Method 1. Direct fermentation method
Using glucose as raw material, breeding auxotrophs and feedback inhibition and repression in the synthesis of structural analogues, the acid production rate of L-threonine was 18g/L, Corynebacterium glutamicum, the acid production rate was 14g/L, The rate of Salmonella marcescens was 14g/L.
Glucose [Brevibacterium flavum, Corynebacterium glutamicum, Bacillus marcescens, etc.] → L-threonine
Threonine obtained by chemical synthesis is a mixture of four optical isomers, namely DL-threonine. The amino acid that constitutes the protein is L-threonine, so it is necessary to separate the threonine body from the other body, and then further split the optical isomers to obtain L-threonine.
Using copper glycinate to react with acetaldehyde under alkaline conditions, a similar aldol condensation reaction occurs, and two mixtures of threonine copper and allosomes are synthesized. According to their stability and solubility, they can be separated by decoppering DL-threonine is extracted from the body, and L-threonine is finally resolved. The small-scale synthesis and scale-up production processes are introduced respectively as follows.
Glycine preparation Mix 189g (2mol) of monochloroacetic acid and 2100ml (3.3mol) of formaldehyde solution, cool to below 10°C, add 750ml (10mol) of concentrated ammonia water dropwise, and control the rate of addition so that the temperature does not exceed 10°C. After adding ammonia water, keep warm at 30°C for 4 hours. Concentrate under reduced pressure to 300-400ml, crystals precipitate out. After washing and drying to obtain crude glycine, add about 1.5-2 times the volume of water to the crude glycine, heat it to dissolve completely, add 1% activated carbon to decolorize; filter the filtrate, add 2-2.5 times the volume of methanol, and place in Put it in the refrigerator overnight, filter to collect the crystals, and obtain the fine product of glycine. The yield is about 60%-68%.
Preparation of Copper Glycinate Take 100g of glycine, add 7L of water, and heat at 60°C to dissolve it completely. Then slowly add 80g of basic copper carbonate, and keep it warm at 60°C for 1h. Remove unreacted basic copper carbonate by filtration while hot. Collect the filtrate and allow it to cool naturally, and blue needle-like crystals (containing 1 molecule of crystal water) are precipitated. The crystals are collected by filtration, washed, and dried at 60°C to obtain copper glycinate with a yield of 95%-98%.
Preparation of copper threonine Take 52.5g of copper glycinate, add 425ml of methanol and stir to dissolve, then add 80ml of ethanol below 10°C, wait until the temperature does not rise, then add 5g of sodium hydroxide pre-dissolved in 90ml of methanol solution, 60°C Insulation reaction 1h. Filter off the insoluble matter while it is hot, collect the filtrate and add 5.5ml of glacial acetic acid, recover methanol to dryness under reduced pressure, add 75ml of methanol to stir and disperse, then cool overnight. The crystals were collected by filtration, washed and dried to obtain a mixture of threonine copper and other threonine copper, with a yield of 68%-74%.
Copper removal and refining Take 427g of the above threonine copper, add 6L of 10% ammonium hydroxide solution to dissolve it completely, filter and collect the filtrate, adsorb it on 732 cation exchange resin, wash with 2mol/L ammonia water and water until the eluate Until ninhydrin does not develop color. Combine the eluents, concentrate the film to 1.5L, add 3L of ethanol to precipitate crystals, place in the refrigerator overnight, and filter to collect crystals to obtain crude DL-threonine. The yield is 62%-73.8%.
Take 42g of crude DL-threonine, add 126ml of water to heat to dissolve completely, decolorize and filter to get the filtrate, add 252ml of ethanol, cool overnight, filter to get crystals to obtain refined DL-threonine. The yield is 87%-91.3%.
Splitting and refining Mix refined DL-threonine 810g, DL-threonine 90g, and water 2.88L, stir slowly (about 50r/min), and heat to above 95°C to dissolve them all. Then lower the temperature to 40°C, and add 10% of the total amount of DL-threonine as a species of D-threonine, slowly cool to 30°C (1°C every 15 minutes on average), and precipitate crystalline D-threonine, The crystals were collected by filtration and dried at 80°C to obtain D-threonine. Collect the filtrate, drop into DL-threonine (about 150-170g) equivalent to the D-threonine that splits out, keep the total volume constant, the same operation as the resolution of D-threonine (but reduce the temperature When the temperature reaches 40°C, the added seed crystal is L-threonine) to obtain L-threonine. By repeating this operation, D-threonine and L-threonine can be separated.
The corresponding crude D-threonine and L-threonine were combined and refined separately, that is, recrystallized. Take D and L-type threonine respectively, add water 4 times respectively, heat at 90°C until fully dissolved, decolorize with 1% activated carbon, collect the filtrate by filtering while it is hot, add 2 times the volume of ethanol and let it stand for cooling, stir from time to time, and precipitate crystals , to get D-threonine and L-threonine fine products, yield 87.3%-91.6%.
The content of L-threonine is above 95%, [α] 20D-26°–29°, and there is a spot in paper chromatography.
Scaled up production process
Using glycine as a raw material, it reacts with basic copper carbonate to form copper glycinate. At this time, its α-H is active. Under alkaline conditions, copper glycinate reacts with acetaldehyde to undergo a similar cross-aldol condensation reaction to form DL- Copper threonine (contains threonine and red body), and then the threonine DL-threonine is separated by ion exchange and decopper, and finally the D-threonine and L-threonine are separated by induced crystallization . It reacts as follows:
Preparation of Copper Glycinate In a 500L reaction tank, put 50kg of glycine, add 350L of water, keep 40kg of basic copper sulfate at 60°C for 1h, filter and discard the unreacted copper salt, collect the filtrate, let it stand for cooling overnight, and filter to obtain crystals, 60 ℃ drying to get blue copper glycinate.
Preparation of Copper Threonine In a 1000L reaction tank, put 75kg of copper glycinate, then add 600L of methanol, stir to dissolve, add 120L of acetaldehyde, 90L of 5%KOH methanol solution, stir and keep at 60°C for 1h, and filter out the insoluble matter , add 5.5L glacial acetic acid to the filtrate, recover methanol to dryness under reduced pressure, add 75L water to stir and disperse, then stand at 5°C overnight, filter to collect crystals, wash, and drain to obtain mixed threoninate copper with threonine and allosomes.
Ion exchange, decopper separation to obtain DL-threonine Put 40kg of threonine copper and 1000L of 10% ammonia water into a 2000L reaction tank respectively, stir to dissolve it, filter and take the filtrate in a 732 cation exchange column (400mm× 200mm), and then washed successively with 2mol/L ammonia water and deionized water. Combine the eluents, concentrate the film to 150L, add 300L ethanol, stir, cool to 5°C and crystallize overnight. The crystals were collected by filtration and dried at 80°C to obtain crude DL-threonine.
Concentrate, refine and take 40kgDL-threonine crude product into 500L reaction tank,
