Fermentation preparation method of lysine
A technology of lysine and fermentation methods, applied in the directions of fermentation, enzymes, microorganism-based methods, etc., can solve problems such as no enlightenment mutation, and achieve the effects of stable quality, improved fermentation yield, and no potential safety hazards.
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Embodiment 1 2
[0035] Embodiment 1 Preparation of dihydrodipicolinate synthase variant gene
[0036]According to the sequence we designed, we commissioned Shanghai Sangon Biotechnology Co., Ltd. to synthesize the variant gene encoding dihydrodipicolinate synthase through commercial channels and constructed it into the Escherichia coli-Corynebacterium shuttle plasmid pMS2 (available from the American Type Microorganisms Collection (ATCC), item number ATCC 67189). The cloning process was carried out according to the "Molecular Cloning Experiment Guide" and the operation guide of the commercial reagents used. The brief process is as follows:
[0037] Synthesize nucleic acid fragments of dihydrodipicolinate synthase variant genes by an automatic DNA synthesizer, phosphorylate the 5' ends of these nucleic acid fragments with T4 polynucleotide kinase (purchased from TaKaRa Company), and then equimolar ratio The five nucleic acid fragments were mixed and denatured at 65°C for 5 minutes, annealed a...
Embodiment 2
[0039] The fermentation experiment of embodiment 2 coryneform bacteria
[0040] The pMS2-dap plasmid is transferred into L-lysine-fermenting coryneform bacteria engineering bacteria (available from the American Type Microorganism Collection (ATCC), product number ATCC 31269) by electroporation method, and its brief process is: the coryneform bacteria Shake culture in 50 mL LB liquid medium until OD500 reaches 0.7, collect the cells by centrifugation, wash with 0°C pre-cooled 10% (V / V) glycerol solution, and resuspend the cells in 200 μL pre-cooled 10% (V / V) glycerol solution. V) In glycerol solution, add pMS2-dap plasmid, mix well, transfer to 0.1cm electric shock cup, conduct electric shock at 1.8kV for 5ms, then immediately add 1mL liquid LB containing 0.5% (M / M) glucose for culture Incubate at 42°C for 5 minutes, spread on solid LB medium containing 100 μg / mL ampicillin and 35 μg / mL kanamycin, and culture at 30°C for 36 hours. After the total DNA was extracted from the tra...
Embodiment 3
[0042] The preparation of embodiment 3L-lysine feed additive
[0043] The fermentation broth prepared in Example 2 was separated by a Suntar-III ultrafiltration membrane (available from Suntar Membrane Technology Co., Ltd.). Then, spray the filtrate directly into the fluidized bed dryer in the form of mist and keep the tail gas temperature at 80±3°C, and collect the discharged particles. Sieve the discharged particles, send the particles with a particle size greater than or equal to 1.5mm to the crusher for crushing, and then mix the crushed particles with the particles with a particle size smaller than 1.5mm obtained by screening and spray them into the fluidized bed again Keep the tail gas temperature in the dryer at 80±3°C, collect the discharged particles and sieve out the particles with a particle size of less than 1.5mm, which are L-lysine feed additives. After the particles with a particle size greater than 1.5mm are crushed again, they are mixed with the discharged pa...
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