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

Aldoxime dehydratase and application thereof in catalytic synthesis of aromatic nitrile compounds

A technology of dehydratase and compound, applied in the field of biocatalysis research

Active Publication Date: 2021-09-14
HANGZHOU NORMAL UNIVERSITY
View PDF3 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Domestic research on aldoxime dehydratase is rarely reported

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
  • Aldoxime dehydratase and application thereof in catalytic synthesis of aromatic nitrile compounds
  • Aldoxime dehydratase and application thereof in catalytic synthesis of aromatic nitrile compounds
  • Aldoxime dehydratase and application thereof in catalytic synthesis of aromatic nitrile compounds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Construction of recombinant expression plasmid pROxdF1

[0036]The aldoxime dehydratase Oxd gene ( figure 1 ), the nucleotide sequence of the gene is shown in SEQ ID NO.1. The nucleotide sequences of primers Oxd_UP and Oxd_down are respectively:

[0037] 5'-CGCGGATCCGATGGAATCTGCAATCGATAAACA-3';

[0038] 5'-ATAAGAATGCGGCCGCCTAGTTGGAACTGACTTTTGC-3'.

[0039] The Oxd-F1 gene was double-digested with Bam HI and Not I, and the digested gene fragment was recovered; at the same time, the expression plasmid pRSFDuet-1 was double-digested with Bam HI and Not I, and the plasmid fragment after digestion was recovered. T4 ligase was used for ligation, and the ligated product was transformed into the cloning host E.coli DH5α. Use LB solid plates containing kanamycin resistance to select positive transformants, and the sequencing results show that the recombinant plasmid after the gene sequence is correct is the recombinant expression plasmid pROxdF1, which is stored at...

Embodiment 2

[0040] Construction and catalyst preparation of embodiment 2 genetically engineered bacteria BL21-pROxdF1

[0041] The recombinant expression plasmid pROxdF1 constructed in Example 1 was transformed into the expression host E. coli BL21(DE3) to obtain the genetically engineered bacterium BL21-pROxdF1. BL21-pROxdF1 was inoculated into liquid LB medium (38 μg / mL kanamycin) in a 10 mL test tube, and cultured with shaking at 37° C. and 200 rpm for 12 to 16 hours. The culture solution was inoculated into 50 mL of liquid auto-induction medium (38 μg / mL kanamycin) in a 250 mL conical flask at an inoculum amount of 1-5%, and cultured with shaking at 37°C and 200 rpm for 2-3 hours. When the strain density OD 600 When the value reaches 0.6-0.8, adjust the culture temperature to 18°C, and continue to culture at 200rpm for 12h.

[0042] The cells are collected by centrifugation for 5-10 minutes under the condition of 5000-10000×g, which is used as a catalyst for dehydrating and synthesi...

Embodiment 3

[0043] Example 3 Characterization of Aldoxime Dehydratase OxdF1

[0044] With 5mM 3-pyridine aldoxime as the substrate, in 0.5mL of 0.1M potassium phosphate buffer (pH 6.0) reaction system, add 50μL of genetically engineered bacteria BL21-pROxdF1 resting cells, and measure its catalytic activity in the range of 20~55℃ vitality, such as image 3 As shown, the optimum reaction temperature is 35 °C.

[0045] With 5mM 3-pyridine aldoxime as the substrate, add 50 μL of genetically engineered bacteria BL21-pROxdF1 resting cells in buffer systems with different pH values ​​(pH4.0-9.5), and measure its catalytic activity at 35 °C. like Figure 4 As shown, the optimum reaction pH is 7.0.

[0046] Place the resting cells of the genetically engineered bacteria BL21-pROxdF1 in water baths at 30 and 35°C respectively, and measure their catalytic activity after keeping them for different times, as shown in Figure 5 As shown, its half-life reaches about 3.8 hours at 30°C.

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 aldoxime dehydratase and application thereof in catalytic synthesis of aromatic nitrile compounds, and belongs to the field of biological catalysis researches. The preparation method comprises the following steps: inserting an aldoxime dehydratase gene from Pseudomonas putida F1 into an expression vector, and constructing a recombinant expression plasmid; and introducing the aldoxime dehydratase into a host cell to construct a genetically engineered bacterium so as to realize efficient co-expression of the aldoxime dehydratase. Through the catalytic action of the engineering bacteria, the aim of efficiently producing the aromatic nitrile from the aldoxime compound can be achieved. The aldoxime dehydratase has the advantages of good catalytic stability, high catalytic activity and the like, is expected to become an excellent industrial catalyst for preparing aromatic nitrile by a biological method, and promotes upgrading of a nitrile compound synthesis process.

Description

technical field [0001] The invention relates to the field of biocatalysis research, in particular to an aldoxime dehydratase genetically engineered bacterium and a method for using the bacterium to catalyze and synthesize aromatic nitrile compounds in water phase without cyanide source. Background technique [0002] Organic nitriles (R-C≡N) are an important class of chemical raw materials and intermediates, and are widely used in pharmaceutical synthesis and polymer polymerization. For example, adiponitrile is the raw material for the production of nylon 66, acrylonitrile is the raw material for the production of polyacrylamide and nitrile rubber, and 3-cyanopyridine is the intermediate for the synthesis of nicotinamide. With the in-depth study of medicinal chemistry, the introduction of cyano groups into small molecule drugs is one of the important strategies for the modification of medicinal chemical structures. According to statistics, the DrugBank database contains more...

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): C12N9/88C12N15/60C12N15/70C12N1/21C12P13/00C12R1/19
CPCC12N9/88C12N15/70C12Y499/01C12P13/00
Inventor 裴晓林陈芝吉郑浩腾肖勤洁毛斐滢
Owner HANGZHOU NORMAL UNIVERSITY
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