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Method for producing coenzyme Q10 by fermenting genetic recombinant bacteria

A technology of gene recombination and bacterial fermentation, applied in the field of production of coenzyme Q10, can solve the problems of difficult large-scale industrial production and low yield of strains, and achieve the effects of increased yield, low cost of raw materials and stable performance

Active Publication Date: 2014-05-28
HUNAN KEYUAN BIO PRODS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] However, the yields of the existing strains applied to the production of coenzyme Q10 are all low, and it is difficult to realize large-scale industrial production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] This embodiment includes the following steps:

[0027] 1. Amplification of the target gene

[0028] Include the following steps:

[0029] (1) According to the NAD kinase gene published by GenBank ppn K sequence, primers are designed as follows:

[0030] ppn K F Xba I: 5'-GC TCTAGA ATGACTGCACCCACGAACG-3'

[0031] ppn K R Sal I: 5'-GC GTC GAC TTACCCCGCTGACCTGG-3'

[0032] Restriction enzymes are underlined Xba I and Sal I restriction site. Primers were synthesized by a gene company in Shanghai.

[0033] (2) Rhodobacter sphaeroides Extraction of genomic DNA. PCR amplified ppn K fragments were recovered by nucleic acid electrophoresis.

[0034] (3) Link the target fragment recovered from the gel with pMD19-T Simple Vector, and the composition of the linking system is as follows:

[0035] Reagent volume pMD19-T Simple Vector 0.2 μL (about 50 ng) PCR product after adding A 4.8 μL (about 300 ng) 10 × Ligation buffer ...

Embodiment 2

[0041] This embodiment includes the following steps:

[0042] (1) Culture of recombinant bacteria

[0043] The obtained 15 strains of recombinant bacteria were inoculated in the seed medium, each in three parallels, and cultured on a shaker at 32°C and 180 rpm for 84 hours.

[0044] (2) Product extraction

[0045] Use 6 M hydrochloric acid to adjust the pH of the fermentation broth to 3.5, then add 6 M sodium hydroxide to adjust the pH to 7.0; collect the bacterial liquid and centrifuge at 8000 rpm for 10 min; Reflux extraction for 2 hours, the extract was centrifuged at 8000 rpm for 10 min, the supernatant was concentrated to dryness, dissolved in absolute ethanol, filtered and tested.

[0046] (3) HPLC detection

[0047] Sample detection method (refer to Chinese Pharmacopoeia coenzyme Q10 detection method)

[0048] Chromatographic column: BDS C18 5μm 250mm×4.6mm

[0049] Mobile phase: methanol: absolute ethanol = 50:50

[0050]Detection wavelength: 275 nm

[0051] Flo...

Embodiment 3

[0056] This embodiment includes the following steps:

[0057] Through orthogonal experiments, the optimal fermentation medium for No. 3 recombinant strain was determined (g / L): glucose 40, corn steep liquor 20, yeast powder 15, sodium nitrate 5, potassium dihydrogen phosphate 1.5, magnesium sulfate heptahydrate 0.5 , manganese sulfate monohydrate 0.01, ferrous sulfate heptahydrate 0.01, biotin 1×10 -5 , pH7.0.

[0058] The starting bacteria and the recombinant bacteria were inoculated in the seed medium respectively, and after 24 hours of shaking culture at 32°C, they were transferred to the optimized fermentation medium. Each group was set up in three parallels, and the inoculation amount was 2wt% of the medium, 32 Cultivate with shaking for 84 hours.

[0059] Carry out extraction and HPLC detection by the method of embodiment 2, the result shows, the growth rate of recombinant bacterial strain and final bacterial body weight are all higher than starting bacterial strain, a...

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Abstract

The invention discloses a method for producing coenzyme Q10 by fermenting genetic recombinant bacteria. According to the method, an NAD (Nicotinamide Adenine Dinucleotide) kinase gene is transplanted into a strain of efficient expression reducing coenzyme Q10. The culture conditions of recombinant bacteria optimization are inclined activation, seed culture and fermental cultivation. According to the method disclosed by the invention, the recombinant bacteria constructed by genetic engineering means are strong in stability; the yield of the coenzyme Q10 of the recombinant bacteria is 1.63g / L and increased by 21.6 percent compared with an original strain; the coenzyme Q10 can meet the requirement on industrial production.

Description

technical field [0001] The invention relates to a method for producing coenzyme Q10, in particular to a method for producing coenzyme Q10 by fermenting gene recombinant bacteria. Background technique [0002] Coenzyme Q is a naturally occurring fat-soluble quinone compound, which mainly exists in the plasma membrane of prokaryotic cells and the inner mitochondrial membrane of eukaryotic cells. Structurally, it is composed of a benzene ring and isoprenoid side chains of different lengths. Due to the different encodings of polyisoprene pyrophosphate synthase in different organisms, the types of coenzyme Q produced by different organisms are different. Coenzyme Q10 is an electron transporter in the respiratory chain, has anti-oxidation function, and is widely used in the comprehensive treatment of cardiovascular diseases, hepatitis, cancer, AIDS, etc. and to improve human immunity. [0003] At present, there are three main production methods of coenzyme Q10: animal and plant ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/66C12N15/74C12R1/01
Inventor 陶帅刘志强
Owner HUNAN KEYUAN BIO PRODS
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