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Method for obviously improving soluble expression quantity of linoleate isomerase in recombinant escherichia coli

A technology of recombinant Escherichia coli and linoleic acid isomerase, applied in the field of genetic engineering, can solve problems such as high separation cost and chemical pollution

Inactive Publication Date: 2018-05-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, conjugated linoleic acid is mainly synthesized by chemical methods, which not only causes serious chemical pollution, but also synthesizes a mixture of conjugated linoleic acid with various structures, and the cost of downstream separation is very high

Method used

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  • Method for obviously improving soluble expression quantity of linoleate isomerase in recombinant escherichia coli
  • Method for obviously improving soluble expression quantity of linoleate isomerase in recombinant escherichia coli
  • Method for obviously improving soluble expression quantity of linoleate isomerase in recombinant escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0032] Pick a single colony from a fresh plate containing E.coli BL21(DE3)(pET-22b(+) / pai), and culture it in ampicillin LB medium with a final concentration of 100μg / ml at 37°C and 200rpm OD 600 = 0.4-0.6, take it out from the shaker, and centrifuge in ice bath for 30 minutes. With 0.1mol / L CaCl 2 Wash the cells 3 times. Add pG-KJE8, pGro7, pTf16, pKJE7 and pG-Tf2 plasmids to competent cells, heat shock in 42°C water bath for 90s after 30 minutes in ice bath, add 1ml of LB medium, incubate at 37°C for 1 hour, and spread the corresponding resistance plate, and after it grows out, pick the correct transformants to obtain recombinant E.coli BL21(DE3)(pET-22b(+) / pai)(pG-KJE8), E.coli BL21(DE3 )(pET-22b(+) / pai)(pGro7), E.coli BL21(DE3)(pET-22b(+) / pai)(pTf16), E.coli BL21(DE3)(pET-22b(+) / pai)(pKJE7), E. coli BL21(DE3)(pET-22b(+) / pai)(pG-Tf2).

example 2

[0034] Pick single colonies of different recombinant bacteria from the plate in LB medium containing corresponding antibiotics, cultivate overnight as seed liquid, inoculate 2% inoculum in 50mL fermentation medium, and add a final concentration of 1.5mg / ml arabinose or 10ng / ml tetracycline induced the expression of molecular chaperones. To be cultured to OD 600 When = 1.5, add IPTG to a final concentration of 0.1 mmol, lower the temperature to 20° C. and culture at 200 rpm for 20 h, collect bacterial cells and ultrasonically break the wall to detect enzyme activity.

example 3

[0036] Pick a single colony of recombinant bacteria E.coli BL21(DE3)(pET-22b(+) / pai)(pGro7) from the plate in LB medium containing ampicillin and chloramphenicol, and use it as a seed solution after overnight culture. Inoculate 2% of the inoculum into 50 mL of fermentation medium, and at the same time add L-arabinose at a final concentration of 0, 1.5 mg / ml, 3.0 mg / ml, and 4.0 mg / ml to induce the expression of the chaperone protein GroEL-GroES. To be cultured to OD 600 When =1.5, add IPTG to a final concentration of 0.1 mmol, lower the temperature to 20° C., and culture at 200 rpm for 20 h, and collect bacterial cells to detect enzyme activity by ultrasonically breaking the wall.

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Abstract

The invention discloses a method for obviously improving soluble expression quantity of linoleate isomerase in recombinant escherichia coli, and belongs to the field of genetic engineering. Accordingto the method, recombinant escherichia coli E.coli BL21(DE3)(pET-22b(+) / pai) is used as a starting strain; through co-expression of molecular chaperone proteins and assistance of the linoleate isomerase folding, soluble expression level of the linoleate isomerase is improved, so that enzyme activity of the linoleate isomerase is improved. Finally, a molecular chaperone system GroEL-GroES is preferably obtained, so that the enzyme activity of the linoleate isomerase is improved by 56 percent.

Description

technical field [0001] The invention relates to a method for significantly increasing the soluble expression level of linoleic acid isomerase in recombinant Escherichia coli, belonging to the field of genetic engineering. technical background [0002] As a newly discovered nutrient, conjugated linoleic acid has almost become a panacea for preventing diseases of modern civilization in the health food industry in Europe and America. From anti-cancer to prevention of cardiovascular disease, diabetes, and weight control, It is almost an indispensable healthy food for modern people living in the 21st century. At present, conjugated linoleic acid is mainly synthesized by chemical methods, which not only causes serious chemical pollution, but also synthesizes a mixture of conjugated linoleic acid with various structures, and the cost of downstream separation is very high. In recent years, the focus of research has gradually shifted to the use of biological methods to synthesize sp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12N15/70
CPCC12N9/90C12N15/70C12Y502/01005
Inventor 诸葛斌黄孟楠陆信曜宗红
Owner JIANGNAN UNIV
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