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Panax japonicus saponin glucoside hydrolase and application thereof in production of notoginsenoside R1

A technology of glycoside hydrolase and ginsenoside, applied in the production of ginger-like notoginseng saponin R1, the field of glycoside hydrolase

Active Publication Date: 2022-03-01
SHANGHAI UNIV OF T C M
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no experimental verification and report on the protein-related functions involved in the present invention, and there is no report on the use of this enzyme to prepare ginger-like notoginsenoside R1 through bio-enzymatic conversion with ginsenoside Ro as a substrate

Method used

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  • Panax japonicus saponin glucoside hydrolase and application thereof in production of notoginsenoside R1
  • Panax japonicus saponin glucoside hydrolase and application thereof in production of notoginsenoside R1
  • Panax japonicus saponin glucoside hydrolase and application thereof in production of notoginsenoside R1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1, cloning of bamboo ginseng saponin glycoside hydrolase and its coding gene

[0056] Genomic DNA extracted from Paenibacillus lactis was used as a template, and 5′-CGCGGATCCATGAGAAACCATACTTTAGATACG-3′(Forward) and 5′-CCCAAGCTT TCAGCTTCTACGGTATCTCTTC-3′(Reverse) were used as primers for polymerase chain reaction PCR amplification.

[0057] PCR system: 12.5 μL of 2×Taq Mixture, 0.5 μL of upstream and downstream primers (10 μm), 0.5 μL of genome and ddH 2 O 11.5 μL.

[0058] The PCR conditions were as follows: pre-denaturation at 95°C for 3 minutes, followed by 30 cycles at 95°C for 30 s, 58°C for 30 s, and 72°C for 2 min; finally, extension at 72°C for 10 min.

[0059] The above PCR products were analyzed by agarose gel electrophoresis, the gel was tapped and the target band was recovered with a kit.

[0060] The recovered products were digested with the pET-28a(+) vector with restriction endonucleases BamHI and HindIII respectively (37°C, 6h); the digested ...

Embodiment 2

[0062] Embodiment 2, expression and purification of recombinant PlGH03

[0063] Inoculate the single colony of BL21(DE3) / pET-28a(+)-Pl3 obtained above into LB liquid medium containing kanamycin (final concentration: 50 μg / mL), culture at 37°C for 12 hours, and take 1 mL of bacteria solution was added to 100 mL of fresh LB liquid medium (containing kanamycin at a final concentration of 50 μg / mL), and cultured at 37°C until OD 600 When it reached 0.4, IPTG (final concentration: 0.2mM) was added to the culture medium, and induction culture was continued at 16°C for 24h. Collect the fermentation broth, and centrifuge at 8000rpm for 5min to collect the bacteria. Wash twice with 50 mL of normal saline, and collect the cells by centrifugation.

[0064] Resuspend the bacteria with 10mL solution A (10mM pH7.4 sodium phosphate buffer, containing 20mM imidazole, 500mM NaCl). In an ice-water bath, crush the bacterial cells with an ultrasonic crusher (400W, work for 4s and pause for 6s,...

Embodiment 3

[0068] The optimal temperature of embodiment 3, recombinase PlGH03

[0069] Take a certain amount of recombinant enzyme PlGH03 and p-nitrophenyl-β-D-glucoside with a final concentration of 2 mM, add pH 8.050 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer to a volume of 0.5 mL, and place in 25- After reacting in a water bath at 55°C for 5 min, add 0.5 mL of 1 mM Na 2 CO 3 The solution terminated the reaction, and the absorbance value at 405 nm was measured, and the highest activity was taken as 100%. The relative activity results are shown in Table 2, and the enzyme has the highest reactivity at 50°C.

[0070] The optimal temperature of table 2 recombinase PlGH03

[0071]

[0072]

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Abstract

The invention discloses panax japonicus saponin glucoside hydrolase and application of the panax japonicus saponin glucoside hydrolase in production of gingerous notoginsenoside R1. The glucoside hydrolase is a protein with an amino acid sequence as shown in SEQ ID NO. 2. Experiments prove that the panax japonicus saponin glucoside hydrolase disclosed by the invention has the effect of efficiently converting ginsenoside Ro to generate notoginsenoside R1. Experiments prove that the panax japonicus saponin glucoside hydrolase also has the effects of efficiently converting panax japonicus saponin IVa to generate calendula officinalis glycoside E, efficiently converting panax japonicus saponin IV to produce aralia elata saponin VI, and efficiently converting pseudo ginsenoside RT1 to generate a corresponding C-28 deglucosylation product.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a glycoside hydrolase and its application in the production of notoginseng saponin R1. Background technique [0002] Araliaceae Ginseng plants (such as Panax ginseng, Panax notoginseng, Panax japonicus, etc.) have extremely high medical value and a long history of clinical application. Modern chemical and pharmacological studies have shown that triterpenoid saponins are one of the main chemical components of Panax ginseng medicinal plants (collectively referred to as "ginsenosides"), which have very significant pharmacological activity and potential new drug development value, such as preventing Or treat cardiovascular and cerebrovascular diseases, hyperlipidemia and other diseases (Chem. Rev. 2012, 112: 3329-3355; J. Pharm. Pharmacol. 2006, 58: 1007-1019). [0003] According to the classification of saponins, ginsenosides can be mainly divided into dammarane-type saponins and olean...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12P33/00
CPCC12N9/2402C12P33/00Y02A50/30
Inventor 王如锋杨小林王峥涛
Owner SHANGHAI UNIV OF T C M
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