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Glycosylation application of dextransucrase and method for preparing phenyl ethyl caffeate glycoside from dextransucrase

A technology of phenethyl caffeate and dextran, which is applied in the field of enzyme chemistry, can solve problems such as difficulties in the application of phenethyl caffeate, and achieve the effects of rich products, good anti-inflammatory activity, and good water solubility

Pending Publication Date: 2019-08-13
HEFEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the above problems, the present invention provides a method and application of catalyzing phenethyl caffeate with dextran sucrase to synthesize phenethyl caffeate glycoside with good water solubility and anti-inflammatory activity, and solves the problem of phenethyl caffeate. The difficult problem of application also provides a new application for dextran sucrase, breaking the notion that dextran sucrase can only specifically act on the enzyme receptor in the technical field; in addition, the method of the present invention can simultaneously Obtain at least two caffeic acid phenethyl ester glycosides, the method is simple, the product is abundant, the cost is low, and the universality is wide

Method used

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  • Glycosylation application of dextransucrase and method for preparing phenyl ethyl caffeate glycoside from dextransucrase
  • Glycosylation application of dextransucrase and method for preparing phenyl ethyl caffeate glycoside from dextransucrase
  • Glycosylation application of dextransucrase and method for preparing phenyl ethyl caffeate glycoside from dextransucrase

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Experimental program
Comparison scheme
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Embodiment 1

[0073] The preparation of embodiment 1 dextran sucrase

[0074] 1. Strain culture

[0075] The genetically engineered bacteria dex-YG-thMU01 (preservation number CGMCC No.12438) was inoculated into LB medium containing 50 μg / mL kanamycin, and cultured on a shaking table at 37°C and a rotational speed of 250r / min for 16h. Obtain the seed culture solution.

[0076] Use a pipette gun to draw 2ml of bacterial solution in the obtained seed culture solution and add it to medium A (glycerol 5g / L, glucose 5g / L, MgSO 4 ·7H 2 O 0.1mmol / L, peptone 10g / L, KNO 3 10g / L, Na 2 HPO 4 12H 2 O 17.1g, KH 2 PO 4 3g / L, NH 4 Cl 1g / L), cultured on a shaking table at 37°C and 250r / min to obtain a bacterial solution for enrichment culture.

[0077] 2. Induce enzyme production

[0078] Use distilled water to dilute the enriched bacterial solution 10 times. When the OD600 of the diluted solution reaches 0.25, about 3.5 to 4 hours, add 500 μL IPTG to induce enzyme production for about 4 hours ...

Embodiment 2

[0079] The preparation of embodiment 2 caffeic acid phenethyl ester glycosides

[0080] 1. Dextran sucrase catalyzes phenethyl caffeate

[0081] Add 0.5g (0.5%w / v) phenethyl caffeate and 30g (30%w / v) sucrose to a 250ml Erlenmeyer flask respectively, then add DMSO containing 10% (v / v), 100ml pH5. 5 of the acetic acid-calcium acetate buffer solution, after mixing, add 100 U of the crude dextran sucrase enzyme solution prepared in Example 1 to the conical flask, and catalyze the mixed solution in the conical flask at a temperature of 25°C After 24 hours, the product obtained after catalysis was mixed with an equal volume of ethanol solution for alcohol precipitation treatment, thereby removing the glucan glycoside by-products, collecting the supernatant, and filtering the supernatant through a microporous membrane with a pore size of 0.22 μM Afterwards, obtain the saccharified derivative of caffeic acid phenethyl ester, carry out high performance liquid chromatography analysis b...

Embodiment 3

[0085] Identification and structural analysis of embodiment 3 glycoside products

[0086] 1. Identification of glycoside products

[0087] In order to verify that the obtained glycoside products are caffeic acid phenethyl ester monoglycoside and caffeic acid phenethyl ester diglycoside, the application for the above preparation process reactant caffeic acid phenyl ethyl ester, glucose and reaction product caffeic acid phenyl ethyl ester saccharified derivatives Mass spectrometry was carried out separately, and the results of mass spectrometry were as follows: figure 2 shown.

[0088] according to figure 2 The analysis results show that the molecular mass of phenylethyl caffeate is 283, and the molecular mass of glucose is 180. When the glucose molecule is combined with phenethyl caffeate, a molecule of water will be removed, so the molecular mass of the monoglycoside product is 445. The molecular weight of the glycoside product is 607, which is the same as figure 1 The p...

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Abstract

The invention discloses application of dextransucrase to glycosylation of phenyl ethyl caffeate and a method for preparing phenyl ethyl caffeate glycosides from the dextransucrase, and relates to thefield of the enzyme chemical industry. The dextransucrase is added to a buffering solution with the phenyl ethyl caffeate and a glycosyl donor for catalysis treatment, and the phenyl ethyl caffeate glycosides can be obtained after purification. By means of the method, the blank of the application of the dextransucrase to the glycosylation of the phenyl ethyl caffeate is filled up; the prepared phenyl ethyl caffeate glycosides are high in water solubility and anti-inflammation activity, and the problem that phenyl ethyl caffeate is difficult to apply is solved; in addition, by means of the method, the two types of phenyl ethyl caffeate glycosides can be obtained at the same time, the method is simple, products are rich, cost is low, and universality is high.

Description

technical field [0001] The invention relates to the technical field of enzyme chemistry, in particular to the application of dextran sucrase and a method for preparing catalyzed phenethyl caffeate. Background technique [0002] Dextransucrase is a kind of dextransucrase that uses sucrose as a substrate to synthesize high-molecular dextran by hydrolyzing and transferring glucose groups. This enzyme belongs to the GH70 family according to its structure and properties. At present, 342 strains of dextransucrase have been found in the GH70 family. Although dextran sucrase has some highly conserved regions and homologous structural domains, the types of glucan glycosidic bonds it synthesizes are quite different, and the application fields of glucans with different bond types are also different. It can be seen that not only The properties of different dextran sucrase enzymes are different, and their functions are also areas of long-term exploration by those skilled in the art. Wan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/46
CPCC12P19/46
Inventor 张洪斌李瑶余晓琴张宇馨胡雪芹杨静文
Owner HEFEI UNIV OF TECH
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