Application of cyclodextrin glycosyltransferase in production of alpha-glycosyl hesperidin

A glycosyltransferase and cyclodextrin technology, applied in the directions of glycosyltransferase, transferase, microorganism-based methods, etc., can solve the problems of low substrate conversion rate, low conversion rate, not much, etc. The effect of high conversion rate, fast catalytic rate and high amount of substrate feeding

Inactive Publication Date: 2019-04-19
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some cyclodextrin glycosyltransferases have been reported for modifying hesperidin with strong enzyme specificity, mild conditions and no “three wastes”, there are not many related patent protection methods, which may be due to these The enzymatic conversion rate is not high, the substrate conversion rate is low, etc.

Method used

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  • Application of cyclodextrin glycosyltransferase in production of alpha-glycosyl hesperidin
  • Application of cyclodextrin glycosyltransferase in production of alpha-glycosyl hesperidin

Examples

Experimental program
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Effect test

Embodiment 1

[0021] Embodiment 1, preparation produces the bacterium agent of α-glycosyl hesperidin

[0022] 1. Construction of Escherichia coli that highly expresses thermostable cyclodextrin glycosyltransferase

[0023] Referring to the Thermoanaerobacterium thermosulfurigenes reported in the NCBI database (NCBI accession number NO.P26827.2), the sequence was sent to the gene synthesis company (Wuxi Qinglan Biotechnology Co., Ltd.) for codon optimization , artificially synthesized cyclodextrin glycosyltransferase coding gene cgt02 (nucleotide sequence shown in SEQ ID NO.1, amino acid sequence shown in SEQ ID NO.2), and cloned between NcoI / HindIII of expression vector pET22b , to obtain the recombinant plasmid pET22b-cgt02.

[0024] The transformation method of the recombinant plasmid pET22b-cgt02 is as follows: Take 5 μL of the recombinant plasmid pET22b-cgt02 and add it to 100 μL of E.coli Rosetta TM (DE3) For competent cells, flick the tube wall several times to mix well, and place i...

Embodiment 2

[0029] The application of embodiment 2 bacterial agents in the production of α-glycosyl hesperidin

[0030] 1. Activity detection of bacterial agents

[0031] Take 2g of hesperidin in a round-bottomed flask (including a rotor), add 40ml of pre-configured 1mol / L NaOH aqueous solution, shake well to completely dissolve the solid particles of hesperidin, and gradually add 2mol / L HCl solution dropwise to adjust the system 10g of dextrin is added thereto, and the whole system is mixed uniformly by using a magnetic stirring device; the broken bacterial liquid obtained by adding 40ml of high-pressure homogenization (the fermented liquid of Example 1 is directly used for high-pressure homogenization and made), adjust The pH is 6.0; finally add deionized water to make up to 100ml total volume, and catalyze in an oil bath at 75°C for 1h. Sampling was used to analyze the content of substrate hesperidin and product glucosyl hesperidin by high performance liquid chromatography.

[0032] ...

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Abstract

The invention discloses application of cyclodextrin glycosyltransferase in production of alpha-glycosyl hesperidin. An active component of a bacterial agent for producing alpha-glycosyl hesperidin isescherichia coli IEF-cgt02, the cyclodextrin glycosyltransferase with high thermal stability can be efficiently synthesized in cells, and dextrin is adopted as a glycosyl donor to efficiently catalyzea glycosylation reaction of hesperidin. 2-15% of alpha-glycosyl hesperidin is produced by biocatalysis of the bacterial agent, the reaction time is controlled within 24 hours, and the conversion rateof hesperidin reaches 70% or above. The cyclodextrin glycosyltransferase has the advantages of high substrate feeding amount, catalysis rate and substrate conversion rate and is suitable for industrial production of alpha-glycosyl hesperidin.

Description

(1) Technical field [0001] The invention relates to the application of a cyclodextrin glycosyltransferase in the production of α-glycosyl hesperidin. (2) Background technology [0002] Hesperidin (Hesperidin), also known as hesperidin and hesperidin, is a glycoside formed by hesperetin and rutinose. It is a dihydroflavone derivative. It is widely found in legumes, Labiatae, Disciformes, and Rutaceae citrus plants. It has the functions of maintaining normal osmotic pressure of blood vessels, reducing vascular fragility, enhancing capillary toughness, and shortening bleeding time. Hesperidin not only has the effect of lowering cholesterol in the human body, but also has the effect of anti-virus and improving immunity. [0003] Hesperidin has poor water solubility and a bitter taste, which greatly limits its application. Through glycosylation modification, its solubility in water can be increased by more than 50 times, greatly improving its solubility, and improving the appli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/60C12N9/10C12N15/54C12R1/19
CPCC12N9/1074C12P19/60C12Y204/01019
Inventor 陈小龙许敏朱林江陆跃乐范永仙
Owner ZHEJIANG UNIV OF TECH
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