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Application of alpha-L-rhamnosidase derived from bacteria in efficient production of hesperetin-7-O-glucoside

A technology of rhamnosidase and glucoside, applied in the application, glycosylase, enzyme and other directions, can solve the problem of non-specific hydrolysis bond type of rhamnosidase, achieve broad application prospects, simple separation and simple steps Effect

Active Publication Date: 2020-11-17
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] Aiming at the deficiencies of the prior art, especially the deficiencies in the research on highly expressible rhamnosidase derived from bacteria, and the problem that the existing rhamnosidase hydrolysis bond type is not specific, the present invention provides a bacterial The application of source α-L-rhamnosidase in efficient production of hesperetin-7-O-glucoside

Method used

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  • Application of alpha-L-rhamnosidase derived from bacteria in efficient production of hesperetin-7-O-glucoside
  • Application of alpha-L-rhamnosidase derived from bacteria in efficient production of hesperetin-7-O-glucoside
  • Application of alpha-L-rhamnosidase derived from bacteria in efficient production of hesperetin-7-O-glucoside

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: Cloning, expression and purification of α-L-rhamnosidase FjRha gene

[0029] Using the genomic DNA of Flavobacter johnii (CGMCC No.1.8922) from the China General Microorganism Culture Collection Center (http: / / www.cgmcc.net / ) as a template, according to the genome of Flavobacter johnii in the NCBI database The gene sequence of the predicted rhamnosidase (GenBank accession No.ABQ07113.1) was designed with upstream and downstream primers P1 and P2 for PCR amplification. The PCR amplification product was detected and purified by 0.8% agarose gel electrophoresis, and passed The PCR recovery kit is recovered to obtain the gene fragment of Flavobacterium johnsonii α-L-rhamnosidase FjRha;

[0030] Wherein, the sequences of the upstream and downstream primers P1 and P2 are as follows:

[0031] P1: 5'-AATT GCTAGC AATTGTTCCAGAGTTTGTTTT-3', the underline indicates the NheI restriction site,

[0032] P2: 5'-GCCG GAATTC CTAAACCGCTTTTCCATTT-3', the underline indicate...

Embodiment 2

[0041] Example 2: Enzymatic properties of recombinant α-L-rhamnosidase FjRha

[0042] 2.1 Enzyme activity assay

[0043] Take 20 μL of enzyme solution, add 60 μL of 2 mM pNP-Rha solution (pH 6.5, prepared in 50 mM sodium phosphate buffer), react at 37°C for 10 min, add 120 μL of 5 mM sodium carbonate solution to terminate the reaction, OD 405 nm detects absorbance. The measured enzyme activity of the recombinant α-L-rhamnosidase FjRha enzyme solution was 1U / mg enzyme protein.

[0044] Enzyme activity is defined as: 1 μmol of p-nitrophenol is released by hydrolyzing pNP-Rha per minute, which is 1 enzyme activity unit (U).

[0045] 2.2 Determination of specificity of enzyme hydrolysis substrate

[0046] Using pNP-Rha, hesperidin, naringin and rutin as substrates to detect the hydrolysis activity of recombinant α-L-rhamnosidase FjRha on artificial substrate pNP-Rha and natural substrates containing different rhamnosidic bonds .

[0047] The specific reaction of substrate hyd...

Embodiment 3

[0060] Example 3: Recombinant α-L-rhamnosidase FjRha catalyzes hesperidin to generate hesperetin-7-O-glucoside

[0061] In 10mL, 50mM Tris-HCl buffer, hesperidin final concentration 5mM (3g / L), 8U / mL recombinant α-L-rhamnosidase FjRha, pH 7.0, react at 37°C, shake 100rpm for 20h , to get the reaction solution.

[0062] 3.1 TLC analysis of hesperetin 7-O-glucoside

[0063] Take 1 μL of the above reaction solution, spot the sample at the origin of the TLC silica gel plate, develop the layer system (chloroform: ethyl acetate: methanol: water = 3:3:1:0.2, volume ratio) to perform chromatography on the sample on the silica gel , blow dry after chromatography, the color developer is anisaldehyde-sulfuric acid solution, blow dry after dyeing, and heat the alcohol lamp to develop color. TLC analysis results such as image 3 As shown, a product point consistent with the mobility and color reaction of hesperetin 7-O-glucoside appeared in the reaction solution, and it was preliminaril...

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Abstract

The invention relates to application of alpha-L-rhamnosidase derived from bacteria in efficient production of hesperetin-7-O-glucoside. The nucleotide sequence of the alpha-L-rhamnosidase FjRha is shown as SEQ ID NO.1, and the gene is obtained by cloning from a genome of flavobacterium johnsonii. The alpha-L-rhamnosidase FjRha can specifically hydrolyze an artificial substrate and a natural substrate containing alpha -1, 6 rhamnoside bonds, and can efficiently and specifically catalyze hesperidin to prepare hesperetin-7-O-glucoside, and the conversion rate of catalyzing 3g / L hesperidin to generate the hesperetin-7-O-glucoside under the conditions that the enzyme dosage is 8U / mL, the pH value is 7.0 and the temperature is 37 DEG C is up to 95% or above. The method has the advantages of simple steps, low cost, mild conditions, environmental friendliness and the like, and has a wide application prospect.

Description

technical field [0001] The invention relates to the application of α-L-rhamnosidase derived from bacteria in the high-efficiency production of hesperetin-7-O-glucoside, which belongs to the technical field of sugar engineering. Background technique [0002] α-L-rhamnosidase (α-L-rhamnosidase, EC 3.2.1.40), can specifically hydrolyze the terminal α-L-rhamnose of glycosides, glycolipids and other natural products, widely distributed in nature, microorganisms is the main source of the enzyme. α-L-rhamnosidase can effectively hydrolyze the terminal rhamnosidic bonds of many natural glycosides, such as ginsenoside, naringin, hesperidin and rutin, and can be used as a biocatalyst to debitter fruit juice , wine aroma, or biotransformation of rutin and other flavonoids to produce products with higher application value. It is an important glycoside hydrolase in the fields of food and medicine. [0003] The research on the catalytic application of α-L-rhamnosidase mainly focuses on ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/60C12N9/24C12N15/56C12N15/70
CPCC12N9/2402C12N15/70C12P19/60C12Y302/0104
Inventor 肖敏季璇徐莉张婷婷
Owner SHANDONG UNIV
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