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Method for performing secretory expression of glucose oxidase based on optimization of metabolic engineering, recombinant bacterium and application thereof

A technology of glucose oxidase and glucose, applied in the direction of microorganism-based methods, genetic engineering, oxidoreductase, etc., can solve the problems of unsatisfactory yield and enzyme activity, GOD inactivity, difficulty in separating and purifying intracellular enzymes, etc.

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

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Problems solved by technology

[0010] 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 performing secretory expression of glucose oxidase based on optimization of metabolic engineering, recombinant bacterium and application thereof
  • Method for performing secretory expression of glucose oxidase based on optimization of metabolic engineering, recombinant bacterium and application thereof
  • Method for performing secretory expression of glucose oxidase based on optimization of metabolic engineering, recombinant bacterium and application thereof

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Experimental program
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Embodiment 1

[0120] Embodiment 1, the construction of recombinant bacterial strain

[0121] 1. Construction of recombinant bacteria G / GODM

[0122] In order to realize the secreted expression of glucose oxidase in Pichia pastoris, the inventors selected the pPIC9K vector. Using pUC57-GOD as a template, by PCR, introduce EcoR 1 at the 5' end of the GOD sequence, introduce Not 1 at the 3' end, digest with enzymes, and connect with pPIC9K cut with the same enzyme to construct the GOD secretion expression vector pPIC9K-GOD, construction flow chart Such as 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 EcoR Ⅰ and Not 1 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 E. coli DH...

Embodiment 3

[0130] Embodiment 3, the performance investigation of recombinant bacteria

[0131] Pick the verified transformants G / GMS3 and G / GMM1, and inoculate them into a test tube containing 3mL of YPG medium. After culturing for about 18 hours, transfer them to a 250mL shake flask containing 25mL of BMGY medium, and enrich the culture until 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 zwf1, sol3, gdh3, mdh1 on the growth of recombinant bacteria, changes in enzyme activity, etc.

[0132] 1. Cell growth

[0133] Depend on Figure 9 It can be seen that the growth trend of the recombinant bacteria G / GMZ1, G / GMS3, G / GMG3, and G / GMM1 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, indicating tha...

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Abstract

The invention relates to a method for performing secretory expression of glucose oxidase (GOD) based on the optimization of metabolic engineering, a recombinant bacterium and application thereof. The invention provides a method which can efficiently secretorily express GOD and improve the enzyme activity thereof. The GOD and malic acid dehydrogenase 1 coding gene (mdh1) or 6-phosphogluconolactonase coding gene (sol3) is co-expressed in a yeast strain, so that the efficient expression of the GOD is realized. Meanwhile, the invention also provides a recombinant bacterium for efficiently secreting the GOD built by adopting 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 optimizing secretion and expression of glucose oxidase based on metabolic engineering, 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. The blood glucose meter made of glucose oxidase can conveniently detect the fluctuation level of blo...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/55C12N15/81C12N1/19C12R1/84
CPCC12N9/0006C12N9/18C12N15/815C12Y101/01037C12Y101/03004C12Y301/01031
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
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