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Method for screening bacterial strains with ability of converting massively existing ginsenoside into rare ginsenoside

A technology of ginsenosides and bacterial strains, applied in the field of bacterial strains of ability

Inactive Publication Date: 2014-12-24
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The conversion efficiency of the existing biotransformation method for producing rare ginsenosides needs to be further improved to meet the needs of industrial production

Method used

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  • Method for screening bacterial strains with ability of converting massively existing ginsenoside into rare ginsenoside
  • Method for screening bacterial strains with ability of converting massively existing ginsenoside into rare ginsenoside
  • Method for screening bacterial strains with ability of converting massively existing ginsenoside into rare ginsenoside

Examples

Experimental program
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Effect test

Embodiment 1

[0050] Example 1. Method for screening strains having the ability to convert abundant ginsenosides into rare ginsenosides by constructing mutant libraries of strains containing existing proteins.

[0051] 1. Construction of mutant library

[0052] 1. Extract the genomic DNA of Sulfolobus sulfolobus DSM1616.

[0053] 2. Using the genomic DNA extracted in step 1 as a template, perform PCR amplification with a primer pair composed of F1 and R1, and recover the PCR amplification product (about 1470bp).

[0054] F1: 5'-atg gctagc atgtactcatttccaaatagc-3';

[0055] R1: 5'-gtg ctcgag ttagtgccttaatggctttac-3'.

[0056] The reaction procedure of PCR amplification: pre-denaturation at 95°C for 4min; 30 cycles of 95°C for 45s, 55°C for 30s, 72°C for 1min30s; extension at 72°C for 10min.

[0057] 3. The PCR amplification product of step 2 was double-digested with restriction endonucleases NheI and XhoI, and the enzyme-digested product was recovered.

[0058] 4. The pET-28a(+) vect...

Embodiment 2

[0101] Example 2. A method for screening mutant proteins with the ability to convert abundant ginsenosides into rare ginsenosides by constructing mutant libraries of existing protein-encoding genes.

[0102] 1. Take the crude enzyme liquid obtained from 5 strains of step 2 of Example 1 and 5 wild-type control strains obtained from step 3 of step 2 of Example 1, and conduct affinity chromatography.

[0103] Affinity chromatography adopts nickel ion affinity chromatography column (produced by Novagen Company, product number is 70666-3, column volume is 1 ml). For the specific elution process, first wash with 3ml of washing buffer (containing 20mM imidazole, 300mM NaCl, pH8, 50mM Tris-HCl buffer) to remove impurities, and then use 2ml of elution buffer (containing 250mM imidazole, 300mM NaCl, pH8, pH8). , 50mM Tris-HCl buffer), washed and collected the post-column solution, dialyzed (the dialysate was MC buffer at pH 5.5, consisting of a final concentration of 200 mM disodium hyd...

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Abstract

The invention discloses a method for screening bacterial strains with the ability of converting massively existing ginsenoside into rare ginsenoside. The method comprises the following steps: (1) inserting a gene A into an expression plasmid and transforming into a host bacterium so as to obtain a wild bacterium; carrying out error PCR by using the gene A as a template, inserting the product of error PCR into the expression plasmid and transforming into the host bacterium to obtain single colonies, wherein all the single colonies form a mutant bacterial strain library and the gene A is a known encoding gene of a protein with the ability of converting massively existing ginsenoside into rare ginsenoside; (2) respectively culturing the wild bacterium and bacteria in the mutant bacterial strain library and collecting fermentation products; (3) acting the fermentation products on the massively existing ginsenoside to obtain a reaction liquid; (4) screening by a DNS method; and (5) carrying out (a) thin layer chromatography and / or (b) HPLC. The method disclosed by the invention has the advantages of efficiency, rapidness and high throughput and the like.

Description

technical field [0001] The present invention relates to a method for screening strains having the ability to convert abundant ginsenosides into rare ginsenosides. Background technique [0002] Ginseng is a traditional Chinese medicinal material. According to modern pharmacological research reports, ginseng has the functions of enhancing human immunity, improving nutrition, anti-aging and reducing fatigue. Studies have found that some rare ginsenosides (such as ginsenoside Rg 3 , Ginsenoside Rh 2 , Ginsenoside Compound K, etc.) has high biological activity, is easier to be absorbed by the human body, has medical effects such as promoting the decline of malignant tumor cells, curbing the spread or deterioration of tumors, and has high production value and application prospects. [0003] Rare ginsenosides are low or absent in natural ginseng extracts. The methods for preparing rare ginsenosides in vitro usually include chemical methods and biotransformation methods. 1 , Gi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12N9/42C12N15/56C12P33/20C12R1/19
Inventor 梁朝宁熊丹丹唐双焱
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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