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Beta-glucosidase as well as preparation method and application thereof

A glucosidase and nucleotide technology, which is applied in the fields of genetic engineering technology and biomedicine, can solve the problems of less by-products, difficult to control product quality, influence of product purity and yield, etc., and achieves strong conversion ability and thermal stability. Excellent and highly expressive effect

Active Publication Date: 2015-05-13
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In view of the existing research on the transformation of ginsenoside Rd and related patents, it is found that there are limitations: (1) most of the biotransformation of ginsenosides is microbial transformation, but the quality of the products obtained by microbial transformation is difficult to control, which has a negative impact on the product. The purity and yield of the product have a great influence, and the specificity of the enzyme treatment is strong, and the by-products are few, so the purpose of improving the yield and purity of the product can be achieved by exploring a single high-efficiency glycoside hydrolase; (2) in the preparation of In the study of ginsenoside Rd, it can be obtained by catalyzing ginsenoside Rb1 with β-glucosidase, but it has not been obtained by catalyzing ginsenoside Rb2 with microbial enzymes, and no β-glucosidase that can catalyze ginsenoside Rb2 has been found

Method used

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  • Beta-glucosidase as well as preparation method and application thereof
  • Beta-glucosidase as well as preparation method and application thereof
  • Beta-glucosidase as well as preparation method and application thereof

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preparation example Construction

[0040] The preparation method of β-glucosidase described in the present invention refers to the separation and purification of induced expression, inducing and culturing the expression host bacteria containing the recombinant plasmid at 37°C without adding IPTG, collecting the bacteria by ultrasonic crushing, and taking the supernatant for affinity chromatography get the fusion protein.

[0041] The present invention also provides a method for preparing ginsenoside Rd, specifically, the β-glucosidase of the present invention simultaneously enzymolyzes ginsenosides Rb1 and Rb2 at pH 6.0 and 90°C to prepare ginsenoside Rd.

[0042] The β-glucosidase of the present invention has multiple functions, including β-glucosidase activity and α-L-arabinopyranosidase activity, and can hydrolyze β-1,6-glucoside on the 20th C of ginsenoside Rb1 bond and α-1,6-arabinopyranoside bond on the 20th C of Rb2 to prepare ginsenoside Rd. After 5 minutes of reaction, ginsenoside Rb1 was transformed ...

Embodiment 1

[0044] Embodiment 1: the acquisition of β-glucosidase gene of the present invention and the construction of recombinant plasmid pET-TPEBGL1

[0045] 1.1 Culture of Thermotoga petrophila DSM 13995

[0046] 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.

[0047] 1.2 Genomic DNA extraction

[0048] (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.

[0049] (2) Resuspend the bacteria in 9.5 mL TE buffer, add 0.5 mL 10% sodium dodecyl sulfate (SDS) and 50 μL ...

Embodiment 2

[0064] Embodiment 2: the preparation of β-glucosidase of the present invention

[0065] The recombinant plasmid pET-TPEBGL1 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 / mL). 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.

[0066] Since the recombinant plasmid pET-TPEBGL1 contains a His-tag tag, it was purified by His·Bind Purification Kit (purchased from Novagen) to obtain a purified recombinant enzyme. The specific operation process:

[0067] A....

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Abstract

The invention provides a beta-glucosidase as well as a preparation method and an application thereof. The amino acid sequence of beta-glucosidase is shown as SEQ ID NO.1. The beta-glucosidase has excellent thermal stability, can resist high temperature, has higher beta-1,6-glucosidic bond hydrolysis capacity as well as higher alpha-1,6-arabinopyranoside bond hydrolysis capacity and has higher transformation capacity for ginsenoside Rb1 and Rb2. After the beta-glucosidase is incubated with the ginsenoside Rb1 and Rb2 for certain period, ginsenoside Rb1 and Rb2 are almost transformed into ginsenoside Rd completely. According to a preparation method of TPEBGL1, high-efficiency expression can be realized without an inducer IPTG (isopropyl beta-D-1-thiogalactopyranoside).

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 of enzymatic conversion of multi-component ginsenosides Rb1 and Rb2 to prepare ginsenoside Rd. Background technique [0002] Ginseng (Panax ginseng C.A. Meyer) is a perennial medicinal plant of the genus Panax in the family Araliaceae. With the advancement of modern separation and analysis techniques, the active ingredients in ginsenosides have also been deeply analyzed, and there are more than 50 kinds of ginsenoside monomers that have been isolated and identified. [0003] In recent years, ginsenoside Rd has received widespread attention because of its unique pharmacological activity. Its unique renal protection function and specific function of blocking receptor-dependent calcium ion channels are not found in other monomers. At the same time, ginsenosi...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/63C12P33/20
CPCC12N9/2445C12P33/20C12Y302/01021
Inventor 赵林果解静聪赵东霞萧伟丁岗王振中
Owner NANJING FORESTRY UNIV
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