High-temperature resistant flavonoid aglycone invertase and applications thereof

A flavonoid aglycone converting enzyme and high temperature resistant technology, which is applied in the fields of genetic engineering technology and biomedicine, can solve the problems of unsatisfied catalytic conditions, unsatisfactory β-glucosidase catalytic efficiency, etc., and achieves strong catalytic conversion ability and thermal stability. good effect

Active Publication Date: 2015-07-29
NANJING FORESTRY UNIV +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the catalytic efficiency of these β-glucosidases is not ideal enough, and the catalytic conditions cannot meet the needs of some practical applications.
Therefore, we belie

Method used

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  • High-temperature resistant flavonoid aglycone invertase and applications thereof
  • High-temperature resistant flavonoid aglycone invertase and applications thereof
  • High-temperature resistant flavonoid aglycone invertase and applications thereof

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preparation example Construction

[0039] The preparation method of the high-temperature-resistant flavone aglycone converting enzyme described in the present invention, the separation and purification of induced expression specifically includes inducing and cultivating the expression host bacteria containing the recombinant plasmid at a suitable concentration of the inducer (IPTG) at 30°C, collecting the bacteria and ultrasonically crushing them, and taking The fusion protein was obtained by affinity chromatography of the supernatant.

[0040] The present invention also provides a method for preparing flavone aglycone, specifically, the high-temperature-resistant flavone aglycone converting enzyme of the present invention enzymatically hydrolyzes four typical flavone glycosides (isoquercetin, Astragalin, daidzin, genistin) were hydrolyzed to prepare corresponding flavonoid aglycones (quercetin, kaempferol, daidzein and genistin).

[0041] The high-temperature-resistant flavone aglycon converting enzyme SiBGL o...

Embodiment 1

[0043] Example 1: Acquisition of high temperature resistant flavone aglycone converting enzyme gene of the present invention and construction of recombinant plasmid pET-SiBGL

[0044] Codon optimization of high temperature resistant flavone aglycon converting enzyme SiBGL gene and construction of recombinant plasmid pET-SiBGL

[0045] according to known Sulfolobus islandicus The whole genome sequence (accession number: NC_012588.1) was screened to obtain the gene fragment encoding the high-temperature-resistant flavone aglycone converting enzyme SiBGL of the present invention. Due to the codon preference of this extreme thermophile and the expression host Escherichia coli BL21(DE3 ) have a certain difference, which may affect the high-efficiency expression of the extremely heat-resistant flavone aglycone convertase protein, so we optimized the codons of the gene encoding the target protein according to the codon preference of the host bacteria, and optimized the coding The m...

Embodiment 2

[0046] Example 2: Preparation of the high temperature resistant flavonoid aglycon converting enzyme SiBGL of the present invention

[0047] The recombinant plasmid pET-SiBGL was transformed into Escherichia coli JM109(DE3) host bacteria (purchased from Novagen), on LB plates containing kanamycin (50 μg / mL) (LB medium: tryptone 10 g / L, yeast Extract 5 g / L, NaCl 5 g / L, agar 15 g / L) after culturing overnight at 37°C, pick the transformants into 200 mL of LB medium (50 μg / mL kanamycin) at 37°C, Shake culture at 200 rpm until OD600 is 0.6, add a final concentration of 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) inducer, culture at 30°C for 6 h, and use a high-speed refrigerated centrifuge to distill the culture solution Centrifuge at 13,000 rpm for 15 min at 4°C to collect the cells.

[0048] Since the recombinant plasmid pET-SiBGL contains a His-tag tag, it was purified by His·Bind Purification Kit (purchased from Novagen) to obtain a purified recombinant enzyme. Specific ...

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Abstract

The invention discloses a high-temperature resistant flavonoid aglycone invertase and applications thereof. Amino acid sequence of the high-temperature resistant flavonoid aglycone invertase is represented by SEQ ID No.1. Thermal stability of the high-temperature resistant flavonoid aglycone invertase is excellent; the high-temperature resistant flavonoid aglycone invertase is capable of enduring high temperature, and possesses relatively excellent catalytic conversion capability on flavone glycosides of different mother nucleus types and different glucosidic bond types. After a certain time of incubation of the high-temperature resistant flavonoid aglycone invertase with typical flavone glycosides (isoquercetin, astragalin, daidzin, and saprosma ternatum glucoside), it is observed via detection that almost all of the flavone glycosides are converted into corresponding flavonoid aglycones (quercetin, kaempferol, isoflavoues aglycone, and genistein).

Description

technical field [0001] The invention belongs to the field of genetic engineering technology and biomedicine, and specifically relates to a high-temperature-resistant flavone aglycone converting enzyme and its application, in particular to enzymatic conversion of various flavone glycoside compounds to generate corresponding flavone aglycone. Background technique [0002] Flavonoids are pharmacologically active ingredients widely present in plants. Flavonoids with different structural types show different physiological activities and functions due to the differences in the type and bond type of the parent nucleus and the sugar group connected. There are more than 1500 different types of flavonoids isolated and identified. Most flavonoids in nature exist in their glycosylated form. Studies have shown that the glycoside form of flavonoids is much better than its aglycon form in terms of solubility, and has greater advantages in oral administration and absorption. However, it...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/70C12P17/06
Inventor 赵林果解静聪刘思宁
Owner NANJING FORESTRY UNIV
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