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A kind of glucosidase composition and the method for preparing icarigenin by enzymatic method

A technology of icariin and icariin, which is applied in the direction of biochemical equipment and methods, enzymes, hydrolytic enzymes, etc., can solve the problems of low product yield, long hydrolysis time, uncontrollable sequence, etc. High efficiency, short enzymatic hydrolysis time, and good substrate solubility

Active Publication Date: 2021-07-23
JIANGSU KANION PHARMA CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, compound cellulase or β-glucosidase is used to combine enzymatic hydrolysis and acid hydrolysis to convert icariin into icariin, but the acid hydrolysis reaction process is not easy to control, with many by-products and low product yield. low, resulting in higher product costs; or convert icariin into icariin by a commercial β-glucosidase, although the enzyme can obtain icariin, but its hydrolysis time Long (24 hours), the highest enzymolysis yield is only about 55%. According to the enzymolysis effect and enzyme specificity, it can be seen that the β-glucosidase used comes from traditional microbial fermentation, and its enzyme is not pure β-glucosidase. Glucosidase; another study uses naringinase to obtain icariin from icariin, and adds icariin to a 30% to 70% ethanol system to adjust the pH (4 to 8), and the system reaches a certain level. Temperature (40~70°C), temperature control reaction under stirring for 30 hours
However, naringinase is a compound enzyme prepared by microbial fermentation. The specific characteristic is mainly to specifically hydrolyze naringin. The proportion of the enzyme is uncontrollable, the order of action is uncontrollable, and the reaction time is long and the efficiency is low.

Method used

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  • A kind of glucosidase composition and the method for preparing icarigenin by enzymatic method
  • A kind of glucosidase composition and the method for preparing icarigenin by enzymatic method
  • A kind of glucosidase composition and the method for preparing icarigenin by enzymatic method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] 1.1 Synthesis of α-L-rhamnosidase gene derived from Aspergillus terreus CCF 3059, plasmid construction and preparation of recombinant enzyme

[0062] 1.1.1 Construction of recombinant plasmid pPICZαA-MRha

[0063] The whole gene was synthesized and the Aspergillus terreus CCF 3059 α-L-rhamnosidase gene (SEQ ID NO: 9) was optimized through yeast codon preference, and it was connected to the pPICZαA plasmid, and the obtained recombinant plasmid was named pPICZαA-MRha.

[0064] 1.1.2 Preparation of recombinant enzyme

[0065] The recombinant plasmid pPICZαA-MRha was extracted, linearized by Sac I, introduced into Pichiapastoris KM71H (Novagen) by electroporation, and positive clones were screened. After being inoculated in YPD medium for activation, transfer to BMGY medium to continue activation, and collect OD 600 The 2.0-3.0 bacterium was transferred into BMMY medium, and placed on a shaker at 30° C. at 180 rpm to induce the expression of α-L-rhamnosidase. Add sterile...

Embodiment 2

[0100] Example 2. Screening of β-glucosidase

[0101] 2.1 Cloning of β-glucosidase of GH3 family derived from Thermotoga thermarum DSM 5069 and preparation of recombinant enzyme

[0102] 2.1.1 Culture of Thermotoga thermarum DSM 5069

[0103] Thermotoga thermarum DSM 5069 was purchased from the DSMZ Culture Collection (www.dsmz.de). Number: DSM 5069. The medium formula is: 5g / L soluble starch, 1g / L yeast powder, 1.5g / L KH 2 PO 4 , 4.2g / LNa 2 HPO 4 x 12H 2 O, 3.4g / L NaCl, 1g / L MgSO 4 ×7H 2 O, 0.76g / L EDTA, 1mL / L trace elements, 0.5g / LNa 2 S·9H 2 O, 0.5g / L Cysteine ​​HCl, 1mg / L resazurin, adjust the pH to 7.0, boil with nitrogen, remove oxygen, put the culture medium into an anaerobic bottle for sterilization under anaerobic conditions. Trace element (1000×) formula: FeCl 3 2.0g / L;H 3 BO 3 0.05g / L; ZnCl 2 0.05g / L; CuCl 2 2H 2 O 0.03g / L; MnCl 2 4H 2 O 0.05g / L; (NH 4 ) 2 MoO 4 0.05g / L; AlKSO 4 2H 2O 0.05g / L. ) was inoculated with a syringe according to...

Embodiment 3

[0193] Example 3. α-L-rhamnosidase and β-glucosidase enzyme activity assay

[0194] Using p-nitrophenol-α-rhamnoside (pNP-R) as a substrate, the hydrolyzed p-nitrophenol had a color reaction with sodium carbonate, and the absorbance of the product was measured at a wavelength of 405nm. 100μL reaction system includes 75μL 100mM buffer solution with optimal pH, 20μL 5mM substrate, mix well and add 5μL diluted enzyme solution after preheating, react at the optimal temperature for 10min, then add 0.3mL 1M NaCO 3 Terminate the reaction, mix well and measure with a microplate reader under the condition of 405nm. At the same time, a control with enzyme solution without substrate and a control with substrate without enzyme solution were made.

[0195] Using p-nitrophenol-β-glucoside (pNP-G) as a substrate, the hydrolyzed p-nitrophenol had a color reaction with sodium carbonate, and the absorbance of the product was measured at a wavelength of 405nm. 100μL reaction system includes 90...

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Abstract

The invention relates to the technical fields of enzyme engineering and biomedicine, in particular to a glycosidase composition and a method for enzymatically preparing icariin. The present invention screens and obtains an α-L-rhamnosidase that can effectively degrade rhamnose residues on the mother nucleus structure of icariin and a high-temperature-resistant β-glucoside that can efficiently degrade glucose residues in its mother nucleus structure Enzyme, followed by double-enzyme conversion to efficiently prepare icariin with fully maintained original biological activity. In the composition provided by the invention, the optimal reaction temperature of the two glycosidases is relatively high, the solubility of the substrate is good, no cosolvent is needed, and the enzymolysis time is short and the yield is high. Experiments show that the molar conversion rate of icariin is 98.3% and the yield of icariin is 90% when the glycosidase composition provided by the invention is used to enzymatically hydrolyze icariin.

Description

technical field [0001] The invention relates to the technical fields of enzyme engineering and biomedicine, in particular to a glycosidase composition and a method for enzymatically preparing icariin. Background technique [0002] Icaritin is a polyhydroxy flavonoid monomer component in Epimedium genus Berberidaceae, which has estrogen-like effects, regulates immunity, promotes cardiomyocyte regeneration and differentiation, and promotes osteoarthritis. Quality protection, impotence, anti-liver damage and delay liver fibrosis, anti-tumor and other pharmacological activities. At present, it has entered clinical research as a new class of anti-liver cancer drug (alcoradine). Icariin is mainly extracted from the traditional Chinese medicine Epimedium, but the content of Icariin in Epimedium is extremely low, and the separation and extraction process is complicated and the cost is high. In the future, the international market demand for icariin will be great, so the method for...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/24C12N9/42C12P17/06
Inventor 萧伟赵林果葛林丁岗裴建军王振中
Owner JIANGSU KANION PHARMA CO LTD
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