Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Recombinant escherichia coli expressing alpha-L-rhamnosidase and application of recombinant escherichia coli

A technology for recombining Escherichia coli and rhamnosidase is applied in the fields of genetic engineering and biomedicine, and can solve the problems of restricting industrial production and utilization, long fermentation time, difficulty in separation and purification, etc.

Active Publication Date: 2019-07-30
陕西量维生物工程有限公司
View PDF0 Cites 15 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The defects of too long fermentation time and difficult separation and purification limit the industrial production and utilization of Aspergillus fermentation method to produce α-L-rhamnosidase

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Recombinant escherichia coli expressing alpha-L-rhamnosidase and application of recombinant escherichia coli
  • Recombinant escherichia coli expressing alpha-L-rhamnosidase and application of recombinant escherichia coli
  • Recombinant escherichia coli expressing alpha-L-rhamnosidase and application of recombinant escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 AnRhaE gene synthesis and recombinant expression of α-L rhamnosidase Escherichia coli construction

[0036] According to the codon optimization of the rhamnoside hydrolase AnRhaE gene of Aspergillus nidulans in the NCBI database, the gene synthesis was performed by Jinweizhi Company, and the final gene sequence is shown in SEQ ID NO.2.

[0037]The primer pair designed to amplify the AnRhaE sequence was F: TTA AGA AGG AGA TAT ACC ATG GCA ATGTCT TTG TCT ATA TCT GGT GT, R: GTG CGG CCG CAA GCT TGT CGACAC CTAAAG TTG ATTCGA ATC TAT. The synthetic sequence was used as a template, and the primer pair was used for PCR amplification, and the Primer Star MasterMix (Takara company) high-fidelity pfu enzyme was selected for pre-denaturation at 95°C for 3 minutes; the amplification stage was 30 cycles, followed by 95°C for 10s , 53°C, 5s, 72°C, 2min; extension 72°C, 5min. The PCR product was purified, and the vector pET28a was subjected to Nco I and Sal I double enzyme dig...

Embodiment 2

[0038] Example 2 Inducing recombinant Escherichia coli to ferment and produce α-L rhamnosidase

[0039] Streak the constructed recombinant Escherichia coli BL21(DE3) / pET28a-AnRhaE (using the strain BL21(DE3) / pET28a transformed with an empty vector as a control) on an LB plate containing a kanamycin concentration of 50 μg / mL, 37 Cultivate at ℃ for 12h. Pick a single colony and transfer it into 5 mL of LB liquid medium containing kanamycin at a concentration of 50 μg / mL, and shake at 220 rpm for 12 hours at 37°C. Transfer to 100 mL of TB liquid medium containing 50 μg / mL of kanamycin according to the inoculum size of 1%, and shake at 220 rpm at 37°C until OD 600 The value is 0.8. Isopropylthiogalactopyranoside (IPTG) was added to the culture to a final concentration of 0.5 μmol / L, and the shaking culture was continued at 25° C. and 220 rpm for 16 h.

[0040] At the end of the culture, draw 1 mL of the culture to measure the final OD 600 The cells were collected by centrifuga...

Embodiment 3

[0041] Example 3 Recombinant AnRhaE Hydrolyzes Epimedin C Rhamnoside-α-1,2-Rhamnoside Bond

[0042] The recombinant α-L-rhamnosidase enzyme liquid was prepared, and the enzyme activity was measured by pNPR. Pipette 40 μL of recombinant α-L-rhamnosidase enzyme solution into a 1.5 mL centrifuge tube, add 10 μL of Epimedin C solution (concentration 1 g / L, dissolved in absolute ethanol), and add 150 μL of deionized water. The catalytic reaction was carried out at 37°C, and 200 μL of absolute ethanol was added to the centrifuge tube every 60 minutes to terminate the reaction. The reaction solution was centrifuged at 12000 × g for 10 min, filtered through a 0.22 μm organic filter membrane, and analyzed by HPLC (see figure 2 ). Recombinant α-L-rhamnosidase has the activity of hydrolyzing the 3-position rhamnoside-α-1,2-rhamnoside bond of Epimedin C. When the added activity is 460U / L, it can completely catalyze the hydrolysis of 1g / L in 90min Epimedin C produces icariin (see ima...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a recombinant escherichia coli expressing alpha-L-rhamnosidase and application of the recombinant escherichia coli, and belongs to the field of genetic engineering technology and biological medicine. A sequence with the coding sequence shown in SEQ ID NO.2 is subjected to heterologous expression in escherichia coli BL21(DE3), induction is carried out with 0.5 micromole / L IPTG, and recombinant rhamnoside hydrolase with vitality of 5.6 U / L is obtained. The recombinase has the specific activity of hydrolyzed rutin, hesperidin, naringin, neohesperidin and epimedin C rhamnoside. When the additive amount of the recombinase is 460 U / L, complete catalytic hydrolysis is carried out on 1 g / L epimedin C within 90 minutes to generate unique product icariin.

Description

technical field [0001] The invention relates to a recombinant Escherichia coli expressing α-L-rhamnosidase and its application, belonging to the technical fields of genetic engineering and biomedicine. Background technique [0002] α-L-rhamnosidase (EC3.2.1.40) is a hydrolase that can specifically hydrolyze rhamnosidic bonds and is widely distributed in nature. The enzyme can specifically and efficiently hydrolyze a variety of glycosides with α-rhamnosidic bonds, such as naringin, rutin, hesperidin and epimedin C. C) etc., releasing rhamnose. α-L-rhamnosidase is mainly derived from microorganisms, and there are many kinds of α-L in bacteria (Bacillus, Lactobacillus, Monas, etc.) and fungi (Aspergillus, Trichoderma, Penicillium, etc.) -Rhamnosidase has been reported. [0003] α-L-rhamnosidase is widely used in food, medicine and other fields. In the food industry, α-L-rhamnosidase is the main active ingredient of naringinase and hesperidinase, which can effectively remove...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N1/21C12N9/24C12P19/60C12R1/19
CPCC12N9/2402C12P19/60C12Y302/0104
Inventor 周景文陈坚吕云斌曾伟主堵国成
Owner 陕西量维生物工程有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com