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Glucose oxidase gene Glox, protein, pichia pastoris and preparation and application of pichia pastoris

A technology of glucose oxidase and gene, which is applied in the field of preparation and construction of engineering bacteria, can solve problems such as difficult purification, and achieve the effects of improving economic benefits, single bands, and reducing costs

Pending Publication Date: 2018-05-08
河北省微生物研究所有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The problem that the above-mentioned prior art exists is that the large-scale production of GOD adopts Aspergillus niger or Penicillium fermentation widely, but in the process of producing GOD by Aspergillus niger and Penicillium fermentation, the production of a large amount of miscellaneous proteins such as catalase, cellulase and amylase presents considerable difficulties in purification
[0009] 2. The strain used in the patent literature with application number CN106591249A is Aspergillus niger, and the fermentation process of Aspergillus niger has been optimized, but in the process of producing glucose oxidase by mold fermentation, a large amount of catalase, cellulase and amylase, etc. The existence of hybrid enzymes brings considerable difficulties to purification

Method used

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  • Glucose oxidase gene Glox, protein, pichia pastoris and preparation and application of pichia pastoris
  • Glucose oxidase gene Glox, protein, pichia pastoris and preparation and application of pichia pastoris
  • Glucose oxidase gene Glox, protein, pichia pastoris and preparation and application of pichia pastoris

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Construction and Identification of Embodiment 1 Recombinant Bacteria

[0050] Select the Penicillium expansum CICC 40658 previously preserved in the laboratory to obtain a strain with stable and high enzyme activity, and use the gene of this strain as a template to design primers to obtain the gene Glox. The purified PCR product was connected with the shuttle vector pMD-AOX by the Gibson method. After the connection was completed, the E. coli DH5α competent cells were transformed, and the white positive clones on the transformation plate were picked to obtain the recombinant expression plasmid pMD-AOX containing the Glox gene. -Glox. Validation by double enzyme digestion.

[0051] The recombinant plasmid pMD-AOX-Glox was electroporated to transform Pichia pastoris X33 competent cells to obtain genetically engineered bacteria Pichia pastoris X33-Glox.

[0052] The transformation of Pichia pastoris adopts the electroporation method: take X33 and culture overnight, pick ...

Embodiment 2

[0053] Enzyme activity assay and protein electrophoresis of embodiment 2 recombinant bacteria

[0054] The Pichia pastoris CGMCC No.13358 obtained in Example 1 was used as the production strain, and after activation, it was cultivated to OD at 30°C and 200rpm. 600 ≈1.2 seed culture solution was transferred to YPCS medium with 2% inoculum, and cultivated at 30°C and 200rpm; cultivated in YPCS medium to OD 600 When ≈1.2, the yeast cells were transferred to YPCS induction medium, and 1% methanol was added every day to continuously induce expression for 72 hours.

[0055] culture medium

[0056] The seed and slant medium are YPD medium: 2% tryptone, 1% yeast extract, 2% glucose; 2% agar is added to the slant medium.

[0057] YPCS medium: tryptone 2%, yeast extract 1%, casein hydrolyzate 2%, sorbitol 0.5%.

[0058] A strip was obtained by protein electrophoresis (SDS-PAGE) Figure 4 A protein band with a molecular weight of approximately 80 kDa is shown.

[0059] Glucose oxida...

Embodiment 3

[0066] The verification of embodiment 3 recombinant bacterial strains enzyme activity on 10L fermenter

[0067] A. Inoculate Pichia pastoris CGMCC No.13358 in sterile YPD medium for 12-16 hours;

[0068] B. Transfer the bacteria cultured in step A to a sterile BMGY medium with a volume of 100mL at a volume ratio of 3% inoculum, and shake the flask to OD at a temperature of 30°C 600 ≈6-7;

[0069] C. Inoculate the cultured strains in step B in a fermenter filled with sterile BSM medium at a volume ratio of 10%, under the conditions of 30°C, 550 rpm, and pH=5 Cultivate to cell OD 600 ≈90;

[0070] D. Adding 50% glycerol, containing 12mL PTM1 per liter of glycerol, the flow rate is 12mL / h / L, and the temperature is 30°C, culture for 4h, OD 600 ≈150-170, stop feeding for 0.5-1h, and confirm that the glycerol is exhausted;

[0071] E. Add the inducer methanol according to the flow rate of 3.0mL-5.0mL / h / L, each liter of methanol contains PTM112mL, the pH is maintained at 6.0-6.5...

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Abstract

The invention belongs to preparation of new genes and establishment of engineering bacteria, and particularly relates to a glucose oxidase gene Glox and preparation, establishment and application of pichia pastoris. 5c / end ATGAAGCTCCTTGGCCTCC and 3c / end CTAATTAACAGTAGCGTTGTAATC serve as specific primers, penicillium expansum DNA serves as a template, the glucose oxidase Glox gene is obtained through a PCR amplification method, and the pichia pastoris engineering bacteria are successfully established. The problem that currently, a glucose oxidase gene is recombined for heterologous expressionis solved, the full-length gene Glox and a shuttle expression carrier established by the Glox gene are obtained, the gene Glox is further converted into a pichia pastoris X33 strain, and a strain better secretorily expressing glucose oxidase than an original strain is obtained through screening and identification. The method is simple and easy to carry out, the cost is low, and a good foundationis laid for large-scale production of glucose oxidase.

Description

technical field [0001] The invention belongs to the preparation of new genes and the construction of engineering bacteria, in particular to a glucose oxidase gene Glox, protein, Pichia pastoris and its preparation and application. Background technique [0002] Glucose oxidase is an aerobic dehydrogenase that can specifically catalyze β-D-glucose to generate gluconic acid and hydrogen peroxide. The catalytic properties of glucose oxidase enable it to remove glucose, deoxidize, sterilize and other functions, and it is safe, non-toxic and without side effects. [0003] Glucose oxidase is widely distributed in animals, plants and microorganisms. Due to the strong ability of mold to produce enzymes, the currently used research and industrial strains mainly include Aspergillus niger and Penicillium strains. The presence of the purification brings considerable difficulties. Penicillium extensa belongs to a kind of Penicillium, which also has such problems. In recent years, the ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/04C12N1/19C12N15/81C12R1/84
CPCC12N9/0006C12N15/815C12Y101/03004
Inventor 高庆华胡美荣董聪王玥王庆庆陶勇罗同阳王云鹏马清河
Owner 河北省微生物研究所有限公司
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