1 and G / GMP3 is very close to that of the original bacteria G / GODM, and the average specific growth rate μ is 0.039h -1 Left and right, i...

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Abstract

The invention relates to a method for secretorily expressing glucose oxidase, and recombinant bacteria and applications thereof, and discloses a method capable of effectively promoting GOD expression and improving the enzyme activity. The method can be realized by co-expressing a GOD gene and a PDI1 gene or a PDI3 gene in saccharomycetes. Furthermore, the invention further provides efficiently secreted recombinant bacteria constructed by taking Pichia pastoris as a host.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and more specifically, the invention relates to a method for promoting the secretion and expression of glucose oxidase, recombinant bacteria and applications thereof. Background technique [0002] Glucose oxidase (Glucose oxidase, GOD) is widely used in food, chemical industry, medicine, biotechnology and other fields. [0003] Based on the specificity of the catalytic reaction of glucose oxidase, it can be used in biosensors. Glucose oxidase is the core component of the sensor used to detect the concentration of glucose in the blood. The GOD fixed on the sensor electrode can convert a small amount of glucose in the blood into easily measured hydrogen peroxide. The more hydrogen peroxide detected by the platinum electrode, the The electrical signal is stronger. [0004] The blood glucose meter made of glucose oxidase can conveniently detect the fluctuation level of blood sugar of diabeti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/81C12N1/19C12R1/84
CPCC12N9/0006C12N15/815C12Y101/03004
Inventor 钱江潮魏东升王泽建段广东吴凡储炬庄英萍张嗣良肖慈英黎亮
Owner EAST CHINA UNIV OF SCI & TECH