The threonine (L-threonine) obtained by chemical synthesis is a mixture of four optical isomers, namely DL threonine. The amino acid constituting the protein is L-threonine, so it is necessary to separate the threonine from the other body, and then further carry out optical isomer resolution to obtain L-threonine.
Tobacco: BU, 22; FC，21； Synthesis: It can be obtained by hydrolyzing and refining protein (such as casein), or by synthesizing methanol and mercury acetate in acrylic acid derivatives.
Under alkaline conditions, copper glycine reacts with acetaldehyde to produce a similar aldol condensation reaction to synthesize two mixtures of threonine copper: threonine and other. According to their different stability and solubility, DL threonine can be separated from threonine by decoppering, and finally L-threonine can be separated. The production processes of small scale synthesis and scale-up are introduced as follows.
Small scale synthesis process:
Glycine preparation 189g (2mol) of monochloroacetic acid and 2100ml (3.3mol) of formaldehyde solution were mixed and cooled to below 10 ℃. 750ml (10mol) of concentrated ammonia water was added dropwise, and the dropping speed was controlled so that the temperature did not exceed 10 ℃. After adding ammonia water, keep the temperature at 30 ℃ for 4h. Concentrate to 300-400ml under reduced pressure, and crystallize. After washing and drying, the crude glycine is obtained. Add 1.5-2 times of water to the crude glycine, heat it to make it completely soluble, and add 1% activated carbon to decolorize it; Filter the filtrate, add 2-2.5 times the volume of methanol, put it in the refrigerator overnight, filter and take the crystal, and obtain the refined threonine (L-threonine). The yield is about 60% - 68%.
Preparation of copper glycine 100g of glycine was added with 7L of water and heated at 60 ℃ to make it completely soluble. Then slowly add 80g of basic copper carbonate and keep it at 60 ℃ for 1h. Remove unreacted basic copper carbonate by hot filtration. Collect the filtrate and let it cool naturally to separate blue acicular crystals (containing one molecule of crystal water), filter and crystallize, wash and dry at 60 ℃ to obtain copper glycine in 95% - 98% yield.
Preparation of copper threonine 52.5g of copper glycine was added 425ml of methanol to stir for dissolution, then 80ml of ethanol was added below 10 ℃, after the temperature did not rise, 5g of sodium hydroxide was added to dissolve in 90ml of methanol solution in advance, and the reaction was conducted at 60 ℃ for 1h. Filter away the insoluble matter while it is hot, collect the filtrate and add 5.5ml of glacial acetic acid. Reduce the pressure to recover methanol until it is dry, add 75ml of methanol to stir and disperse, and then cool overnight. After filtration, collection and crystallization, washing and drying, a mixture of threonine and allothreonine copper was obtained with a yield of 68% - 74%.
Remove copper and refine 427g of threonine copper, add 6L of 10% ammonia hydroxide solution to dissolve it completely, filter and collect the filtrate, adsorb it with 732 cation exchange resin, and wash it with 2mol/L ammonia water and water until the eluent does not show color to ninhydrin. Combine the eluent, concentrate the film to 1.5L, add 3L ethanol to crystallize it, put it in the refrigerator overnight, filter it for crystallization, and obtain crude DL threonine. The yield was 62% - 73.8%.
Take 42g of crude DL threonine, add 126ml of water to make it completely soluble, decolorize and filter the filtrate, add 252ml of ethanol, cool overnight, filter and crystallize to obtain refined DL threonine. The yield is 87% - 91.3%.
Resolution and refining Mix 810g of refined DL threonine, 90g of DL threonine and 2.88L of water, slowly stir (about 50r/min), and heat to above 95 ℃ to dissolve them all. Then cool down to 40 ℃, add 10% of the total DL threonine dosage of D-threonine, slowly cool down to 30 ℃ (1 ℃ drop every 15min on average), separate crystalline D-threonine, filter and crystallize, and dry with 80 ℃ to obtain D-threonine. Collect the filtrate, put in the same amount of DL threonine (about 150-170g) as the resolved D-threonine, keep the total volume unchanged, and perform the same operation as the resolution of D-threonine (but when the temperature is reduced to 40 ℃, the crystal seed added is L-threonine) to obtain L-threonine. After repeated operation, D-threonine and L-threonine can be separated.
The corresponding D-threonine crude product and L-threonine crude product are combined for refining, i.e. recrystallization. Take D-threonine and L-threonine respectively, add 4 times of water respectively, heat them at 90 ℃ until they are completely dissolved, decolorize them with 1% activated carbon, filter the filtrate while it is hot, add ethanol twice the volume of ethanol to cool them, stir them from time to time, and crystallize them to obtain D-threonine and L-threonine fine products, with the yield of 87.3% - 91.6%.
The content of L-threonine is more than 95%[ α] 20D-26 ° - 29 °, paper chromatography shows a spot.
Enlarged production process:
Glycine is used as raw material to react with basic copper carbonate to produce copper glycine α- H is active. Under alkaline conditions, similar cross aldol condensation reaction can occur between copper glycine and acetaldehyde to generate DL threonine copper (including threonine and red body). Then DL threonine is separated from threonine by ion exchange and copper removal. Finally, D-threonine and L-threonine are separated by induced crystallization. The reaction is as follows:
The preparation of copper glycine is carried out in a 500L reaction tank. 50kg glycine is added, 350L water is added, and 40kg basic copper sulfate is kept at 60 ℃ for 1h. The unreacted copper salt is filtered and discarded, the filtrate is collected, left to cool overnight, filtered and crystallized, and dried at 60 ℃ to obtain blue copper glycine.
The preparation of copper threonine is carried out in a 1000L reaction tank. 75kg of copper glycine is added, 600L of methanol is added, and stirred to dissolve it. Then 120L of acetaldehyde and 90L of 5% KOH methanol solution are added. After stirring and holding at 60 ℃ for 1h, insoluble matters are filtered. 5.5L of glacial acetic acid is added to the filtrate, and methanol is recovered at reduced pressure until it is dry. 75L of water is added, stirred and dispersed, and left overnight at 5 ℃. Crystallization is filtered, washed, and dried to obtain threonine copper mixed with threonine and others.
DL threonine is obtained by ion exchange and copper removal. Put 40kg of threonine copper and 1000L of 10% ammonia respectively into 2000L reaction tanks, stir to dissolve them, and filter the filtrate into 732 cation exchange column (400mm × 200mm), and then wash with 2mol/L ammonia water and deionized water successively. Combine the eluent, concentrate the film to 150L, add 300L ethanol, stir, and cool to 5 ℃ for crystallization overnight. The crude DL threonine was obtained by filtering and crystallization, and drying at 80 ℃.
Concentrate and refine 40kg DL threonine crude product and put it into 500L reaction tank.