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

Method for rapidly screening high-yield caffeic acid strain with high throughput

A caffeic acid, high-throughput technology, applied in the field of bioengineering, can solve the problems of difficult extraction of caffeic acid, long time, high cost, etc., and achieve the effects of shortening the color reaction time, high conversion efficiency, and reducing the amount of sampling

Pending Publication Date: 2022-02-15
CHINA PHARM UNIV
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, due to the difficulty in extracting caffeic acid from plants and the high cost, it cannot meet the needs of industrial production
In order to meet the market demand of caffeic acid and achieve high-level production, researchers have used metabolic engineering and other means to ferment Escherichia coli to produce caffeic acid. In the process of fermentation caffeic acid, the acid-producing ability of the production strain determines the production cost As a key link, it is very important to obtain high-yield caffeic acid strains. In the prior art, high-performance liquid chromatography is usually used to detect the content of caffeic acid in the fermentation broth one by one to screen high-yield caffeic acid strains. This screening process takes a long time , The processing steps are cumbersome, the cost is high, and it is difficult to achieve high-throughput screening of high-caffeic acid-producing bacteria

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
  • Method for rapidly screening high-yield caffeic acid strain with high throughput
  • Method for rapidly screening high-yield caffeic acid strain with high throughput
  • Method for rapidly screening high-yield caffeic acid strain with high throughput

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: The method for high-throughput rapid screening of high caffeic acid producing bacteria

[0033] Specific steps are as follows:

[0034] Step (1): Inoculate the strain to be screened into a 96-deep-well plate containing P-5052 medium, and culture it on a shaker at 220 rpm at 37°C for 24 hours; in addition, use the P-5052 medium without strain inoculation as a blank control;

[0035] Step (2): Centrifuge the 96 deep-well plate fermentation broth obtained from the strain culture in step (1) at 14,000 rpm for 2 minutes to obtain the fermentation supernatant;

[0036] Step (3): Take 200 μl of the obtained fermentation supernatants of different strains and blank control P-5052 medium respectively, transfer them to 96 microwell plates, and then add 10 μl of trivalent iron ions to each microwell for reaction Solution, scan the OD value at 680nm with a microplate reader, the strain corresponding to the reaction solution with high OD value is the strain with high c...

Embodiment 2

[0037] Example 2: Establishment of a standard curve for the relationship between caffeic acid output and OD value

[0038] Step (1): Weigh an appropriate amount of caffeic acid standard powder, add an appropriate volume of methanol to prepare a 10 mg / ml caffeic acid mother liquor.

[0039] Step (2): Dilute the 10mg / ml caffeic acid mother liquor with ultrapure water to 1mg / ml, 800μg / ml, 600μg / ml, 300μg / ml, 150μg / ml, 100μg / ml, 50μg / ml, 25μg / ml ml of dilute caffeic acid solution with a concentration gradient, use ultrapure water as a blank control, i.e. 0 μg / ml dilute caffeic acid solution, and use dilute caffeic acid solution as a standard solution;

[0040] Step (3): Selection of the maximum absorption wavelength of the reaction product: transfer 200 μl of the above-mentioned standard solution to a 96 microwell plate, then add 20 μl of ferric ion reaction solution to each well, place at room temperature for 1 min, and develop a color reaction later as Figure 1a As shown, scan...

Embodiment 3

[0048] Example 3: Construction of a library of mutants producing caffeic acid

[0049] Step (1): Using the hydroxylase gene shown in SEQ ID NO.1 as a template, design a PCR primer pair for error-prone PCR amplification, recover and purify the target gene fragment by agarose gel electrophoresis;

[0050] Among them, restriction endonucleases Nde I and Not I restriction endonuclease sites are added to the front and rear ends of the target gene, as shown below:

[0051] Nde I: Restriction sequence and protection base: GGGAATTC

[0052] Not I: Restriction sequence and protected base: AAGGAAAAAAA

[0053] Error-prone PCR amplification system (50 μl) includes: PCR primer pair includes upstream primer such as F-4HPA3H 0.2 μl shown in SEQ ID NO.2, downstream primer such as R-4HPA3H shown in SEQ ID NO.3 0.2 μl, 10 mM mn 2+ 0.6μl, dNTP (25mM) 4μl, dTTP (100mM) 4μl, dCTP (100mM) 4μl, template 0.4μl, 10×PCR buffer 5μl, rTaq enzyme 1μl, 25mM MgCl 2 5 μl with ddH 2 O was made up ...

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 method for rapidly screening a high-yield caffeic acid strain with high throughput, and belongs to the technical field of bioengineering. The method comprises the following steps: fermenting a strain to be screened to obtain a fermented liquor; centrifuging the obtained fermented liquor to obtain a fermentation supernatant; placing the fermentation supernatant on a porous plate; adding a ferric chloride reaction solution into the fermentation supernatant for a chromogenic reaction, and detecting the OD value at 680 nm by using a full-wavelength microplate reader; and selecting caffeic acid with high OD value to produce the strain. According to the high-throughput screening method, the screening efficiency of the strains can be greatly improved, the number of the strains screened in a single batch is greatly increased, and the screening experiment period of the caffeic acid high-yield strains is shortened.

Description

technical field [0001] The invention discloses a high-throughput and rapid screening method for high-yield caffeic acid bacteria, and the invention belongs to the technical field of bioengineering. Background technique [0002] Caffeic acid is a natural phenolic acid compound derived from the phenylalanine pathway in plants. Phenylpropionic acid, especially caffeine, has attracted more and more attention due to its important pharmacological effects such as anti-oxidation, anti-inflammation, anti-cancer, anti-virus, anti-diabetes and anti-depression. [0003] However, because caffeic acid is difficult to extract from plants and the cost is high, it cannot meet the needs of industrial production. In order to meet the market demand of caffeic acid and achieve high-level production, researchers have used metabolic engineering and other means to ferment Escherichia coli to produce caffeic acid. In the process of fermentation caffeic acid, the acid-producing ability of the produc...

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): C12N1/02C12N15/70C12N1/21C12P7/42C12R1/19
CPCC12N1/02C12N15/70C12P7/42Y02A50/30
Inventor 刘人铭孙敬方
Owner CHINA PHARM UNIV
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