Method utilizing oxygenase to oxidize amidogen

Abstract

The invention discloses a method utilizing oxygenase to oxidize amidogen, which utilizes three kinds of nitrogen oxygenases with different sources to achieve biological oxidation of amidogens arranged on aromatic nucleus by adopting aromatic nucleus materials with amidogens as substrates, wherein high yield recombinant strains of three kinds of nitrogen oxygenases are obtained by utilizing genetic engineering technology firstly, then biological enzyme crude products are collected to be directly used for a catalytic reaction, cofactors are not added into a reaction system, and the reaction system adopts the aromatic nucleus materials with the amidogens as the substrates, and effectively catalyzes the amidogens arranged on oxidized aromatic nucleuses. Compared with an existing chemical method for achieving amidogen oxidation, the method utilizing oxygenase to oxidize the amidogen is simple in process, low in cost and safe and controllable in process, and has important application value and excellent economy.

Description

technical field

[0001] The invention belongs to the technical field of biocatalytic conversion, and relates to a method for oxidizing an amino group by using an oxygenase, in particular to a method for oxidizing an aromatic amino group by using an arylamine compound as a substrate and nitrogen oxygenase as a biocatalyst. Process method for highly efficient catalysis of aromatic nitro groups. Background technique

[0002] Under normal circumstances, nitro compounds are considered as a typical environmental pollutant, which will cause soil and groundwater pollution during the synthesis and use. At the same time, nitro compounds have also been found to be related to some DNA damage and cancer, posing a hazard to human health. Therefore, most of the current researches are aimed at the degradation of nitro compounds and the reduction of nitro groups in them.

[0003] However, at the same time, aromatic nitro compounds are also important industrial chemicals that exist relativel...

Examples

Embodiment 1

[0025] Embodiment 1: the construction of the escherichia coli engineering bacterium expressing nitrogen oxygenase gene

[0026] The three sources were respectively Streptomyces thioluteus Nitrogenase gene aur F , derived from Streptomyces venezuelae Nitrogenase gene cmI and from Pseudomonas fluorescens Nitrogenase gene prnD Add NdeI and XohI restriction sites at both ends, and insert the gene fragments into the corresponding restriction sites of the expression vector pET22b by double restriction digestion and ligation, and put them under the control of the T7 promoter to construct the expression of AurF, CmlI, PrnD recombinant plasmid pET22b-AurF, pET22b-CmlI, pET22b-PrnD. Transform pET22b-AurF, pET22b-CmlI, and pET22b-PrnD into E.coliBL21(DE3), respectively, to obtain engineering bacteria E.coliBL21(DE3) / pET22b-AurF,E.coliBL21(DE3) / pET22b-AurF,E. coliBL21(DE3) / pET22b-CmlI, E. coliBL21(DE3) / pET22b-PrnD.

Embodiment 2

[0027] Embodiment 2: Escherichia coli engineering bacteria ferment and produce enzyme

[0028] Separately pick engineered bacteria E. coli BL21(DE3) / pET22b-AurF, E. coli BL21(DE3) / pET22b-CmlI, E. coli BL21(DE3) / pET22b-PrnD was added to LB liquid medium containing 100 μg / mL ampicillin, cultured overnight at 37°C, 200 rpm, and then inoculated into LB liquid medium (containing 100 μg / mL mL ampicillin), cultured at 37°C, 200rpm until OD600 was 0.6-0.8 (about 3h), added the inducer IPTG, induced expression for 6h, centrifuged at 8000rpm, 4°C for 5min, discarded the supernatant, and precipitated for later use.

Embodiment 3

[0029] Example 3: Preparation of Escherichia coli Crude Enzyme Solution Expressing Nitrogen Oxygenase

[0030] Take the bacterium precipitate in Example 2, wash twice with potassium phosphate buffer (100mmol / L, pH7.2), the precipitate after washing is suspended in potassium phosphate buffer, sonicate the cells (130W, ultrasonic 2s, intermittent 2s, a total of 10min), the broken liquid at 8000rpm, 4 ℃ Centrifuge for 20 minutes, and take the supernatant as the crude enzyme solution.

technical field

[0001] The invention belongs to the technical field of biocatalytic conversion, and relates to a method for oxidizing an amino group by using an oxygenase, in particular to a method for oxidizing an aromatic amino group by using an arylamine compound as a substrate and nitrogen oxygenase as a biocatalyst. Process method for highly efficient catalysis of aromatic nitro groups. Background technique

[0002] Under normal circumstances, nitro compounds are considered as a typical environmental pollutant, which will cause soil and groundwater pollution during the synthesis and use. At the same time, nitro compounds have also been found to be related to some DNA damage and cancer, posing a hazard to human health. Therefore, most of the current researches are aimed at the degradation of nitro compounds and the reduction of nitro groups in them.

[0003] However, at the same time, aromatic nitro compounds are also important industrial chemicals that exist relativel...

Examples

Embodiment 1

[0025] Embodiment 1: the construction of the escherichia coli engineering bacterium expressing nitrogen oxygenase gene

[0026] The three sources were respectively Streptomyces thioluteus Nitrogenase gene aur F , derived from Streptomyces venezuelae Nitrogenase gene cmI and from Pseudomonas fluorescens Nitrogenase gene prnD Add NdeI and XohI restriction sites at both ends, and insert the gene fragments into the corresponding restriction sites of the expression vector pET22b by double restriction digestion and ligation, and put them under the control of the T7 promoter to construct the expression of AurF, CmlI, PrnD recombinant plasmid pET22b-AurF, pET22b-CmlI, pET22b-PrnD. Transform pET22b-AurF, pET22b-CmlI, and pET22b-PrnD into E.coliBL21(DE3), respectively, to obtain engineering bacteria E.coliBL21(DE3) / pET22b-AurF,E.coliBL21(DE3) / pET22b-AurF,E. coliBL21(DE3) / pET22b-CmlI, E. coliBL21(DE3) / pET22b-PrnD.

Embodiment 2

[0027] Embodiment 2: Escherichia coli engineering bacteria ferment and produce enzyme

[0028] Separately pick engineered bacteria E. coli BL21(DE3) / pET22b-AurF, E. coli BL21(DE3) / pET22b-CmlI, E. coli BL21(DE3) / pET22b-PrnD was added to LB liquid medium containing 100 μg / mL ampicillin, cultured overnight at 37°C, 200 rpm, and then inoculated into LB liquid medium (containing 100 μg / mL mL ampicillin), cultured at 37°C, 200rpm until OD600 was 0.6-0.8 (about 3h), added the inducer IPTG, induced expression for 6h, centrifuged at 8000rpm, 4°C for 5min, discarded the supernatant, and precipitated for later use.

Embodiment 3

[0029] Example 3: Preparation of Escherichia coli Crude Enzyme Solution Expressing Nitrogen Oxygenase

[0030] Take the bacterium precipitate in Example 2, wash twice with potassium phosphate buffer (100mmol / L, pH7.2), the precipitate after washing is suspended in potassium phosphate buffer, sonicate the cells (130W, ultrasonic 2s, intermittent 2s, a total of 10min), the broken liquid at 8000rpm, 4 ℃ Centrifuge for 20 minutes, and take the supernatant as the crude enzyme solution.

Images