two There are two methods for preparing L-proline. One is direct fermentation, which uses glucose and Brevibacterium flavum mutant or Corynebacterium glutamicum wild strain to obtain L-proline through microbial fermentation; The second is chemical synthesis, which uses glutamic acid as raw material, esterification with anhydrous ethanol under the catalysis of sulfuric acid, and adding triethanolamine to free amino sulfate to obtain glutamic acid - δ - ethyl ester. Then, the metal reducing agent potassium borohydride is used to reduce glutamic acid - δ - ethyl ester to obtain crude proline. Finally, the crude proline can be obtained by separating and purifying it. In the pilot process, 147g of L-glutamic acid was weighed by esterification, put into a three necked bottle, added 1L of absolute ethanol, stirred and cooled to 0 ℃, and then dropped with H two SO four 80ml, stirred at 0-5 ℃ for 1h, continued at room temperature for 1h, and the reaction became clear. Add triethylamine dropwise at 20 ℃ to the pH of 8-8.5, precipitate white crystals, stir at room temperature for 1h, let it stand and cool for 5 ℃, filter, take crystals, wash them with 95% ethanol, dry them in vacuum, and obtain about 141g of glutamic acid - δ - ethyl ester. Melting point 178-180 ℃, yield 80% - 83%. [α]32D+29.8 (C=1g/ml 10% HCl)。 To reduce, put 175g of glutamic acid - δ - ethyl ester into a three necked bottle, add 875ml of distilled water, stir and cool to 5 ℃, and then add KBH in batches four 53.9g, about 1h, reaction 1h at room temperature, reaction 3h at 50 ℃. Cool to 0 ℃, add 6mol/L HCl to adjust to pH4, filter the filtrate to obtain crude L-proline aqueous solution. Separation and purification ion exchange resin alumina column chromatographic separation method, the crude L-proline aqueous solution is put into 732-H+resin exchange column at a flow rate of 4ml/min (10ml resin is required for 1g acid feed). First, wash with distilled water to neutral, then wash with 1mol/L ammonia, and collect the eluent containing L-proline segment (controlled by silica gel G thin layer chromatography). Concentrate the eluent to dryness under reduced pressure, dissolve it with a small amount of water, enter it into a neutral alumina chromatographic column, and then elute it with 60% ethanol aqueous solution (still controlled by silica gel G thin layer chromatography). The collected eluent is concentrated to dryness under reduced pressure, washed several times with anhydrous ethanol, cooled slightly, then added with anhydrous ether, cooled, filtered, crystallized, and vacuum dried to obtain L-proline. Melting point 220-222 ℃ (decomposition), yield about 28%. [α]24D-82.4(C=1g/ml, H two O)。 Salt formation by pentachlorophenol precipitation desorption separation method Place the crude proline aqueous solution in a reaction flask, add pentachlorophenol ethanol solution (0.111mol/70ml ethanol) drop when heated to 50 ℃, mix with heat preservation for 5h, let it cool to 0 ℃, filter to take crystals, wash with a small amount of ice water, drain, dry to obtain double salt, melting point 240-242 ℃, sedimentation rate 95%. Analyze and put 38.4g of double salt into a three necked bottle, add 200ml of distilled water and 20ml of ammonia water, stir at room temperature for 8h, cool to 0 ℃, filter and take the filtrate, decompress and concentrate the filtrate, add 100ml of distilled water, filter and take the filtrate, add activated carbon for decolorization. Extract with ether, separate the water layer, continue to concentrate until dry, decolorize with anhydrous ethanol for several times, add a small amount of anhydrous ethanol to wet, add twice the amount of anhydrous ether, cool and crystallize, filter and crystallize, vacuum dry to obtain L-proline finished product. Scale up the production process, esterify and put 15kg L-glutamic acid and 100L absolute ethanol into 200L reaction tank, cool them to 0 ℃, and add concentrated H drops under mixing conditions two SO four 8.1L, keep it at 0 ℃, stir it for 1h, keep it at 25 ℃, stir it for 1h, then add triethylamine to make pH 8.0-8.5. After mixing for 1h, white precipitates appear. Cool to 5 ℃, filter and take the precipitate, wash with 50L 95% ethanol, dry the precipitate in vacuum at 50 ℃, and obtain L-glutamic acid - δ - ethyl ester. Put the obtained L-glutamic acid - δ - ethyl ester into a 100L reaction tank by reduction, add 70L water, stir and cool to 5 ℃, and add 4.3kg KBH in batches within 1h four , heating and holding 200 ℃, stirring reaction for 1h, heating up 50 ℃, stirring reaction for 3-4h, cooling to 0 ℃, adjusting pH to 4.0 with 6mol/L HCl, filtering the filtrate to obtain L-proline crude solution. Precipitation Put the crude L-proline solution into a 100L reaction tank, heat it to 50 ℃, slowly add 7L 1.5mol/L pentachlorophenol ethanol solution under constant stirring, keep it at 50 ℃ for reaction for 5h, cool it to 0 ℃ to precipitate crystals, filter and take crystals, drain them to get double salt. Analyze and refine, put the double salt into a 100L reaction tank, add 20L of 3% ammonia water, stir at room temperature for 7-8h, cool down to 0 ℃ for filtration, wash the sediment with a small amount of ice water, pump it dry, combine the lotion and filtrate, reduce pressure and concentrate it to dry, mix and dissolve it with 10L deionized water, filter the filtrate, add 0.5% activated carbon, heat it at 70 ℃, stir and decolorize it for 1h, filter the filtrate, let it cool to 0 ℃, Add equal volume of ether for extraction, separate the water layer, decompress and concentrate it to dryness, add 10L absolute ethanol for dehydration for three times, drain it, add 2L absolute ethanol for precipitation, mix it evenly, add 10L ether, cool it to 0 ℃, filter the precipitation, vacuum extract ether, dry it at 80 ℃, and obtain L-proline finished product.
