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

Method for preparing immobilized D-xylonic acid dehydratase

A technology of xylonic acid dehydratase and ferric tetroxide, applied in immobilized enzymes, biochemical equipment and methods, immobilized on or in inorganic carriers, etc., can solve problems such as inactivation, and achieve efficient encapsulation, high The effect of enzymatic catalytic activity, convenient magnetic recovery performance

Inactive Publication Date: 2020-12-11
南京凯诺生物科技有限公司
View PDF1 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, magnetic nanomaterials modified with viscous materials such as polydopamine can achieve better adsorption and immobilization of enzymes. However, this immobilized form of enzyme protein is still exposed to external media and is easily inactivated by environmental changes.

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The construction of embodiment 1 recombinant plasmid

[0025] The primers at the 5' end and 3 end of the D-xylonate dehydratase xylD gene introduce restriction sites BamHI and XhoI, perform double enzyme digestion on the xylD gene and PRSFDuet-1, and then connect the xylD gene to the PRSFDuet-1 vector (Refer to Example 1 in "CN201711190972.1 A method for synthesizing 1,2,4-butanetriol by enzymatic reaction").

Embodiment 2

[0026] The construction of embodiment 2 recombinant strains

[0027] The recombinant plasmid constructed in Example 1 was transformed into Escherichia coli BL21 (DE3) competent cells, and spread on the LB solid medium of kanamycin (Kana) with a final concentration of 50 mg / L, and cultured at 37°C Afterwards, colony PCR verification was performed. The correct bacterial strain verified by colony PCR is then sequenced and verified, and its nucleotide sequence is shown in SEQ ID NO.2 in a method for synthesizing 1,2,4-butanetriol in CN201711190972.1, which will eventually be correct The strains were stored in a glycerol with a final concentration of 25vt% in a -80°C ultra-low temperature freezer.

Embodiment 3

[0028] Embodiment 3 culture method

[0029] 3.1 Plate culture

[0030] Take out the glycerol bacteria stored in the -80°C refrigerator, streak on a plate containing 50 mg / L Kana, and then culture the plate in a 37°C incubator for 12 to 14 hours.

[0031] 3.2 Seed liquid culture

[0032] Pick a single colony on the plate and inoculate it into 10 mL of culture medium containing 50 mg / L Kana, and culture on a shaker at 200 rpm at 37°C for 8-10 hours.

[0033] 3.3 Shake flask fermentation culture

[0034] The seed solution was inoculated into 100 mL of LB liquid medium containing 50 mg / L Kana, the inoculum amount was 1 vt%, 200 rpm, and cultured at 37°C. When the OD600 is 0.6-0.8, add IPTG with a final concentration of 1 mmol / L for induction, cool down to 30°C and incubate for 12 hours, then collect the bacteria by centrifugation.

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

PropertyMeasurementUnit
particle diameteraaaaaaaaaa
Login to View More

Abstract

The invention discloses a method for preparing immobilized D-xylonic acid dehydratase. The method comprises the following steps of: (1) mixing the buffer solution of D-xylonic acid dehydratase with ferroferric oxide magnetic nanoparticles modified by polydopamine to obtain mixed liquid; and (2) adding dopamine into the mixed liquid, standing, and carrying out centrifugation, wherein an obtained precipitate is the immobilized D-xylonic acid dehydratase. By use of the method disclosed by the invention, a magnetic nanometer material of a nuclear shell structure is used for efficiently packaging the D-xylonic acid dehydratase, and an obtained enzyme catalyst has convenient magnet recovery performance, long-acting catalysis service life, good enzyme activity stability and higher enzyme catalysis activity. A whole preparation process does not need expensive equipment and complex preparation technologies, a nano-enzyme preparation synthesis step is simplified, and the activity of the enzyme in the preparation process can be guaranteed to a maximum extent.

Description

technical field [0001] The invention relates to the field of immobilized enzymes, in particular to a method for preparing immobilized D-xylonate dehydratase. Background technique [0002] D-xylonic acid dehydratase is involved in many important metabolic pathways in microbial organisms and can catalyze the dehydration reaction of xylonic acid. The synthesis of many high value-added chemicals by microorganisms, such as ethylene glycol, triols, and tetraols, requires the participation of D-xylonate dehydratase. However, in the actual production process, xylonate dehydratase faces the disadvantages of long-time reaction and unsuitable catalytic environment, which leads to the decrease of enzyme reaction rate and enzyme inactivation. Therefore, the use of immobilized nanoparticles to achieve embedding and encapsulation of D-xylonate dehydratase can meet the needs of D-xylonate dehydratase in the actual production process, thereby improving the reaction rate and stability of xyl...

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
Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N11/089C12N11/14
CPCC12N9/88C12N11/14C12Y402/01082C12N11/089
Inventor 陈可泉许晟王昕
Owner 南京凯诺生物科技有限公司
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