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Production and purification method of glucose dehydrogenase by taking FAD (Flavin Adenine Dinucleotide) as prothetic group

A glucose dehydrogenase, FAD-GDH technology, applied in the field of purification and glucose dehydrogenase production, can solve the problems of protein purification and scale-up production difficulties, and achieve good thermal stability

Inactive Publication Date: 2018-09-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The heterologous expression of glucose dehydrogenase from fungi in Escherichia coli often requires co-expression with molecular chaperones to achieve soluble expression, and protein purification and scale-up production are difficult

Method used

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  • Production and purification method of glucose dehydrogenase by taking FAD (Flavin Adenine Dinucleotide) as prothetic group
  • Production and purification method of glucose dehydrogenase by taking FAD (Flavin Adenine Dinucleotide) as prothetic group
  • Production and purification method of glucose dehydrogenase by taking FAD (Flavin Adenine Dinucleotide) as prothetic group

Examples

Experimental program
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Effect test

Embodiment 1

[0056] The construction of embodiment 1 genetically engineered bacteria

[0057] (1) The obtained FAD-GDH gene SEQ ID NO: 1 is connected with the plasmid pTrc99A to obtain the expression vector pTrc99A-GDH;

[0058] (2) Transforming the recombinant vector pTrc99A-GDH into Escherichia coli BL21(DE3) to obtain the Escherichia coli recombinant bacteria.

Embodiment 2

[0059] Example 2 Effect of 7.5L fermenter with constant flow and feeding medium on the production of glucose dehydrogenase by fermentation

[0060] The method for producing glucose dehydrogenase by recombinant Escherichia coli fermentation tank fermentation is carried out in a 7.5L tank, and the process is divided into three stages:

[0061] The first stage: Inoculate single colonies of recombinant E. coli in Example 3 into a 150mL Erlenmeyer flask equipped with 30mL LB medium, 200rpm, 37°C shaking culture for 8h, as a primary seed fermentation liquid;

[0062] The second stage: take 15 mL of the primary seed fermentation liquid and inoculate it in a 500 mL Erlenmeyer flask equipped with 300 mL of fermentation medium, 200 rpm, shake at 37°C for 8 hours, and use it as the secondary seed fermentation liquid;

[0063] The third stage: put the activated seed liquid into the fermenter in batches by 10% by volume, and when the DO value rises rapidly, the dissolved oxygen rises rapid...

Embodiment 3

[0065] Example 3 Effect of 7.5L fermenter with constant flow and feeding medium on the production of glucose dehydrogenase by fermentation

[0066] The method for producing glucose dehydrogenase by recombinant Escherichia coli fermentation tank fermentation is carried out in a 7.5L tank, and the process is divided into three stages:

[0067] The first stage: Inoculate single colonies of recombinant E. coli in Example 3 into a 150mL Erlenmeyer flask equipped with 30mL LB medium, 200rpm, 37°C shaking culture for 8h, as a primary seed fermentation liquid;

[0068] The second stage: take 15 mL of the primary seed fermentation liquid and inoculate it in a 500 mL Erlenmeyer flask equipped with 300 mL of fermentation medium, 200 rpm, shake at 37°C for 8 hours, and use it as the secondary seed fermentation liquid;

[0069] The third stage: put the activated seed liquid into the fermenter in batches according to the volume fraction of 10%. When the DO value rises rapidly, the dissolved...

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Abstract

The invention discloses a production and purification method of glucose dehydrogenase by taking FAD (Flavin Adenine Dinucleotide) as a prothetic group, and belongs to the field of bioengineering. Theproduction and purification method is characterized by expressing the glucose dehydrogenase sourced from burkholderia cepacia by taking Escherichia coli as a host, carrying out batch feeding, controlling the temperature in stages, and expressing the glucose dehydrogenase. According to the production and purification method disclosed by the invention, the activity of the glucose dehydrogenase is upto 22200.0U / L, and the mycelia content is up to 69.8g / L; a crude enzyme solution is separated and purified through three-step chromatography, so that recombinase of which the specific enzyme activityis 10<9>U / mg, the property problem of enzyme is solved, and the recombinase is suitable for industrial production.

Description

technical field [0001] The invention relates to a method for producing and purifying glucose dehydrogenase with FAD as a prosthetic group, belonging to the field of bioengineering. Background technique [0002] Blood glucose level is the main and routine detection index for clinical diagnosis of diabetes. Glucose oxidase is the most widely used raw material enzyme in glucose detection kits or glucose sensors, but its catalytic activity is easily limited by dissolved oxygen, which will cause measurement errors. At present, it is found that glucose dehydrogenase has the potential to replace glucose oxidase, because it does not use oxygen as an electron acceptor and is not limited by dissolved oxygen, so the error of the detection result is smaller. [0003] Glucose dehydrogenase can be divided into four categories according to the type of prosthetic group or coenzyme: (1) NADP + Glucose-6-phosphate dehydrogenase (EC1.1.1.49 referred to as G6PDH) as a coenzyme (2) with NAD +...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N9/04C12R1/19
CPCC12N9/0006C12N15/70C12Y101/9901
Inventor 张玲杨海麟林荣
Owner JIANGNAN UNIV
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