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Preparation of glutamic acid through three stages of fermentation

A glutamic acid and fermenter technology, which is applied in the field of three-stage fermentation preparation of glutamic acid, can solve the problem of no report on polymerase sigma-32 research, and achieve the effects of no safety hazard, stable quality, and increased fermentation yield

Active Publication Date: 2012-08-15
NINGXIA EPPEN BIOTECH
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Problems solved by technology

Although wild-type RNA polymerase sigma-32 has been published (see NCBI (http: / / www.ncbi.nlm.nih.gov) protein and gene accession number AAB18436.1; also see Chinese Patent Application No. 96193336) , but studies on variants of the polymerase sigma-32 have not been reported

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  • Preparation of glutamic acid through three stages of fermentation
  • Preparation of glutamic acid through three stages of fermentation
  • Preparation of glutamic acid through three stages of fermentation

Examples

Experimental program
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Effect test

Embodiment 1

[0028] The preparation of embodiment 1 RNA polymerase sigma-32 factor variant gene construct

[0029] According to the sequence we designed, we commissioned Shanghai Sangon Biotechnology Co., Ltd. to synthesize the gene encoding RNA polymerase sigma-32 factor variant 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:

[0030] Synthesize nucleic acid fragments of the RNA polymerase sigma-32 factor variant gene by an automatic DNA synthesizer, phosphorylate the 5' ends of these nucleic acid fragments with T4 polynucleotide kinase (purchased from TaKaRa), and then mix in an equimolar ratio The five nucleic acid fragments were denatured at 65° C. for 5 m...

Embodiment 2

[0032] The fermentation experiment of embodiment 2 coryneform bacteria

[0033] The pMS2-sigma plasmid is transferred into L-glutamic acid fermenting coryneform bacteria engineering bacteria (available from the American Type Microorganism Collection (ATCC), product number ATCC 13869) by electroporation method, and its brief process is: the coryneform bacteria Shake culture in 50 mL LB liquid medium until OD500 reaches 0.8, 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-sigma plasmid, mix well, transfer to 0.1cm electric shock cup, conduct electric shock at 1.5kV 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 f...

Embodiment 3

[0035] The tertiary fermentation example 1 of embodiment 3 glutamic acid

[0036] First-level preparation: the coryneform bacterium engineered bacterium that the conversion of embodiment 2 has pMS2-sigma plasmid inserts 20 cubic meters of fermentors with 0.5% inoculum (the medium formula wherein is: 650 kilograms of glucose, 150 kilograms of sugarcane molasses , corn syrup 500 kg, K 2 HPO 4 60 kg, MgSO 4 ·7H 2 O 9 kilograms, 12 grams of biotin, and 9 grams of vitamin B1, the volume was adjusted to 18 cubic meters with water), and cultured at 35° C. with saturated aeration for 8 hours to increase the cell density.

[0037] Second-level preparation: inject the nutrient solution of 20 cubic meters of fermentation tanks into 50 cubic meters of fermentation tanks (wherein the culture medium formula is the same as above) with 5% inoculum, cultivate 10 hours in 35 ℃ of saturated aerations, make the thalline suitable for producing acid.

[0038] The third level of preparation: t...

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Abstract

The invention provides a method for fermenting L-glutamic acid, which comprises the following steps of: inoculating engineering bacteria expressing an RNA polymerase sigma-32 factor variant into a first fermentation tank for culture, inoculating the obtained culture solution into a second fermentation tank for culture, and inoculating the obtained culture solution in an inoculation amount into a third fermentation tank; and continuously adding sugar and nitrogen into the third fermentation tank under the condition of stagewise temperature change.

Description

technical field [0001] The invention belongs to the field of amino acid fermentation, in particular, the invention relates to a method for fermenting L-glutamic acid, which comprises inserting engineering bacteria expressing RNA polymerase sigma-32 factor variants into a first fermenter for cultivation and obtaining the The culture solution is inoculated in the second fermenter for cultivation, and the inoculum amount of the obtained culture solution is inoculated in the third fermenter for cultivation, and then sugar and nitrogen sources are continuously fed into the third fermenter under the condition of stepwise temperature change. In addition, the present invention also provides products produced by the method and the like. Background technique [0002] L-glutamic acid is an important amino acid raw material, which has been widely used as seasoning and food additive. At present, the production of L-glutamic acid is mainly through the fermentation of microorganisms, such...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/14C12R1/15
Inventor 马吉银陈崇安孟刚曹洪程耀东刘鑫
Owner NINGXIA EPPEN BIOTECH
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