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Genetically engineered bacterium for producing glucose oxidase as well as construction and application thereof

A technology of glucose oxidase and genetically engineered bacteria, which is applied in the direction of oxidoreductase, fungi, and microbial-based methods, can solve the problems of high cost, complicated separation and extraction, and achieve the effect of reducing production costs

Active Publication Date: 2013-03-27
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 proteins are produced, and the separation and extraction are complicated and costly

Method used

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  • Genetically engineered bacterium for producing glucose oxidase as well as construction and application thereof
  • Genetically engineered bacterium for producing glucose oxidase as well as construction and application thereof
  • Genetically engineered bacterium for producing glucose oxidase as well as construction and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Construction and Identification of Embodiment 1 Recombinant Bacteria

[0021] According to the gene of Aspergillus niger CCTCC NO: M2011291 screened and preserved in our laboratory, primers were designed to obtain the GOD gene, and the purified GOD gene and vector pPIC9K were digested with restriction endonucleases SnaB I and Not I, and T4DNA The ligase was ligated overnight at 16°C, and the ligated product was transformed into the host strain JM109 by chemical transformation method, and the transformed bacteria liquid was spread on the LB plate containing kanamycin (30 mg / mL), cultured overnight at 37°C, and GOD-F and GOD-R was used as a primer to carry out colony PCR to identify positive clones, and finally the recombinant expression plasmid pPIC9K-GOD containing GOD gene was obtained, which was verified by double enzyme digestion.

[0022] The recombinant plasmid pPIC9K-GOD was electroporated to transform Pichia pastoris GS115 competent cells to obtain genetically en...

Embodiment 2

[0024] Enzyme activity assay and protein electrophoresis of embodiment 2 recombinant bacteria

[0025] Use the yeast engineered bacteria obtained in Example 1 as the production strain, after activation, cultivate it to OD at 30°C and 200rpm 600 The seeds between 1.6-1.7 are transferred to the basic fermentation medium with 2% inoculum, and cultured at 30°C and 200rpm; when the OD value is 1.2-1.5, the yeast cells are transferred to BMMY medium to induce protein generation.

[0026] Medium: YPD medium (1L) for seed and slant medium: tryptone 20g, yeast extract 10g, glucose 20g; slant medium with agar 20g; basic fermentation medium is BMGY medium (1L): tryptone 20g, yeast extract 10g, glycerin 10mL, YNB 13.4g, 100mM phosphate buffer (pH 6.0); induction medium is BMMY medium (1L): tryptone 20g, yeast extract 10g, methanol 8mL, YNB 13.4 g, 100mM phosphate buffer (pH 6.0);

[0027] A protein band with a molecular weight of about 68kDa was obtained by protein electrophoresis (SDS...

Embodiment 3

[0028] The enzymatic property research of embodiment 3 recombinant protein

[0029] The enzymatic activity of the recombinant GOD glucose oxidase at different temperatures and the thermostability at a gradient temperature of 20-80°C were determined. The results show that the optimal temperature of recombinant GOD is 40°C, and it has good thermal stability at 20-60°C. The recombinant GOD is incubated at 60°C for 3h, and the enzyme activity can still reach 80%. After 3 hours of incubation, more than 98% of the enzyme activity remains, and after exceeding 80°C, the enzyme activity is seriously lost ( figure 2 ).

[0030] The pH mainly affects the activity of the enzyme by changing the spatial structure of the enzyme and affecting the dissociation of the active center group of the enzyme molecule. The activity of the recombinant GOD glucose oxidase was tested at its optimum temperature and in a buffer system with pH 2.0-10.0 to determine its optimum pH. The results showed that...

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Abstract

The invention discloses a genetically engineered bacterium for producing glucose oxidase as well as a construction method and an application thereof, and belongs to the technical field of genetic engineering. A recombinant DNA (Deoxyribonucleic Acid) technology is used for cloning and connecting an Aspergillus niger glucose oxidase (GOD) gene to a Pichia pastoris expression carrier pPIC9K and converting Pichia pastoris GS115; screening and identifying to obtain recombinant Pichia pastoris GS115-pPIC9K-GOD which can produce the glucose oxidase with the higher activity and has the preservation number of CCTCC (China Center For Type Culture Collection) NO: M2012266. The glucose oxidase expressed by the bacterial strain has the enzyme activity of 52 U / mL in a shaking bottle and the enzyme activity is improved by about 24 times as compared with the enzyme activity of wild funguses, so as to lay a good foundation for the large-scale production of the glucose oxidase.

Description

technical field [0001] The invention relates to a genetic engineering bacterium producing glucose oxidase and its construction and application, in particular to an engineering bacterium induced to express glucose oxidase gene and its construction method and application. Background technique [0002] Glucose oxidase is one of the most important tool enzymes in the biological field. Since Updike and Hicks immobilized 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 ...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N9/04C12R1/84C12R1/685
Inventor 陈坚顾磊张娟堵国成沈依娜李梦洁李婷王丹丁雪殷政
Owner JIANGNAN UNIV
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