(1) in L Lysine Secom hydrochloride, add purified water (deionized water), the weight ratio of L-lysine hydrochloride and purified water is 1: 1, mix homogeneously and dissolve completely, place stand-by;
(2) Upper column adsorption
The lysate obtained in step (1) is added to the ion exchange column equipped with the cation exchange resin at a flow rate of 13 L/min, until the resin cation exchange resin all adsorbs lysine, and the effluent of the column does not contain lysine. , then wash with purified water by the resin in the exchange column, wash to no Cl in the effluent of the column (the pH of the effluent is 7 at this time);
Then use 2mol/L NH4OH (ammonia) to elute L Lysine Secom, NH4OH flows through the ion exchange column at a flow rate of 13L/min, when the effluent pH value of the ion exchange column rises to 8.0, the effluent begins to have indene The triketone reaction means that L-lysine begins to flow out, and the effluent is collected; when the pH value of the effluent of the ion exchange column rises to 9.0 to 12, the color of the ninhydrin reaction is dark, and the L-lysine content is high at this time, and the elution is The collection can be stopped until there is no ninhydrin reaction in the effluent;
(4) Concentration under reduced pressure
Concentrate the collected solution under reduced pressure at 0.2Mpa and 60～62°C to catch ammonia, when concentrated to 1/3 of the volume of the original collected solution, add activated carbon with 3.5% of the weight of the concentrated solution at this time, decolorize, filter, and crystallize by cooling to obtain L ‑Lysine.
The purity of the product detected and calculated by a potentiometric titrator was 98.5%.
Preparation of free L Lysine Secom solid: Weigh 1.00kgL Lysine Secom hydrochloride and add it to 5.0L purified water, stir to dissolve and clarify, then add the solution to 7.20kg of activated strong acid cation exchange resin column, rinse the resin column slowly, After 2-3 hours of rinsing, continue to rinse with purified water until neutral and free of chloride ions (0.1mol/L silver nitrate monitoring and extensive pH test paper). Add 11.1kg of 5% v/v ammonia water to the resin column, and slowly rinse the resin column for about 2-3 hours. When the pH value is greater than 10 during the flushing process, start to collect the alkaline eluent, and stop until the pH value decreases to about 10. collect. Concentrate the collected solution under reduced pressure at 60°C (-0.10～-0.08Mpa), concentrate water and ammonia until no droplets drip out, continue to concentrate and turn into a white solid, add 8L of absolute ethanol to the system, Continue beating and crystallization, beating for 3h, all solids are precipitated, centrifuging and filtering out the solids, until no droplets drip out, spread the filter cake on an enamel tray, vacuum dry (60±5℃, -0.08～-0.1Mpa) for 8h, The product was cooled to room temperature, sealed in a sealed bag and stored in a cool place to obtain 776 g of free L Lysine Secom solid, yield 96.95%, purity: 99.62%, HNMR (D2O): 3.223-3.188 (CHN), 2.848-2.794 (2H/ CH2N), 1.542-1.523, 1.276 (-CH2CH2CH2-). LCMS(M+1): 147.1127.
The fermentation method of L Lysine Secom includes three steps of lysine primary seed culture, lysine secondary seed culture and lysine fermentation culture. The specific operation of each step is as follows:
1. Lysine primary seed culture
Prepare lysine primary seed medium. In this example, the lysine primary seed medium includes sucrose 20g/L, magnesium sulfate 0.5g/L, potassium dihydrogen phosphate 1.5g/L, ammonium sulfate 9g/L, yeast extract 5g/L, threonine 0.5g/L, methionine 0.5g/L, monosodium glutamate 7g/L, sodium pyruvate 0.5g/L, calcium acetate 1g/L.
The prepared lysine primary seed medium was adjusted to pH 6.0 with 20% (volume ratio) potassium hydroxide solution, and the medium was heated to 121° C. 36°C and kept constant. Start stirring at 300rpm, introduce sterile air according to the ventilation ratio of 1:0.4, adjust the tank pressure to 0.05Mpa, and adjust the pH to 6.7 with ammonia water and keep it constant, and adjust the dissolved oxygen to 100%.
The lysine-producing Escherichia coli shake flask seeds were connected to the lysine primary seed medium at 1‰ of the volume of the lysine primary seed medium for cultivation. Oxygen 30-50%, after culturing for 10 hours, samples were taken every 2 hours for microscopic examination and total sugar was measured. When the microscopic examination of the bacteria was normal and the total sugar dropped to 10g/L, the culture was stopped, and the lysine mature first-class seed liquid was obtained.
2. Lysine secondary seed culture
Prepare lysine secondary seed medium. In this example, the lysine secondary seed medium includes glucose 70g/L, magnesium sulfate 1g/L, potassium dihydrogen phosphate 1g/L, ammonium sulfate 10g/L, corn steep liquor hydrolyzate 1g/L, hair hydrolyzate 1g /L, beet molasses 10ml/L, threonine 0.4g/L, methionine 0.4g/L, calcium acetate 2g/L.
The prepared lysine secondary seed medium was adjusted to pH 6.2 with ammonia water, the medium was heated to 121 °C by steam, sterilized for 20 min, and the circulating water was opened to cool down to 37 °C and maintained constant. Start stirring at 300rpm, introduce sterile air according to the ventilation ratio of 1:0.4, adjust the tank pressure to 0.05Mpa, adjust the pH value to 6.6 with ammonia water and keep it constant, and adjust the dissolved oxygen to 100%. The mature lysine first-level seed liquid obtained in step (1) is received in the lysine second-level seed medium by 2% of the volume of the lysine second-level seed medium and cultivated. , The tank pressure is controlled by 30-50% of dissolved oxygen. After culturing for 10 hours, samples are taken every 2 hours for microscopic examination and reducing sugar. Secondary seed solution.
3. Lysine fermentation culture
Prepare lysine fermentation medium. In this example, the lysine fermentation medium includes glucose 20g/L, magnesium sulfate 0.5g/L, phosphoric acid 0.3/L, ammonium sulfate 9g/L, corn steep liquor hydrolyzate 0.5g/L, hair hydrolyzate 0.5g/L , Beet Molasses 10ml/L, Betaine 0.5g/L, Calcium Acetate 1g/L.
Adjust the pH of the prepared lysine fermentation medium to 5.6 with 20% potassium hydroxide, heat the medium to 121°C with steam, sterilize it for 20min, and open circulating water to cool down to 36°C and keep it constant. Start stirring at 400 rpm, introduce sterile air according to the ventilation ratio of 1:0.3, adjust the tank pressure to 0.07Mpa, adjust the pH value to 6.7 with ammonia water and keep it constant, and adjust the dissolved oxygen to 100%. The mature lysine secondary seed liquid obtained in step (2) is received in the lysine fermentation medium by 10% of the volume of the lysine fermentation medium and cultivated. Dissolved oxygen 25-40%.
During the culturing process, samples were taken every 2h to measure the reducing sugar concentration and ammonia nitrogen concentration of the fermentation broth. When the reducing sugar concentration in the fermentation broth dropped to 4g/L, the 60% (m/v) glucose solution was added and the reduction of the fermentation broth was maintained. The sugar concentration is about 4g/L. When the ammonia nitrogen concentration in the fermentation broth drops to 1g/L, 40% (m/v) ammonium sulfate solution is added and the ammonia nitrogen concentration in the fermentation broth is maintained at about 1g/L.