A method for producing long-chain glycosylated sophoric acid

A technology of sophorin and glucoside, which is applied in the field of enzyme engineering and fermentation engineering, can solve the problems of low efficiency of long-chain sophoroside and limitation of the output of long-chain glycosylated sophoroside, and achieve content improvement, high yield, The effect of optimization of preparation conditions

Active Publication Date: 2022-04-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] However, the efficiency of the existing cyclodextrin glucosyltransferase for synthesizing long-chain sophoroside is relatively low, and the efficiency for synthesizing short-chain glycosyl sophoroside is relatively high, which greatly limits the use of cyclodextrin glucosyltransferase to synthesize long-chain glucoside. Yield of chain glycosylated sophoridosides

Method used

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  • A method for producing long-chain glycosylated sophoric acid
  • A method for producing long-chain glycosylated sophoric acid
  • A method for producing long-chain glycosylated sophoric acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Preparation and expression of different cyclodextrin glucosyltransferases

[0057] Specific steps are as follows:

[0058] Chemically synthesize the gene encoding cyclodextrin glucosyltransferase (the nucleotide sequence is shown in SEQ ID NO.1); after the obtained gene and the pET-20b (+) plasmid are digested with double enzymes (NdeI and XhoI) Perform ligation, transform Escherichia coli JM109, spread the transformation product on LB solid medium, culture at 37°C for 8 hours, pick transformants on LB solid medium, insert them into LB liquid medium for culture, and extract after culturing at 37°C for 10 hours Plasmid, the plasmid is sequenced to obtain the correct sequenced recombinant plasmid pET20b-CGT; the sequenced correct recombinant plasmid pET20b-CGT is transformed into Escherichia coli E.coli BL21 (DE3), and the recombinant Escherichia coli pET20b-CGT / E. coli BL21.

[0059] Using the whole plasmid PCR technology, the obtained recombinant plasmid pE...

Embodiment 2

[0073] Example 2: Product Specificity of Different Cyclodextrin Glucosyltransferases to Different Glycosylated Sophoricrosides

[0074] Specific steps are as follows:

[0075] Sophoridin (purchased from Sigma Company) was dissolved in dimethyl sulfoxide (DMSO) to prepare a final concentration of 1g / L sophoridin solution; the water-soluble starch was dissolved in PBS buffer (50mM, pH 7) prepared in a maltodextrin solution with a final concentration of 40g / L; the lyophilized mutants S77N, Y195S, Y195I, S77N, S77N / Y195S and the pure enzymes of S77N / Y195I obtained in Example 1 were dissolved in PBS buffer respectively solution (50mM, pH 7) to prepare a CGTase enzyme solution with a final concentration of 15U / L; respectively take 200μL sophoridin solution, 600μL water-soluble starch solution and 200μL CGTase enzyme solution and mix them in a 2mL small tube with a cover, and put them in a 40 ℃, 120rpm shaker and shake slowly for 20-24h to obtain a reaction solution.

[0076] Detec...

Embodiment 3

[0083] Example 3: Effect of Reaction Temperature on Yield of Long-chain Glycosylated Sophoridin

[0084] Specific steps are as follows:

[0085] On the basis of Example 2, the mutant S77N / Y195I was selected, and the reaction temperatures were replaced by 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, and 60°C, respectively.

[0086] Referring to Example 2, short-chain glycosylated sophorosides in the reaction solution were detected by HPLC (herein, the short-chain glycosylated sophoridosides were monoglycosylated sophoroside, diglycosylated sophoroside and trisaccharide Sophoroside) and long-chain glycosylated sophoroside (herein long-chain glycosylated sophoroside is tetraglycosylated sophoroside, pentaglycosylated sophoroside and hexaglycosylated sophoroside The molar content of the mixture of glycosides), and calculate the ratio (% ), the test results are shown in Table 3.

[0087]It can be seen from Table 3 that when the temperature is 45-50°C, the yield of long-chain glycosylated...

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Abstract

The invention discloses a method for producing long-chain glycosylated sophoridoside, which belongs to the technical field of enzyme engineering and fermentation engineering. The invention provides a method for producing long-chain glycosylated sophoridosides, in which cyclodextrin glucosyltransferase mutants S77N, Y195S, Y195I, S77N, S77N / Y195S and S77N / Y195I are used, so that the long-chain The content of chain glycosylated sophoric acid was significantly increased. By optimizing the reaction conditions, the content of long-chain glycosylated sophoroside in the reaction solution was increased to 9.8g / L, and the long-chain glycosylated sophoric acid in the reaction solution was reduced to 9.8g / L. The ratio of the glycoside content to the total glycosylated sophoridoside content in the reaction solution is increased to 45%.

Description

technical field [0001] The invention relates to a method for producing long-chain glycosylated sophoridoside, which belongs to the technical field of enzyme engineering and fermentation engineering. Background technique [0002] Sophorin, the fruit of Sophora japonica, a leguminous plant, is widely distributed in nature, mainly found in soybeans, mung beans, alfalfa, oats, barley, rye, wheat, corn and other cereal plants. It is a weak estrogen-like effect The non-estrogen compounds have high application prospects in the fields of medicine and health care. [0003] Modern pharmacological studies have shown that sophoridoside has a good effect on lowering alanine aminotransferase in patients with chronic hepatitis, and can also inhibit ear edema induced by croton oil and paw edema induced by carrageenan. Clinical studies have also confirmed that sophoridoside can be used to prevent and treat the symptoms of postmenopausal osteoporosis in women. Sophora japonica soaked lamb i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P19/44C12R1/19
CPCC12N9/1074C12Y204/01019C12N15/70C12P19/44
Inventor 韩瑞枝柴宝成姜钰琳倪晔
Owner JIANGNAN UNIV
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