Method for extracting CEL I nuclease in celery

A nuclease and celery technology, applied in the field of biochemistry, can solve the problems of low total yield, low yield, and recovery rate of only 1.63%, and achieve the effect of improving recovery rate

Inactive Publication Date: 2009-09-23
GUANGZHOU UNIVERSITY
View PDF0 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method requires multiple precipitation extractions, ion exchange chromatography, gel filtration, and repeated ultrahigh-speed centrifugation, resulting in a very low output per unit time
In addition, the total yield of this method is also very low, about 7kg of celery stems containing 350g protein can only be extracted to obtain 0.1 μg/μL of CEL I nuclease 3ml (NucleicAcides Res, 26, 4597-4602, 1998)
[0004] Subsequently, Yang et al. improved the above-mentioned method (PCT/US01/05502), adopted α-methylmannoside to overcome the problem of aggregation reaction bet

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 extracting CEL I nuclease in celery
  • Method for extracting CEL I nuclease in celery
  • Method for extracting CEL I nuclease in celery

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0021] 1. Preparation

[0022] Weigh 100 kg of celery (ie parsley) purchased on the market, remove the leaves, add 8 L of buffer A, pound into a homogenate, and squeeze out 74 L of filtrate. Use 1M Tris aqueous solution to adjust the pH of the filtrate to 8.0 (the amount of Tris solution is 7.5L), then keep it warm at 50°C for 20 minutes and then cool it in ice water; centrifuge at 5000×g for 40 minutes, remove the residue, and take the supernatant Slowly add ammonium sulfate powder (about 144g per liter) to 25% saturation while stirring, centrifuge at 5000×g for 40min, remove the precipitate, collect supernatant, add ammonium sulfate powder to 80% saturation while stirring (approximately 390 g per liter was added), centrifuged at 5000×g for 40 min, removed the supernatant, and retained the precipitate. The pellet was dissolved in 8 L of buffer B and dialyzed against the same Tris buffer for desalting. After desalting, 200 ml of concanavalin (activated and balanced with buff...

example 2

[0030] Weigh 100 kg of celery (ie Chinese celery) purchased on the market, remove the leaves, add 12 L of buffer solution A, pound into a homogenate, and squeeze out 77 L of filtrate. Use 1M Tris aqueous solution to adjust the pH of the filtrate to 8.0 (the amount of Tris solution is 7.3L), then keep it warm at 50°C for 15 minutes and then cool it in ice water; centrifuge at 3000×g for 20 minutes, remove the residue, and take the supernatant Slowly add ammonium sulfate powder (about 144g per liter) to 25% saturation while stirring, centrifuge at 3000×g for 20min, remove the precipitate, collect supernatant, add ammonium sulfate powder to 80% saturation while stirring (Add about 390g per liter), centrifuge at 3000×g for 20min, remove the supernatant, and keep the precipitate. The precipitate was dissolved in 8.2 L of buffer B and dialyzed against buffer B for desalting. After desalting, 200 ml of concanavalin was added to the solution (the used concanavalin had been activated ...

example 3

[0032] Weigh 100 kg of celery purchased in the market, remove the leaves, add 10L of buffer A, mash into a homogenate, and squeeze out the filtrate. Adjust the pH of the filtrate to 8.0 with 1M Tris aqueous solution, then incubate at 50°C for 18 minutes and then cool in ice water; centrifuge at 4000×g for 30 minutes, remove the residue, and take the supernatant; while stirring, slowly add ammonium sulfate powder to 25% saturation, centrifuge at 4000×g for 30min, collect the supernatant, add ammonium sulfate powder to 80% saturation while stirring, centrifuge at 4000×g for 30min, remove the supernatant, and keep the precipitate. The pellet was dissolved in 7 L of buffer B and dialyzed against the same Tris buffer for desalting. After desalting, 200 mL of concanavalin (activated and equilibrated with buffer B) was added to the solution, and stirred overnight at 4°C to fully combine the enzyme with concanavalin. Let stand for 40 minutes, remove the supernatant part by suction fi...

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 relates to a method for extracting CEL I nuclease in celery and discards measures adopting a plurality of high-speed or ultra-speed centrifugation and a plurality of gel filtration and ion exchange chromatography in the prior art. The method comprises the following steps: adding a small amount of sodium sulfite to extract so as to reduce and clear colored substances in the extract; adopting a thermal denaturation physical method to rapidly clear a great amount of foreign protein with poor temperature toleration; utilizing the characteristic that CEL I is the combination of glycoprotein and activated concanavalin to combine CEL I nuclease and concanavalin so as to remove the foreign protein and further purify the CEL I; and finally, selecting DEAE-Sepharose FF as a suitable medium for the CEL I by once chromatography purification. Accordingly, the extraction method replaces the time-consuming and expensive measures adopting a plurality of high-speed even ultra-speed refrigerated centrifugation, a plurality of gel filtration and ion exchange chromatography, and the like and achieves the aims that the CEL I nuclease is rapidly extracted from the celery and purified.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to the preparation of enzymes. Background technique [0002] CEL I nuclease is a single-strand specific nuclease with an isoelectric point of 6.0-6.5 and a molecular weight of 43kDa. Its catalytic activity requires Mg 2+ and Zn 2+ Participation, belonging to one of the S1 endonuclease family. CEL I nuclease is the only enzyme found in the biological world that can accurately cut the mismatched sites in heteroduplex DNA. It plays an important role in genetic research and application fields and has broad application prospects. [0003] At present, the acquisition route of CEL I nuclease is mainly extracted from celery. People such as Oleykowski reported a kind of method extracting CEL I nuclease from celery at first in 1998, and this method method is as follows: first by continuous ammonium sulfate precipitation, then through concanavalin A-agarose column extraction (with [ α]-d+-manno...

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): C12N9/22
Inventor 郭培国李荣华
Owner GUANGZHOU UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap