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Alpha-L-rhamnosidase mutant enzyme, gene and expression preparation method

A technology of rhamnosidase and mutant enzymes, which is applied in the field of genetic engineering, can solve the problems of low yield and complex process, and achieve the effect of simple steps, broad application prospects, and improved conversion rate

Active Publication Date: 2021-07-09
JIMEI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To prepare hesperetin 7-O-glucose from the crude extract of citrus flavonoids containing hesperidin or neohesperidin, the raw material needs to be purified into hesperidin or neohesperidin first, and then passed through α-L-mouse Liglusidase hydrolysis, and then further purification into hesperetin 7-O-glucose, requires 2 times of purification and 2 times of concentration and crystallization to prepare hesperetin 7-O-glucose, the process is complicated and the yield is low

Method used

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  • Alpha-L-rhamnosidase mutant enzyme, gene and expression preparation method

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Embodiment 1: Construction of α-L-rhamnosidase mutant enzyme S303R encoding gene expression vector

[0021] Escherichia coli DH5α containing the WT (pPIC9K-r-Rhal) plasmid was inoculated and cultured in 30 mL LB liquid medium containing 1 mg / mL ampicillin resistance at 37° C. for 16 h. Use the small plasmid extraction kit to extract the WT ((pPIC9K-r-Rha1) plasmid according to the instructions. Use the site-directed mutagenesis kit of TOYOBO Biotechnology Co., Ltd. to construct mutants, and the steps are as follows:

[0022] ①Reverse PCR

[0023] Dilute the synthesized primers to 10 μM, adjust the concentration of the template plasmid pPIC9K-r-Rha1 to 50 ng / μL, and the reverse PCR reaction system is as follows: 17.5 μL of sterile water, 2.5 μL of 10× Buffer for iPCR; 2.5 μL μL of 2mMdNTPs; 0.75 μL of primers S303R-F, S303R-R; 0.5 μL of template plasmid; 0.5 μL of KOD-Plus. PCR reaction conditions: pre-denaturation at 94°C for 2 minutes, denaturation at 98°C for 10 sec...

Embodiment 2

[0032] Example 2: Expression and purification of α-L-rhamnosidase WT and mutant enzyme S303R using recombinant expression strains

[0033] Inoculate the strain prepared in Example 1 with 1% inoculum in 50 mL of YPD liquid medium for strain activation, shake culture at 30°C for 16 hours; then inoculate the activated strain into 100 mL of BMGY medium with 1% inoculum , 30°C, 200rpm, after culturing for 16 hours, measure and confirm that its OD600 reaches the range of 3.0-5.0; centrifuge for 10 minutes to collect all the bacteria, discard the supernatant, transfer all the bacteria to 100mL BMMY medium, 30°C, culture for 7 days, During the culture period, 0.5% anhydrous methanol was added to the medium every 24 hours; after the culture was finished, the supernatant was collected by centrifugation to be the enzyme solution.

[0034] The crude enzyme solution of α-L-rhamnosidase WT and mutant enzyme S303R was collected, concentrated by ultrafiltration through a 30kDa membrane, and s...

Embodiment 3

[0035]Example 3 α-L-rhamnosidase WT and S303R substrate specificity research

[0036] Using 0.5mmol / L naringin, hesperidin, rutin naringin, and citrusin as substrates, the substrate-specific hydrolysis rate of purified WT and S303R was determined, and the reaction system was: 1mL 0.5mmol / L Naringin, hesperidin, rutin naringin, citrusin, 980μL of 0.02mol / L citric acid-phosphate buffer (pH 4.0), incubate at 60°C for 10min, then quickly add 20μL of enzyme solution, After reacting for 10 minutes, put it into boiling water at 100° C. and boil for 10 minutes to terminate the reaction. Use a 1mL syringe to take 1mL of the reaction solution, inject it into a 1.5mL liquid phase bottle through a 0.22 μm water phase filter membrane, and finally measure the residual substrate concentration by Agilent 1260 liquid chromatography. The determination of the residual substrate concentration shows that the substrates of different enzymes The blank control of specific conversion rate was inactiv...

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Abstract

The invention discloses alpha-L-rhamnosidase capable of specifically converting hesperidin to generate hesperetin-7-O-glucoside, a gene and an expression preparation method of the alpha-L-rhamnosidase, a coding nucleotide sequence of the alpha-L-rhamnosidase is shown as SEQ ID NO: 1, and an amino acid sequence of the alpha-L-rhamnosidase is shown as SEQ ID NO: 2. The enzyme is prepared by performing site-directed mutagenesis on an alpha-L-rhamnosidase r-Rha1 gene from aspergillus niger to obtain a mutant S303R gene, and expressing the mutant S303R gene in pichia pastoris SMD1168. The enzyme only hydrolyzes hesperidin instead of other citrus flavonoids, wherein the hesperidin in crude citrus flavonoids can be specifically hydrolyzed to generate hesperetin-7-O-glucoside, so that the preparation efficiency of the hesperetin-7-O-glucoside is improved, and an important tool enzyme is provided for industrial preparation of the hesperetin-7-O-glucoside.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a sequence-improved α-L-rhamnosidase mutant enzyme (mutant), a coding gene and an expression preparation method. The α-L-rhamnosidase mutant enzyme can be specifically transformed Hesperidin produces hesperetin-7-O-glucoside. Background technique [0002] Hesperetin-7-O-glucoside, molecular formula C 22 h 24 o 11 , which is the product of removing one molecule of rhamnose from hesperidin. It is a derivative of hesperidin / hesperetin, and the non-sugar part is hesperetin. The water solubility of hesperetin 7-O-glucoside is 50 times that of hesperidin, and its bioavailability is better than that of hesperidin. Hesperetin 7-O-glucoside is a new type of low calorific value after ring-opening hydrogenation The sweetener, the monoglucose of hesperetin dihydrochalcone, is a precursor substance of a new type of sweetener. At present, the preparation methods of hesperetin 7-O-glucosid...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/24C12N15/81C12R1/84
CPCC12N9/2402C12N15/815C12Y302/0104
Inventor 倪辉李利君孙江李文静龚建业李清彪姜泽东
Owner JIMEI UNIV
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