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.
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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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