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Method for preparing icaritin through converting total flavones of epimedium by enzyme method

A technology of icariin and enzymatic conversion is applied in the field of catalyzing total flavonoids of Epimedium to prepare icariin by recombinant enzyme system, which can solve the problem that the large-scale production of icariin and epimedium cannot be guaranteed. The yield and efficiency of aglycone needs to be improved, and the ratio of β-glucosidase is uncontrollable, so as to achieve the effects of good solubility, strong biological activity and high temperature.

Active Publication Date: 2017-08-01
NANJING FORESTRY UNIV
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Problems solved by technology

In this method, the ratio of α-L-rhamnosidase and β-glucosidase is uncontrollable, the order of action is uncontrollable, and the reaction time is long (30 hours), and the efficiency is low (this patent only shows that icariin can be generated Yuan)
[0004] Based on the existing research results, it can be found that: (1) the current research process can only use icariin as the substrate to prepare icariin, and it is still unable to prepare the main components of the total flavonoids of Epimedium (icariin, Chaohuoding A, Chaohuoding B and Chaohuoding C) are simultaneously converted into icariin; (2) the yield and efficiency of preparing icariin remain to be improved
(3) In the prior art, in the preparation process, the epimedium extract must first be obtained, then the icariin is isolated, and then converted by enzymatic or microbial methods, which not only greatly increases the cost, but also does not fully and efficiently utilize Chao Huo Ding A, Chao Huo Ding B, Chao Huo Ding C
On the other hand, the problem of obtaining a large amount of icariin resources is very prominent, and it is impossible to guarantee the large-scale preparation of icariin

Method used

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  • Method for preparing icaritin through converting total flavones of epimedium by enzyme method
  • Method for preparing icaritin through converting total flavones of epimedium by enzyme method
  • Method for preparing icaritin through converting total flavones of epimedium by enzyme method

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Embodiment 1

[0024] Embodiment 1: the acquisition of α-rhamnosidase gene of the present invention and the construction of recombinant plasmid pET-TPERha

[0025] 1.1 Culture of Thermotoga petrophila DSM 13995

[0026] Thermotoga petrophila DSM 13995 was purchased from the DSMZ Culture Collection Center (www.dsmz.de) and the number was 13995. The medium formula was: 10g / L starch, 5g / L tryptone, 3g / L yeast extract, 5g / L meat Infusion solution, 10g / L 2-malineethanesulfonic acid, 10mg / L ferric sulfate heptahydrate, 1mg / L resazurin, adjust the pH to 7.2. Inoculate with a syringe according to 0.5% inoculation amount, culture statically at 85° C. for 24 hours, and collect cells.

[0027] 1.2 Genomic DNA extraction

[0028] (1) Statically culture Thermotoga petrophila DSM 13995 for about 24 hours, take 30 mL of bacterial liquid and centrifuge at 4,000 g for 10 min to collect cells.

[0029] (2) Resuspend the bacteria in 9.5 mL TE buffer, add 0.5 mL 10% sodium dodecyl sulfate (SDS) and 50 μL pro...

Embodiment 2

[0041] Embodiment 2: Preparation of thermostable α-rhamnosidase of the present invention

[0042] The recombinant plasmid pET-TPERha was transformed into Escherichia coli JM109 (DE3) host bacteria (purchased from Novagen), and placed on an LB plate containing kanamycin (50 μg / mL) (LB medium: tryptone 10 g / L, yeast extract 5g / L, NaCl 5g / L, agar 15g / L) after culturing overnight at 37°C, pick the transformant into 200mL LB medium (50μg / mL kanamycin) at 37°C, shake at 200rpm until OD600 is 0.6 Add a final concentration of 0.5mM isopropyl β-D-thiogalactopyranoside (IPTG) inducer, culture at 30°C for 6h, and centrifuge the culture solution at 13,000rpm for 15min at 4°C with a high-speed refrigerated centrifuge , to collect bacteria.

[0043] Since the recombinant plasmid pET-TPERha contains a His-tag tag, it was purified by His·Bind Purification Kit (purchased from Novagen) to obtain a purified recombinant enzyme. Specific operation process:

[0044] A. Processing of samples

[...

Embodiment 3

[0059] Example 3: The acquisition of the β-glucoside / β-xyloside bifunctional enzyme gene of the present invention and the construction of the recombinant plasmid pET-DthBGL3

[0060] 3.1 Culture of Dictyoglomus thermophilum DSM 3960

[0061]Dictyoglomus thermophilum DSM 3960 was purchased from the DSMZ Culture Collection Center (www.dsmz.de) and the number was 3960. The medium formula was: potassium dihydrogen phosphate 1.5g / L, disodium hydrogen phosphate dodecahydrate 4.2g / L, chlorine Ammonium chloride 0.5g / L, magnesium chloride hexahydrate 0.38g / L, dihydrate and calcium chloride 0.06g / L, ferric ammonium sulfate hexahydrate 0.04g / L, cobalt chloride hexahydrate 2.9mg / L, molybdic acid dihydrate Sodium 2.4mg / L, sodium selenate pentahydrate 1.7mg / L, tetrahydrate and manganese chloride 2mg / L, zinc sulfate 2.8mg / L, soluble starch 5g / L, peptone 2g / L, yeast extract 2g / L , sodium carbonate 1g / L, cysteine ​​hydrochloride 1g / L, resazurin sodium 1g / L, deoxidize under nitrogen environmen...

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Abstract

The invention relates to a method for preparing icaritin through converting total flavones of epimedium by an enzyme method. The method comprises the step of converting multicomponent flavone glycosides in the total flavones of epimedium by a dual-enzyme system consisting of heat-resistant alpha-L-rhamnosidase and heat-resistant beta-glucosidase, so as to produce the icaritin. According to the method, multicomponent flavone compounds (such as icariin, Epimedin A, Epimedin B and Epimedin C) in the total flavones of epimedium can be almost completely converted into the target product; the optimal acting temperature is high, so that a substrate and intermediate products have good solubility, and a cosolvent is not required; and the enzymolysis time is short, and the yield is high. The icaritin prepared by the method has a remarkable higher propagation inhibiting effect on liver cancers, lung cancers, colon cancers and mammary cancers compared with each monomer of the total flavones of epimedium before conversion.

Description

technical field [0001] The invention belongs to the field of enzyme engineering and biomedicine, and specifically relates to a method for preparing icariin by catalyzing total flavonoids of epimedium with a recombinant enzyme system. Background technique [0002] Icaritin is a polyhydroxy flavonoid monomer component in the Berberidaceae Epimedium plant Epimedium. Its content in Epimedium is extremely low, and it is the hydrolysis product of icariin. , has estrogen-like effects, regulates immunity, promotes cardiomyocyte regeneration and differentiation, promotes bone protection, aphrodisiac, resists liver damage, delays liver fibrosis, and resists tumors. With the discovery of new medicinal effects of icariin at home and abroad, there will be a great demand for icariin in the international market in the future. Therefore, how to develop an efficient and environmentally friendly method for preparing icariin is the focus of current research. [0003] Since icariin is one of ...

Claims

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Application Information

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IPC IPC(8): C12P17/06C12N9/24C12N9/42
CPCC12N9/2402C12N9/2445C12P17/06C12Y302/01021C12Y302/0104
Inventor 赵林果张珊珊解静聪王靖秋吴涛葛林
Owner NANJING FORESTRY UNIV
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