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[Beta]-glucosidase, preparation method and application thereof

A glucosidase and enzymatic hydrolysis technology, which is applied in the enzymatic transformation and preparation of ginsenoside 20-Rg3, in the field of β-glucosidase and its preparation, can solve the problems of time-consuming separation, high cost, and low yield. Achieve the effects of less intermediate products, strong conversion ability and high purity

Active Publication Date: 2015-02-04
JIANGSU KANION PHARMA CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the methods for preparing 20(S)-Rg3 mainly include chemical methods, physical methods and biotransformation methods, among which 20(S)-ginseng It is reported that 20(S)-Rg3 is prepared by chemical synthesis using triol as a raw material, but the reaction steps are cumbersome and the yield is very low. In addition, the raw materials required for this method are also difficult to obtain
With ginsenosides such as Rb1, Rb2, and Rc, which have a higher content in ginseng, Rg3 can also be obtained by physical methods such as heat treatment or acid treatment, but the resulting product is a mixture of 20(S)-Rg3 and 20(R)-Rg3, the product Later separation is time-consuming and expensive
Biotransformation methods mainly include microbial transformation and enzymatic transformation. Microorganisms can produce glycoside hydrolase for the preparation of rare ginsenosides through cultivation. However, the enzymes produced by microorganisms are complex and have different specificities, which makes the products obtained by microbial transformation The quality is difficult to control and has a certain impact on the purity of the product
However, no glycoside hydrolase has been reported for the production of ginsenoside 20(S)-Rg3

Method used

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  • [Beta]-glucosidase, preparation method and application thereof
  • [Beta]-glucosidase, preparation method and application thereof
  • [Beta]-glucosidase, preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0057] Embodiment 1: the acquisition of β-glucosidase gene of the present invention and the construction of recombinant plasmid pET-TPEBGL3

[0058] 1.1 Culture of Thermotoga petrophila DSM 13995

[0059] 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-morpholinethanesulfonic 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.

[0060] 1.2 Genomic DNA extraction

[0061] (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.

[0062] (2) Resuspend the bacteria in 9.5 mL TE buffer, add 0.5 mL 10% sodium dodecyl sulfate (SDS) and 50 μL ...

Embodiment 2

[0077] Embodiment 2: the preparation of β-glucosidase of the present invention

[0078] The recombinant plasmid pET-TPEBGL3 was transformed into Escherichia coli JM109 (DE3) host bacteria (purchased from Novagen), and placed on an LB plate containing ampicillin (50 μg / mL) (LB medium: tryptone 10 g / L, yeast extract 5 g / L L, NaCl 5g / L, agar 15g / L) after culturing overnight at 37°C, pick the transformants into 200mL LB medium (50μg / mL ampicillin) at 37°C, shake at 200rpm until the OD600 is 0.6, add the final The concentration is 0.5mM isopropyl β-D-thiogalactopyranoside (IPTG) inducer, cultivated at 30°C for 6h, centrifuged the culture solution at 13,000rpm for 15min at 4°C with a high-speed refrigerated centrifuge, and collected the bacteria body.

[0079] Since the recombinant plasmid pET-TPEBGL3 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:

[0080] A. Proces...

Embodiment 3

[0096] Embodiment 3: the qualitative determination of β-glucosidase of the present invention

[0097] 1. Determination method of enzyme activity

[0098] Add 85 μL 100 mmol / L citric acid-disodium hydrogen phosphate buffer (pH 5.0) to 5 μL 20 mmol / L p-nitrophenyl β-D glucoside (pNPG) in 100 μL reaction system, first incubate at 90oC for 3 minutes, then add 10 μL enzyme solution (diluted to an appropriate multiple) for 10 minutes, and then add 600 μL of 1 mol / L sodium carbonate solution to terminate the reaction after color development. Absorbance was measured at 405 nm. Enzyme activity unit (U) is defined as: under the assay conditions, the amount of enzyme required to produce 1 μmol p-nitrophenol per minute is 1 enzyme activity unit.

[0099] 2. Determination of the optimum reaction temperature

[0100] In the range of 60-100°C, the enzyme activity was measured every 5°C. The buffer is 100mmol / L citric acid-disodium hydrogen phosphate buffer solution, pH 5.0, the result is...

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Abstract

The invention belongs to the fields of gene engineering technology and biological medicines, particularly relates to a [beta]-glucosidase, a preparation method and an application thereof and especially relates to an application in enzymic-method conversion for preparing ginsenoside 20(S)-Rg3. The [beta]-glucosidase is high-temperature-resistant and can maintain an enzyme activity to be constant almost at 70 DEG C for 3h. The [beta]-glucosidase has a strong conversion capability on ginsenoside Rb1. When being used for incubation with the ginsenoside Rb1 for 60 min, the [beta]-glucosidase enables the ginsenoside Rb1 to be converted almost into the ginsenoside 20(S)-Rg3. The method for preparing the ginsenoside 20(S)-Rg3 is less in intermediate product in an enzymatic conversion reaction and the ginsenoside 20(S)-Rg3 is high in purity after conversion.

Description

technical field [0001] The invention belongs to the field of genetic engineering technology and biomedicine, and specifically relates to a β-glucosidase and its preparation method and application, especially the application in the preparation of ginsenoside 20(S)-Rg3 by enzymatic transformation. Background technique [0002] Ginseng (Panax ginseng C.A. Meyer) is a perennial medicinal plant of the genus Panax in the family Araliaceae. It is a traditional and precious Chinese herbal medicine in my country. It has good curative effects on the treatment of cardiovascular diseases, diabetes, mental diseases and other diseases. Modern pharmacological research has proved that ginsenoside is its main active ingredient [1], and its significant anti-tumor, anti-inflammatory and anti-aging activities have always been a research hotspot. [0003] So far, there are more than 50 kinds of ginsenosides that have been isolated and identified. Among them, the content of ginsenoside monomer co...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/70C12P33/20
CPCC12N9/2445C12P33/005C12P33/20C12Y302/01021
Inventor 赵林果萧伟裴建军丁岗解静聪王振中
Owner JIANGSU KANION PHARMA CO LTD
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