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Method for improving water solubility of dye lignin by utilizing cyclodextrin glucosyltransferase transglycosylation reaction

A glycosyltransferase, glucose-based technology, applied in microorganism-based methods, transferases, biochemical equipment and methods, etc., can solve the problems of poor solubility, low solubility, limited application, etc., and achieve the effect of improving water solubility

Inactive Publication Date: 2017-05-31
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, genistein has strong hydrophobicity, is almost insoluble in water, has poor solubility in general organic solvents, and is easily soluble in organic solvents such as dimethyl sulfoxide
Due to the extremely low solubility of genistein in aqueous solution, it not only limits its application as food additives, cosmetics and other water-soluble products, but also greatly reduces its medicinal effect as oral drugs and intravenous injection agents, limiting its Applications in the pharmaceutical industry

Method used

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  • Method for improving water solubility of dye lignin by utilizing cyclodextrin glucosyltransferase transglycosylation reaction
  • Method for improving water solubility of dye lignin by utilizing cyclodextrin glucosyltransferase transglycosylation reaction
  • Method for improving water solubility of dye lignin by utilizing cyclodextrin glucosyltransferase transglycosylation reaction

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Experimental program
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Embodiment 1

[0019] Embodiment 1: the construction of genetically engineered bacteria producing cyclodextrin glucosyltransferase

[0020] This example takes the total chemical synthesis method as an example for illustration, but the claimed protection of the present invention is not limited to the method of total chemical synthesis only. The construction method of the genetically engineered bacteria producing cyclodextrin glucosyltransferase is as follows: according to the co-α-CGTase gene sequence published by (GenBankaccession no. JX412224), the co-α-CGTase mature enzyme gene sequence is connected by chemical total synthesis Between NcoI and Xhol after pelB of pET-20b(+). Then transfer the recombinant plasmid pET-20b(+) / co-α-CGTase into E.coli BL21(DE3) competent cells by chemical heat shock method to construct the genetically engineered bacteria E.coli BL21(DE3) / pET-20b (+) / co-α-CGTase.

Embodiment 2

[0021] Embodiment 2: Preparation and purification method of cyclodextrin glucosyltransferase

[0022] The genetically engineered bacterium producing cyclodextrin glucosyltransferase was used as the production strain, and after activation, it was inserted into LB liquid medium containing 100 μg / mL ampicillin for overnight culture at 37° C. and 200 rpm. Then the seed liquid was inoculated into the TB liquid medium containing 100 μg / mL ampicillin according to the inoculum amount of 4%. Incubate at 30°C, shaker at 200rpm to OD 600 =0.6, add 0.01mM final concentration of IPTG to induce extracellular expression, and continue to culture and ferment at 25°C, 200rpm shaker for 90h, then centrifuge the fermentation broth at 4°C, 8000rpm for 10min to remove bacteria, and collect cyclodextrin-rich glucose base transferase supernatant. Add 30% solid ammonium sulfate to the supernatant for salting out overnight, centrifuge at 4°C and 8000rpm for 10min, take the precipitate and dissolve it...

Embodiment 3

[0024] Example 3: Cyclodextrin glucosyltransferase catalyzes the glycosylation reaction of genistein

[0025] Using α-cyclodextrin as a glycosyl donor and genistein as a glycosyl acceptor, the glycosylated derivatives of genistein were synthesized under the catalysis of the prepared CGTase. The specific process is as follows: dissolving genistein in dimethyl sulfoxide (DMSO) to prepare a solution with a final concentration of 10 g / L; dissolving α-cyclodextrin in PBS buffer (50 mM, pH 6.5) to prepare a final concentration It is a 10g / L solution; the lyophilized CGTase enzyme powder is dissolved in PBS buffer (50mM, pH 6.5) to prepare an enzyme solution with a final concentration of 15g / L. Then take 300 μL genistein solution, 500 μL α-cyclodextrin solution and 200 μL CGTase enzyme solution and mix them in a 2 mL small tube with a cover, and place them on a shaker at 30°C for 20-24 hours and shake slowly. The reaction solution was analyzed by HPLC. Wherein the HPLC analysis con...

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Abstract

The invention discloses a method for improving the water solubility of dye lignin by utilizing cyclodextrin glucosyltransferase transglycosylation reaction, and belongs to the field of enzyme engineering. A gene (GenBank accession no.JX412224), which is derived from P.macerans strain JFB05-01 (CCTCC NO:M208063) and optimized by a cyclodextrin glucosyltransferase codon, is connected to pET20b(+) plasmid and is transferred to an E.coli BL21(DE3) cell to establish gene engineering bacteria for producing the cyclodextrin glucosyltransferase. The pure cyclodextrin glucosyltransferase, which is obtained through fermentation enzyme production and purification, is applied to catalytic transglycosylation reaction, alpha-cyclodextrin serves as a glycosyl donor, the dye lignin serves as glycosyl receptor, and at least four dye lignin glycosylated derivatives (Glc)n-genistein (n is equal to 1, 2, 3 and 4) are finally obtained, wherein Glc-genistein and (Glc)2-genistein are main products, in particular the water solubility of the (Glc)2-genistein is greatly improved compared with that of the dye lignin.

Description

technical field [0001] The invention discloses a method for improving the water solubility of genistein by utilizing cyclodextrin glucosyltransferase transglycosylation reaction, which belongs to the field of enzyme engineering. Background technique [0002] Soybean isoflavones are a class of secondary metabolites formed during the growth of soybeans, which mainly exist in the cotyledons and hypocotyls of soybean seeds. Among them, genistein (also known as genistein, genistein, etc.) is considered to be the one with the highest activity and function among soybean isoflavones. In legumes, genistein often exists in the form of its glucoside derivative, genistin (also known as 4',5,7-trihydroxyisoflavone-7-glycoside). Genistein has a wide range of pharmacological effects in human and animal cells. The main performances are as follows: 1) It has the effect of chemical anti-cancer (breast cancer and prostate cancer, etc.). Genistein has estrogen-like and anti-hormone effects, ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/10C12P19/18C12P19/44C12R1/19
Inventor 韩瑞枝倪晔姜明杨葛彬彬许国超董晋军
Owner JIANGNAN UNIV
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