Method for preparing high-purity lysine salt by fermentation and product thereof
A kind of technology of lysine sulfate and lysine, applied in the field of amino acid fermentation
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Embodiment 1
[0074] The preparation of embodiment 1 RNA polymerase sigma-32 factor gene construct
[0075] According to the amino acid sequence of the protein accession number AAB18436.1 of NCBI (http: / / www.ncbi.nlm.nih.gov), the inventors designed moderate expression codons (non-expression-optimized codons), and obtained The pathway commissioned Shanghai Sangon Biotechnology Co., Ltd. to synthesize the gene encoding RNA polymerase sigma-32 factor and construct it into E. coli expression plasmid pET-20b(+) (available from Novagen, USA, product number Cat.No.69739-3 )middle. 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:
[0076] Synthesize nucleic acid fragments of the RNA polymerase sigma-32 factor gene by an automatic DNA synthesizer, phosphorylate the 5' ends of these nucleic acid fragments with T4 polynucleotide kinase (purchased from TaKaRa), and then m...
Embodiment 2
[0078] Embodiment 2 Escherichia coli fermentation produces L-lysine
[0079] The L-lysine-producing Escherichia coli strain W3110(tyrA) / pCABD2 constructed by the method described in Chinese Patent No. 94194962 was transformed into a pET-sigma plasmid, and a positive clone was obtained after identification (named W3110(tyrA) / pCABD2 -sigma), the strains W3110(tyrA) / pCABD2 and W3110(tyrA) / pCABD2-sigma were respectively subjected to lysine fermentation experiments with reference to Chinese Patent No. 03120099. In short, the bacterial strains were inserted into liquid LB medium for shaking culture until OD500 reached 0.35, and then inserted into lysine fermentation medium with a 5% inoculum size (the formula per liter of medium was: 100g glucose, 60g (NH 4 ) 2 SO 4 , 50gCaCO 3 , 35mL peptone-B (SoyProteinEnzymaticHydrolysate, purchased from Shanghai Xinran Biotechnology Co., Ltd., the total nitrogen content is not less than 3%), 1gKH 2 PO 4 , 400mgMgSO 4 ·7H 2 O, 200mg L-met...
Embodiment 3
[0081] The refining of embodiment 3L-lysine sulfate product
[0082] The supernatants obtained from strains W3110(tyrA) / pCABD2 and W3110(tyrA) / pCABD2-sigma described in Example 2 were respectively taken out by 1 / 4 volume and purified first: these supernatants were loaded on the SephadexG-10 layer Analyze the column, elute with pure water, collect fractions with a molecular weight between 100 and 200Da, and use HPLC to quantify the content of L-lysine and other components in the fractions, of which: W3110(tyrA) / pCABD2 The composition distribution is: lysine, 92.60%; threonine, 3.23%; serine, 1.70%; glutamic acid, 1.33; methionine, 0.76%; other impurities accounted for 0.38% (mainly inorganic salts) ;The composition distribution of W3110(tyrA) / pCABD2-sigma is: lysine, 80.73%; aspartic acid, 2.39%; threonine, 1.51%; serine, 0.89%; glutamic acid, 3.73%; valine , 1.37%; Methionine, 0.68%; Isoleucine, 0.97%; Leucine, 1.97%; Tyrosine, 1.12%; Phenylalanine, 0.99%; Histidine, 1.50%; ...
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