Method for catalytically synthesizing curcumin glycoside compounds by biological method

A technology of curcumin glycoside and biological method, which is applied in the field of bioengineering and can solve the problems of poor water solubility and low bioavailability of curcumin

Active Publication Date: 2021-08-31
NANJING UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The object of the present invention is to provide a kind of biological method catalyzing the method for synthesizing curcumin glycosides for curcumin bioavailability is low, the problem of poor water solubility at present, At the same time, it solves the problem that plant-derived glycosyltransferase is expressed in Escherichia coli to produce a large number of inclusion bodies, and provides a recombinant bacterium containing glycosyltransferase and sucrose synthase. The target gene is constructed on the expression vector pRSFDuet1 to obtain recombinant The plasmid pRSF-CaUGT2-AtSUS1 was transformed into the host cell Escherichia coli BL21 (DE3) competent cells to obtain the recombinant strain CaUGT2-AtSUS1, the recombinant strain was induced to express, and the supernatant was obtained by ultrasonication and centrifugation to obtain the best soluble expression , and finally efficiently catalyze the substrate curcumin to improve its water solubility

Method used

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  • Method for catalytically synthesizing curcumin glycoside compounds by biological method
  • Method for catalytically synthesizing curcumin glycoside compounds by biological method
  • Method for catalytically synthesizing curcumin glycoside compounds by biological method

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1 Construction of recombinant plasmids

[0031] Select the double gene expression vector pRSFDuet-1, the recombinant plasmid is pRSF-CaUGT2-AtSUS1, in Nco I and EcoR I Insert the optimized sucrose synthase gene AtSUS1 , the sequence is shown in SEQ ID NO:1; in xho I and Nde I inserted the optimized glycosyltransferase gene CaUGT2 , the sequence is shown in SEQ ID NO: 3, and the recombinant plasmid pRSF-CaUGT2-AtSUS1 was obtained to form the co-expression recombinant plasmid pRSF-CaUGT2-AtSUS1. The recombinant plasmid was synthesized by Nanjing GenScript Company. sucrose synthase AtSUS1 Its amino acid sequence is shown in SEQ ID NO: 2, glycosyltransferase CaUGT2 Its amino acid sequence is shown in SEQ ID NO:4.

Embodiment 2

[0033] Example 2 Obtaining of Recombinant Escherichia coli Strains

[0034] The above-mentioned recombinant plasmid pRSF-CaUGT2-AtSUS1 was transformed into Escherichia coli BL21 (DE3) competent cells, and the transformants were spread on LB solid plates containing 50 µg / L kanamycin (NaCl 10 g / L, yeast powder 5 g / L peptone, 10 g / L peptone, 20 g / L agar), and cultured overnight in a 37°C incubator at a constant temperature to obtain a recombinant strain CaUGT2-AtSUS1 containing a dual-enzyme co-expression system.

[0035] The above recombinant plasmid pRSF-UGT76G1-StSUS1 was transformed into Escherichia coli BL21(DE3) competent cells, and the transformants were spread on LB solid plates containing 50 µg / L kanamycin (NaCl 10 g / L, yeast powder 5 g / L peptone, 10 g / L peptone, 20 g / L agar) and cultured overnight in a 37°C incubator at a constant temperature to obtain a recombinant strain UGT76G1-StSUS1 containing a dual-enzyme co-expression system.

Embodiment 3

[0036] Example 3 Induced Expression of Recombinant Strains

[0037] The two recombinant strains constructed in Example 2 were respectively activated, transferred to LB medium, and placed in a shaker at 25-40°C, 200rpm, until the OD 600 When it reaches 0.6, add 0.1 mM inducer isopropyl-β-D-thiogalactoside to induce culture, centrifuge at low temperature (4°C, 7000 rpm, 6 min) and collect the bacteria, wash twice with potassium phosphate buffer. Then add an appropriate amount of potassium phosphate buffer, place it in an ice-water mixture, and use a sonicator to sonicate the bacteria, with the parameters set at Ф6, 300 W, and 30 min. Then centrifuge in a refrigerated centrifuge with the parameters set at 4°C, 8000rpm, for 30 min. The supernatant is taken as the crude enzyme solution, and stored in a 4°C refrigerator for later use.

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Abstract

The invention discloses a method for catalytically synthesizing curcumin glycoside compounds by a biological method. A glycosyl transferase gene CaUGT2 and a sucrose synthase gene AtSUS1 are constructed on an expression vector and are introduced into escherichia coli to obtain a recombinant strain, the soluble expression quantity of the recombinant strain after induced expression is improved compared with that of glycosyl transferase from other plants, and a substrate curcumin can be efficiently catalyzed. A recombinant plasmid is pRSF-CaUGT2-AtSUS1, a sucrose synthase AtSUS1 gene is inserted into Nco I and EcoR I, glycosyl transferase CaUGT2 is inserted into Xho I and NdeI, a co-expression recombinant plasmid pRSF-CaUGT2-AtSUS1 is formed, the recombinant plasmid is transformed into escherichia coli BL21 (DE3) competent cells, and a recombinant strain CaUGT2-AtSUS1 is obtained. After the recombinant strain is subjected to induced expression, the soluble expression quantity of the glycosyl transferase CaUGT2 is increased compared with that of glycosyl transferase from other plants, the conversion rate of catalytic synthesis of curcumin glycoside compounds reaches 98%, curcumin is catalyzed to generate curcumin monoglucoside and curcumin diglucoside, the water solubility of the curcumin monoglucoside and curcumin diglucoside is superior to that of curcumin, and the problem that curcumin is poor in water solubility is solved. The concentration of the substrate curcumin is 75 mM, the concentration of a catalytic substrate is relatively high, and the method is more suitable for food and medicine industries in industrialization.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for catalyzing and synthesizing curcumin glycoside compounds through a biological method. Background technique [0002] Curcumin is a yellow pigment that was first extracted from Zingiberaceae plants and is a relatively rare polyphenolic compound. The crystal of curcumin is orange-yellow, has a bitter taste, is insoluble in water and ether, and easily soluble in glacial acetic acid and alkali solution. There are hydroxyl groups at both ends of the curcumin molecule. Under alkaline conditions, conjugation effects are prone to occur, so when the pH is greater than 8, curcumin will turn from yellow to red. For a long time, curcumin, as a commonly used natural plant dye, has been widely studied and applied in the food industry, mainly in the processing and dyeing of canned food, sausage products, soy sauce and stewed products. With the deepening of curcu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P19/44C12R1/19
CPCC12N9/1062C12N9/1048C12N15/70C12Y204/01013C12P19/44
Inventor 李艳贾红华余杰林磊孙萍徐娇娇
Owner NANJING UNIV OF TECH
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