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Method for producing 4,6-diaminoresorcinol and special-purpose engineering bacterium for the same

A technology of diaminoresorcinol, engineering bacteria, applied in bacteria, DNA/RNA fragments, recombinant DNA technology, etc.

Active Publication Date: 2011-07-27
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The chemical synthesis of DAR was initially successfully developed by the American Dow Chemical Company, using trichlorobenzene as the starting material for synthesis (such as Figure 2), the yield is relatively high, which has played a great role in the industrial production of PBO , but there are problems of difficult access to raw materials and high cost
At present, only Japan's production process of DAR synthesis with trichlorobenzene as the starting material has the industrial production capacity of DAR in the world, but there are also limited raw materials, high prices, difficult treatment of waste acid in the nitrification process, and highly toxic substances are easily produced in the treatment of three wastes, etc. Problem, currently many large companies in Japan are looking for industrialization methods to replace their current process routes
Some domestic scientific research institutes use the synthetic route of resorcinol to synthesize DAR (such as As shown in Figure 3), but the yield is low, the cost is high, and there are also a lot of waste liquid treatment problems in the nitrification process

Method used

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  • Method for producing 4,6-diaminoresorcinol and special-purpose engineering bacterium for the same
  • Method for producing 4,6-diaminoresorcinol and special-purpose engineering bacterium for the same
  • Method for producing 4,6-diaminoresorcinol and special-purpose engineering bacterium for the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, the preparation of the engineering bacterium that contains azanol dismutase

[0038] 1. Construction of Engineering Bacteria Containing Hydroxanil Dismutase Gene

[0039] Using Genomic DNA of Comamonas testosteroni CNB-1 CGMCC No.1028 as a template, a pair of primers were designed to forward HabPf: 5'-GTCC AAGGAGA CCCCTTC C-3' (sequence 1 in the sequence listing) (bold is the EcoRI restriction site; italic is the start codon; the underlined sequence is the ribosome binding site) and reverse primer HabPr: 5'-GTCA TGCGGGAA GTC TGGT-3' (bold is the HindIII restriction site; italic is the stop codon), PCR amplified axanil dismutase gene.

[0040] Amplification program: 94°C 4min, 94°C 1min, 48°C 1min, 72°C 1min, 30 cycles, 72°C 1min; PCR reaction system: 94°C 4min, 94°C 1min, 50°C 1min, 72°C 1min, 30 cycles , 72°C for 1min;

[0041] The amplification system is: 2μl template (100ng); 2μl Primer 1 (10μM); 2μl Primer 2 (10μM); 12μl dNTP (2.5mM each); ...

Embodiment 2、4

[0057] Embodiment 2,4, the production and separation and purification of 6-diaminoresorcinol

[0058] 1. Production of 4,6-diaminoresorcinol

[0059] according to Figure 4 Shown reaction process, carry out catalyzed reaction with the immobilized microsphere of the engineering bacterium that contains the axanil dismutase gene of above-mentioned preparation, according to Figure 6The shown process flow connects each operating unit to build a biocatalytic reaction device, which specifically includes: sample injection bottle (2.0L common reagent bottle, containing 1.0g / L 2-amino-4-nitrophenol and 1.39g / L NH4Cl solution) (1), constant flow pump (2) (BT00-300T, Baoding Lange Constant Flow Pump Co., Ltd.), reduction column (3) (1689 Peyer’s gas absorption tube, tube The outer diameter is 20mm, the outer diameter of the branch pipe is 5mm, and the overall height is 205mm, purchased from Dongguan Tangxia Yongxian Electronic Instrument Factory), calcium alginate immobilized cell pac...

Embodiment 3、4

[0069] Embodiment 3,4, the production and separation and purification of 6-diaminoresorcinol

[0070] 1. Production of 4,6-diaminoresorcinol

[0071] according to Figure 4 Shown reaction process, carry out catalytic reaction with the immobilized microsphere of the engineering bacterium that contains the hydroxylaminobenzene dismutase gene that embodiment 1 prepares, according to Figure 6 The shown process flow connects each operation unit to build a biocatalytic reaction device, and the biocatalytic reaction device is the same as that in Example 2. The immobilized cells occupy 60% of the volume of the catalytic reaction column (4) or (5) filled with the calcium alginate immobilized cells.

[0072] 2.0L stock solution (containing 1.0g / L 2-amino-4-nitrophenol and 1.39g / L NH 4 Cl solution) into the sample bottle (1), under N 2 Under the protection of the substrate, the substrate 2-amino-4-nitrophenol solution was continuously flowed through the zinc particles ( In the redu...

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Abstract

A 4,6-diamino-resorcinol and its special engineering bacterium are disclosed. The process is carried out by converting oxy-phenyl-dismutase gene into colibacillus, fermenting engineering bacterium to log phase, inducing expression culture to obtain engineering bacterium containing oxy-phenyl-dismutase, taking 2-amino-4-oxyamido-phenol as substrate, and catalyzing to obtain final product. It's efficient and has short synthetic route, various raw materials, no need of organic solvent and no environmental pollution.

Description

technical field [0001] The present invention relates to a method for producing 4,6-diaminoresorcinol and its special engineering bacteria, in particular to a method for microbial catalyzed production of 4,6-diaminoresorcinol and its special-purpose production of hydroxyaniline Engineering bacteria of dismutase. Background technique [0002] Poly-p-phenylene-benzobisoxazole fiber (Poly-p-phenylene-benzobisoxazole, referred to as PBO), PBO trade name Zylon (Zylon), is a composite material developed by the United States in the 1980s for the development of the aerospace industry. The material is the most promising member of the polyamide family containing heterocyclic aromatics, and is known as the super fiber of the 21st century. Polyparaphenylene benzobisoxazole fiber is now officially on the market, and the use of single fiber and composite materials is being developed. PBO fiber has excellent mechanical properties and high temperature resistance, and it is regarded as a ne...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/52C12P7/22
Inventor 刘双江杨勇姜嘉彤刘志培姜成英
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI