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

1,3-propylene glycol oxidoreductase isozyme mutant, and gene and application thereof

A technology of propylene glycol and reductase, applied in 1 field, can solve problems such as difficulty in large-scale industrialization, limited improvement, and achieve the effects of improved activity, high catalytic activity, and broad application prospects

Inactive Publication Date: 2012-07-25
UNIV OF SHANGHAI FOR SCI & TECH
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the previous patent applications CN20071017175812 and CN200710171759.6 of the inventor of this patent, it was also disclosed that on the basis of the 1,3-propanediol oxidoreductase isoenzyme gene yqhD, the catalytic 3-hydroxypropanal activity was improved by mutation directed evolution technology. High variant enzyme, but the degree of improvement is limited, and there are still some difficulties in the large-scale industrialization of biotransformation and synthesis of 1,3-propanediol

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
  • 1,3-propylene glycol oxidoreductase isozyme mutant, and gene and application thereof
  • 1,3-propylene glycol oxidoreductase isozyme mutant, and gene and application thereof
  • 1,3-propylene glycol oxidoreductase isozyme mutant, and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Design of Saturation Mutation Primers and Acquisition of PCR Amplified Products

[0016] Upstream primer 5'-ACACCGTGGAANNNTATGTTACCAAACC-3' (SEQ ID NO: 2)

[0017] Downstream primer 5'-GGTTTGGTAACATANNNTTCCACGGTGT-3' (SEQ ID NO: 3)

[0018] Add 100pmol of dNTPs to a 500μL eppendorf tube, 15pmol of each upstream primer and downstream primer, about 2ng pET28α(+)yqhD (obtained for cloning yqhD into pET28α(+) vector, has been reported in many literatures) as template DNA , 2.5U PfuDNA polymerase, 5 μL of MgCl containing 25mmol / L 2 The Pfu PCR buffer was then added to a total volume of 50 μL with sterile distilled water. The reaction parameters were denaturation at 95°C for 1min, followed by 20 cycles at 95°C for 30s, 52°C for 1min, and 72°C for 1min, and then extended at 72°C for 2min to obtain PCR amplification products.

[0019] Mutation library construction

[0020] The above-mentioned PCR amplified PCR product was digested with DpnI for 2 hours, then transformed int...

Embodiment 2

[0024] Fermentative Production of 1,3-Propanediol by Recombinant Escherichia coli

[0025] a) The positive clone containing the 1,3-propanediol reductase isoenzyme variant gene Gln202His obtained in picking Example 1 was inserted into the seed medium containing 40 μg / ml kanamycin, 180r / min, 37 Cultivate overnight on a shaker for 12 hours. Insert the seed solution into the fermentation medium containing 40 μg / mL kanamycin at an inoculum size of 2% (v / v), culture at 180 r / min, shaker at 37°C until the OD of the bacteria 600 About 0.7-0.9, add IPTG to the final concentration of 0.4mmol / L, and add 25g / L substrate 3-hydroxypropanal, at 25°C, 120r / min fermentation culture for 10 hours.

[0026] b) Collect the fermentation broth, centrifuge at 3000-5000r / min for 15-30 minutes, and collect the supernatant. Add an equal volume of organic solvent chloroform to extract 1,3-propanediol, and then use rotary hair growth to concentrate the extract for gas chromatography analysis .

[0027...

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 1,3-propylene glycol oxidoreductase isozyme mutant, and a gene and application thereof. The 1,3-propylene glycol oxidoreductase isozyme mutant, provided by the invention, has activity for catalyzing 3-hydroxypropionaldehyde to generate 1,3-propylene glycol, and is 1,3-propylene glycol oxidoreductase isozyme containing Q202H mutation. A variant gene is measured by an enzyme kinetics curve; in comparison with the 1,3-propylene glycol oxidoreductase isozyme mutant, the catalysis efficiency is remarkably improved, and the wide application prospect is provided for biological production of 1,3-propylene glycol.

Description

technical field [0001] The invention belongs to the technical field of producing 1,3-propanediol chemical raw materials by genetically engineered bacteria in biochemical industry, and in particular relates to a 1,3-propanediol oxidoreductase isozyme that catalyzes 3-hydroxypropionaldehyde to generate 1,3-propanediol Mutants and their uses. Background technique [0002] 1,3-propanediol is an important solvent and chemical raw material, and it can be used as a monomer to synthesize polycondensates such as polyester and polyurethane with excellent performance. Recently, polytrimethylene terephthalate (PTT) synthesized from 1,3-propanediol and terephthalic acid has very unique properties, so it has been paid attention by many foreign research institutions. At present, the patent of chemical synthesis of 1,3-propanediol is owned by Dutch Shell (Shell) chemical company and German Degussa. The patent for biotransformation and synthesis of 1,3-propanediol was obtained by the U.S. ...

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): C12N9/04C12N15/53C12P7/18
Inventor 李红梅王伟洁邓龙华
Owner UNIV OF SHANGHAI FOR SCI & TECH
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