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Method for preparing rare ginsenoside CK through gene combination transformation and application

A ginsenoside and gene combination technology, applied in the field of biomedicine, can solve the problems of low activity of a single enzyme, complicated operation, etc., and achieve the effects of low cost, good selectivity and short conversion period

Pending Publication Date: 2021-03-12
HUNAN INSTITUTE OF ENGINEERING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The present invention aims to overcome the technical defects and deficiencies of existing natural enzymes such as difficulty in obtaining, low single enzyme activity, high selectivity to glycoside compounds such as ginsenoside, and complicated operation, and provides a codon-optimized method for co-expression in Escherichia coli A method for preparing rare ginsenoside CK by transforming ginsenoside Rc with Bacillus subtilis α-L-arabinofuranosidase and Bifidobacterium β-glucosidase, which is simple, efficient and environmentally friendly

Method used

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  • Method for preparing rare ginsenoside CK through gene combination transformation and application
  • Method for preparing rare ginsenoside CK through gene combination transformation and application
  • Method for preparing rare ginsenoside CK through gene combination transformation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] 1. Cultivate recombinant bacteria with Luria-Bertani (LB) liquid medium; the preparation method of LB medium is as follows: take 10g of tryptone, 5g of yeast extract, and 10g of NaCl, add an appropriate amount of water to dissolve, and after the solute is dissolved, use 5mol / L sodium hydroxide (NaOH) to adjust the pH to 7.0, then dilute to 1L with deionized water, and steam sterilize under high pressure at 121°C for 20min.

[0031] 2. The specific method for fermenting and cultivating the recombinant bacteria is as follows: inoculate the recombinant bacteria on solid LB medium containing 50 μg / mL kanamycin, cultivate overnight (8-10 hours) at 37° C., and then pick Inoculate a single colony in LB liquid medium containing 50 μg / mL kanamycin, culture at 37°C until the absorbance OD600 is 0.4-0.6, add 0.1-1mmol / L IPTG for induction for 2-10 hours, and obtain recombinant bacteria fermentation broth.

[0032] 3. Centrifuge the recombinant bacterial fermentation broth at 4°C...

Embodiment 2

[0038] Prepare the BsAbfA (SEQ ID No.1) and BbBgl2 (SEQ ID No.3) genes (codons are not optimized) and add Internal ribosomal-binding site (IRBS) between the two genes by the culture method in Example 1. ) sequence (SEQ ID NO.5) of the recombinant BL21 bacterial liquid, prepared into a bacterial liquid with a cell concentration of 40g / L; dissolving ginsenoside Rc in methanol, and mixing it with acetic acid-sodium acetate buffer solution of pH 4.0-6.0 , to make a concentration of 40g / L ginsenoside Rc substrate solution, and then mix the ginsenoside Rc solution and the recombinant bacterial solution according to the volume ratio of 1:2, the pH is 5.0, react at 40°C for 24 hours, and then place in 80 Inactivate in a water bath at ℃ for 20 minutes, centrifuge at 10,000 rpm for 10 minutes at 25 ℃, and dry the supernatant at 60 ℃ to obtain rare ginsenoside CK.

Embodiment 3

[0040]Prepare the recombinant BL21 bacterium liquid containing only BsAbfA (SEQ ID No.1) gene (codon not optimized) with the cultivation method in Example 1, prepare the bacterium liquid that cell concentration is 40g / L; Dissolve ginsenoside Rc in In methanol, mix with acetic acid-sodium acetate buffer solution at pH 4.0-6.0 to prepare a ginsenoside Rc substrate solution with a concentration of 40g / L, then mix the ginsenoside Rc solution with the recombinant bacterial solution at a volume ratio of 1:2 , pH was 5.0, reacted at 40°C for 24 hours, then inactivated in a water bath at 80°C for 20 minutes, centrifuged at 10,000 rpm for 10 minutes at 25°C, and dried the supernatant at 60°C to obtain rare ginsenoside CK.

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Abstract

The invention discloses a method for preparing rare ginsenoside CK through gene combination transformation and application of the rare ginsenoside CK. According to the invention, glycosidase generatedby using genes of bacillus subtilis str.168 and bifidobacterium breve 689b can be used, so that the ginsenoside Rc can be converted into the rare ginsenoside CK efficiently in vitro.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a method and application of gene combination transformation to prepare rare ginsenoside CK, in particular to a co-expression of Bacillus subtilis α-L-arabinofuranosidase and Bifidobacterium β- A method for preparing rare ginsenoside CK by transforming ginsenoside Rc with glucosidase. Background technique [0002] Ginsenosides are the most important active ingredients in ginseng. Ginsenosides belong to tetracyclic triterpenoids. According to the types and quantities of glycosidic bonds (Glycosidic bonds) at positions C-3, C-6 and C-20 on the aglycone Different, ginsenosides can be divided into dammarane-type and oleanolic acid-type saponins, wherein dammarane-type saponins are further divided into protopanaxadiol-type saponins (PPD-type saponins, mainly including Rb1, Rb2, Rc and Rd, etc. ) and protopanaxatriol saponins (PPT saponins, including Re and Rg1, etc.). According to ...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/70C12N15/62C12N1/21C12P33/20C12R1/19
CPCC12N9/2445C12N9/2402C12N15/70C12P33/20C12Y302/01055C12Y302/01021C12N2800/22C07K2319/00
Inventor 张儒谭时泉张变玲
Owner HUNAN INSTITUTE OF ENGINEERING
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