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Engineered yeasts producing glucose oxidase and construction method and use thereof

A technology of glucose oxidase and yeast engineering, applied in the direction of oxidoreductase, biochemical equipment and methods, applications, etc., can solve the problems of complicated separation and extraction, high cost, etc., and achieve the effect of reducing production cost and significant application value

Inactive Publication Date: 2013-02-13
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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  • Engineered yeasts producing glucose oxidase and construction method and use thereof
  • Engineered yeasts producing glucose oxidase and construction method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Construction and Identification of Recombinant Bacteria

[0020] According to the gene of Aspergillus niger CCTCC NO: M2011291 screened and preserved in our laboratory as a template, primers were designed to obtain the GOD gene. Restriction endonucleases Kpn I and Not I double-digest the purified GOD gene and vector pGAPZα, and use T4 DNA ligase to ligate overnight at 16°C. The ligated product is transformed into the host strain JM109 by chemical transformation, and the transformed strain is spread on On an LB plate containing kanamycin (30 mg / mL), culture overnight at 37°C, and finally obtain the recombinant expression plasmid pGAPZα-GOD containing the GOD gene, which was verified by double enzyme digestion.

[0021] The recombinant plasmid pGAPZα-GOD was electroporated to transform Pichia pastoris X33 competent cells to obtain genetically engineered bacteria, which were identified and named Pichia pastoris X33-pGAPZα-GOX.

[0022] The transformation of Pich...

Embodiment 2

[0023] Example 2 Enzyme Activity Determination and Protein Electrophoresis of Recombinant Bacteria

[0024] The yeast engineered bacteria constructed in Example 1 was used as the production strain. After activation, it was cultivated to OD at 30°C and 200rpm 600 The seeds between 1.6-1.7 were transferred to the basic fermentation medium with an inoculum of 2%, and fermented at 30°C and 200rpm.

[0025] Medium: YPDS medium (1L) for seeds and basic fermentation medium: 20g tryptone, 10g yeast extract, 20g glucose, 168g sorbitol; add 20g agar to the slant medium;

[0026] After the fermentation, a protein band with a molecular weight of about 68kDa was obtained by protein electrophoresis (SDS-PAGE). At the same time, the ability to produce glucose oxidase was verified in a shake flask. The highest enzyme activity was 23U / mL compared with the enzyme activity of wild bacteria (2.2U / mL) increased nearly 11 times.

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Abstract

The invention discloses engineered yeasts producing glucose oxidase and a construction method and use thereof, and belongs to the technical field of genetic engineering. The glucose oxidase(GOD) gene of aspergillus niger is cloned and bonded to a pichia pastoris expression vector pGAPZalpha by recombination DNA technology, the Pichia pastoris X33 is transformed, and finally, the recombinant pichia pastoris X33-pGAPZalpha-GOD producing high-activity glucose oxidase is separated and identified, and the collection number of the recombinant pichia pastoris is CCTCC NO.M2012267. The activity of the glucose oxidase expressed by the strain in shake flask is 23U / mL, which is nearly 11 times higher than that of wild fungi. The engineered yeasts provide a good basis for the mass production of glucose oxidase and the use of the glucose oxidase in starch as a food additive.

Description

technical field [0001] The invention relates to a genetic engineering bacterium producing glucose oxidase, in particular to a yeast engineering bacterium producing glucose oxidase, which belongs to the technical field of genetic engineering. Background technique [0002] Glucose oxidase (GOD) 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 aspects: removing gluco...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/53C12N15/81C12N9/04C12R1/84
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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