Cytochrome P450 epoxidase and application thereof

A technology of cyclooxygenase and epoxy, which is applied in the field of bioengineering to achieve the effects of accelerating the industrialization process, improving the activity of epoxidation, and high catalytic specificity

Active Publication Date: 2021-02-26
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to realize the epoxidation reaction, P450 enzymes often need to mutate specific key residues, and the specificity of catalyzing the epoxidation reaction is even more challenging.

Method used

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  • Cytochrome P450 epoxidase and application thereof
  • Cytochrome P450 epoxidase and application thereof
  • Cytochrome P450 epoxidase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: Expression and purification of CytP enzyme

[0032] Construction of genetically engineered bacteria and protein expression:

[0033] With the nucleotide sequence (shown in SEQ ID NO.2) of the target protein coding gene in Pseudomonas putida KT2440 as a template, utilize F1 and R1 as primers (underlines are respectively BamH I and EcoR I restriction enzyme digestion site) PCR amplification, the amplification conditions are:

[0034] 95°C for 5min, 29 cycles (98°C for 10s, 55°C for 15s, 72°C for 1.5min), 72°C for 5min.

[0035] F1: caaatgggtcgcggatccATGGAGATCC (SEQ ID NO. 3);

[0036] R1: tcgacggagctcgaattcTTACCAAATCAC (SEQ ID NO. 4).

[0037] The cDNA sequence of the CytP gene coding region was obtained, and after the PCR product was recovered, it was ligated with the pET-28a(+) plasmid vector that had been cut with the same double enzymes through homologous recombination to obtain the recombinant expression plasmid pET-28a(+)-p28p, and the recombinant p...

Embodiment 2

[0040] Example 2: Verification of CytP enzyme activity

[0041]Spread the strains preserved from the glycerol tube on the LB solid medium, culture at a constant temperature at 37°C until a single clone grows, pick a single clone into a fresh LB liquid medium, and culture at a constant temperature of 200rpm and 37°C After 12 hours, the activation solution was obtained, and the activation solution was inoculated into fresh TB medium at an amount of 1 mL / 100 mL. After culturing for 2 hours, IPTG with a final concentration of 0.2 mM was added, and the culture was induced at 25°C for 14 hours. After the end, the cells were collected.

[0042] Add 0.2 g whole cells expressing CytP protein and 1 g norbornene (C 7 h 10 O, NBE), 320μL 30% hydrogen peroxide, 2.18mL phosphate buffer (100mmol / L KCL, pH7.4) and 7.5mL ethyl acetate, centrifuge at 5000r / min for 10min after reacting at 25°C for 48h, absorb the upper organic phase, It was dried over anhydrous magnesium sulfate, passed throug...

Embodiment 3

[0047] Example 3: Optimum reaction pH of whole cells

[0048] See Example 2 for the specific implementation, the difference is that 0.2g of whole cells are respectively mixed with pH6.0, pH6.5, pH7.0, pH7.5, pH8.0, pH8.5 and pH9.0 phosphate buffer composition Carry out 48h conversion in 10mL two-phase conversion system (organic phase:water phase=3:1), measure the yield of epoxynorbornane according to the above detection method, and calculate the molar yield of epoxidation. The results show that the epoxidation activity of CytP enzyme increases with the increase of pH in pH6.0-pH8.5, and can reach 10.67g / L at pH8.0, and reaches the peak around pH8.5. Epoxynorbornane The yield was 11.17g / L, the molar yield was 9.54%, and then the epoxidation activity decreased with the further increase of pH. This shows that a higher pH (alkaline environment) is more favorable for the CytP enzyme to catalyze the epoxidation reaction, and the whole cell has better epoxidation activity at pH8.0-8...

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Abstract

The invention discloses a cytochrome P450 epoxidase and application thereof, and belongs to the technical field of bioengineering. The cytochrome P450 epoxidase CytP is expressed in escherichia coli,and norbornene is converted into epoxynorbornane by using a whole cell transformation method. A two-phase catalytic system of the CytP enzyme is optimized from the factors such as the pH, temperature,phase ratio and induction time of the reaction, the limitations of low epoxidation activity and poor catalytic specificity of the previous P450 monooxygenase are overcome, the yield of the epoxynorbornane can reach 36.81g / L, the catalytic characteristic reaches 99%, and the molar yield of the epoxidation reaction reaches 31.46%. Therefore, the previous problems of substrate cost, environmental pollution and the like are greatly reduced, and a foundation is laid for industrial production and green production of the epoxynorbornane.

Description

technical field [0001] The invention relates to a cytochrome P450 cyclooxygenase and an application thereof, belonging to the technical field of bioengineering. Background technique [0002] Epoxynorbornane, also known as 3-epoxypropyl[3,2,1,0 2,4 ] Octane, one of the derivatives of norcisane. As a saturated bridged ring compound, its chemical formula is C 7 h 10 O, the melting point is 126°C. It is formed by bridging a methylene group at the 1 and 4 positions of the carbon skeleton of cyclohexane, and at the same time forming an epoxy structure with an oxygen atom at the 2 and 3 positions. [0003] At present, the main production method of epoxy norbornane is chemical synthesis, mainly hydrogen peroxide method, but although this method has a high yield, it consumes a lot of energy, and at the same time, it will have a toxic effect on the environment and is not in line with green production. , production safety and sustainable development requirements. The preparation ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/02C12N15/70C12N9/02C12R1/19
CPCC12P17/02C12N15/70C12N9/0071
Inventor 刘立明燕宇宋伟陈修来刘佳高聪
Owner JIANGNAN UNIV
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