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A kind of cytochrome p450 epoxidase and its application

A technology of cyclooxygenase and epoxy, which is applied in the field of bioengineering to achieve the effects of speeding up the industrialization process, high catalytic specificity, and reducing production costs

Active Publication Date: 2022-07-22
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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  • A kind of cytochrome p450 epoxidase and its application
  • A kind of cytochrome p450 epoxidase and its application
  • A kind of cytochrome p450 epoxidase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

[0033] Take the nucleotide sequence (SEQ ID NO. 2) of the target protein-coding gene in Pseudomonas putida KT2440 as the template, and use F1 and R1 as primers (underlined are BamH I and EcoR I restriction enzymes respectively). site) PCR amplification, 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. After the PCR product was recovered, it was connected with the pET-28a(+) plasmid vector that had been digested with the same double enzyme through homologous recombination to obtain the recombinant expression plasmid pET-28a(+)-p28p. The recombinant plasmid pET -28a(+)-p2...

Embodiment 2

[0040] Example 2: CytP enzyme activity verification

[0041]The strains stored in the glycerol tube were spread on LB solid medium, and incubated at a constant temperature of 37°C until a single clone was grown. Pick a single clone into a fresh LB liquid medium, and cultured at a constant temperature of 200rpm and 37°C. After 12 h, the activation solution was obtained, and the activation solution was inoculated into fresh TB medium in an amount of 1 mL / 100 mL. After culturing for 2 h, IPTG with a final concentration of 0.2 mM was added, and the culture was induced at 25 °C for 14 h, and the cells were collected after the end.

[0042] 0.2 g of whole cells expressing CytP protein after induction culture, 1 g of norbornene (C 7 H 10 O, NBE), 320 μL of 30% hydrogen peroxide, 2.18 mL of phosphate buffer (100 mmol / L KCL, pH 7.4) and 7.5 mL of ethyl acetate, centrifuged at 5000 r / min for 10 min after reaction at 25°C for 48 h, and sucked the upper organic phase, It was dried over ...

Embodiment 3

[0047] Example 3: Whole Cell Optimum Response pH

[0048] For specific embodiments, see Example 2, the difference is that 0.2 g of whole cells were prepared in phosphate buffer solutions of pH6.0, pH6.5, pH7.0, pH7.5, pH8.0, pH8.5 and pH9.0, respectively. 10 mL of a two-phase transformation system (organic phase: aqueous phase = 3:1) was transformed for 48 h, the yield of norbornane was measured according to the above detection method, and the molar yield of epoxidation was calculated. The results showed that the epoxidation activity of CytP enzyme increased with the increase of pH in pH 6.0-pH 8.5, reaching 10.67 g / L at pH 8.0, and reaching a peak around pH 8.5. The yield was 11.17 g / L with a molar yield of 9.54%, followed by a decrease in epoxidation activity with further increase in pH. This indicates that higher pH (alkaline environment) is more favorable for CytP to catalyze epoxidation reaction, and whole cells have better epoxidation activity at pH 8.0-8.5.

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Abstract

The invention discloses a cytochrome P450 epoxidase and an application thereof, belonging to the technical field of biological engineering. The present invention provides a cytochrome P450 epoxidase CytP. By expressing the cyclooxygenase CytP in Escherichia coli, and using a whole cell transformation method, norbornene is converted into epoxy norbornane. The two-phase catalytic system of CytP enzyme was optimized from the factors such as pH, temperature, comparison and induction time of the reaction, which overcomes the previous limitations of low epoxidation activity and poor catalytic specificity of P450 monooxygenase, which can make epoxidation possible. The norbornane yield reached 36.81 g / L, the catalytic performance reached 99%, and the epoxidation reaction molar yield reached 31.46%. Thereby, the previous problems of substrate cost and environmental pollution are greatly reduced, and a foundation is laid for the industrial production and green production of epoxy norbornane.

Description

technical field [0001] The invention relates to a cytochrome P450 epoxidase and an application thereof, belonging to the technical field of biological engineering. Background technique [0002] Epoxynorbornane, also known as 3-epoxypropyl[3,2,1,0 2,4 ]Octane, one of the derivatives of nortzane. As a saturated bridged ring compound, its chemical formula is C 7 H 10 O, mp 126°C. It is formed by bridging a methylene group at the 1,4 position of the carbon skeleton of cyclohexane, and forming an epoxy structure with an oxygen atom at the 2,3 position. [0003] At present, the main production method of epoxy norbornane is chemical synthesis method, 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, which is not in line with green production. , safety production and sustainable development requirements. The preparation of epoxy norbornane by biological ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12P17/02C12N15/70C12R1/19
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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