Method utilizing oxygenase to oxidize amidogen

An amino oxidation and oxygenase technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, immobilized on or in inorganic carriers, can solve the problems of difficult control of the synthesis process, high risk factor, and high cost. Achieve the effects of safe and green reaction, simple production process and mild reaction process

Inactive Publication Date: 2015-12-02
NANJING UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, most industrial applications use chemical methods to synthesize nitro compounds, which require a mixed acid environment of sulfuric acid and nitric acid. The synthesis process is difficult to control, the cost is high, the pollution is serious, and the risk factor is high.
Compared with the

Method used

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  • Method utilizing oxygenase to oxidize amidogen

Examples

Experimental program
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Effect test

Example Embodiment

[0025] Example 1: Construction of E. coli engineered bacteria expressing nitrogen oxygenase gene

[0026] The 3 types are derived from Streptomycesthioluteus Nitrogen oxygenase gene aurF From Streptomycesvenezuelae Nitrogen oxygenase gene cmlI And from Pseudomonasfluorescens Nitrogen oxygenase gene prnD NdeI and XohI restriction sites are added at both ends, and the gene fragments are inserted into the corresponding restriction sites of expression vector pET22b through double restriction digestion and ligation, and placed 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. coli BL21 (DE3), respectively, to obtain engineered bacteria E. coli BL21 (DE3) / pET22b-AurF, E. coli BL21 (DE3) / pET22b-AurF, E. coliBL21(DE3) / pET22b-CmlI, E.coliBL21(DE3) / pET22b-PrnD.

Example Embodiment

[0027] Example 2: Fermentation of E. coli engineered bacteria to produce enzyme

[0028] Pick engineering bacteria separately E.coli BL21(DE3) / pET22b-AurF, E.coli BL21(DE3) / pET22b-CmlI, E.coli BL21(DE3) / pET22b-PrnD to LB liquid medium containing 100μg / mL ampicillin. After incubating overnight at 37℃ and 200rpm, inoculate 1% (v / v) inoculum into LB liquid medium (containing 100μg / mL). mL ampicillin), culture at 37°C and 200rpm until the OD600 is 0.6~0.8 (about 3h), add the inducer IPTG, and induce expression for 6 hours. Centrifuge the bacterial solution at 8000rpm and 4°C for 5min, discard the supernatant, and precipitate for later use.

Example Embodiment

[0029] Example 3: Preparation of E. coli crude enzyme solution expressing nitrogen oxygenase

[0030] Take the bacterial pellet in Example 2 and wash it twice with potassium phosphate buffer (100mmol / L, pH7.2). The washed pellet is suspended in potassium phosphate buffer, and the cells are treated with ultrasound (130W, ultrasound for 2s, intermittent 2s, 10min in total), the broken liquid is at 8000rpm, 4 ℃ Centrifuge for 20 minutes and take the supernatant to obtain the crude enzyme solution.

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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...

Claims

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Application Information

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IPC IPC(8): C12P13/00C12N11/14C12R1/19
Inventor 陈可泉王璟应晗笑张阿磊马金莲曹逊欧阳平凯
Owner NANJING UNIV OF TECH
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