Plant synthesis
There are two pathways for the synthesis of plant proline: one pathway uses glutamic acid (Glu) as a substrate to synthesize proline, and the other pathway uses ornithine as a substrate to synthesize proline, which is usually stressed in plants. Or in the case of nitrogen deficiency, the main source of proline is the glutamate synthesis pathway. In the case of sufficient nitrogen supply, the main synthesis pathway of proline in plants is synthesis of ornithine as a substrate.
Chemical synthesis
The hydrolysate of protein such as gelatin and casein is treated with ion exchange resin, and then the neutral amino acid part is treated with picric acid or Reineckeatesalt, only L-proline is precipitated, and finally with absolute ethanol It is obtained by recrystallization with isopropanol. It is obtained by fermentation of Corynebacterium acetoacidophilum XQ-3 (selected by the Central Research Institute of Wuxi University of Light Industry) using ammonium chloride as a nitrogen source. The acid production rate is about 60g/L.
There are two methods for preparing L-proline. One is a direct fermentation method, which uses glucose and a mutant strain of Brevibacterium flavum or a wild strain of Corynebacterium glutamicum to obtain L-proline through microbial fermentation; the other is a chemical synthesis method, which uses glutamic acid as a raw material and anhydrous ethanol Esterification occurs under the catalysis of sulfuric acid, and triethanolamine is added to free the aminosulfate to obtain glutamic acid-δ-ethyl ester. Then use the metal reducing agent potassium borohydride to reduce glutamic acid-δ-ethyl ester to obtain crude proline, and finally to separate and purify it to obtain crude proline. Esterification of small-scale trial process Weigh 147g of L-glutamic acid, put it into a three-necked flask, add 1L of absolute ethanol, stir and cool to 0℃, then add 80ml of H2SO4 dropwise, stir at 0–5℃ and react for 1h, and continue the reaction at room temperature After 1h, all the reactions became clear. Add triethylamine dropwise to pH 8–8.5 at 20°C, precipitate white crystals, stir at room temperature for another 1h, let stand and cool at 5°C and filter, take the crystals, wash with 95% ethanol, drain and vacuum dry, get Glutamate-δ-ethyl ester is about 141g. The melting point is 178–180℃, and the yield is 80%-83%. [α]32D+29.8 (C=1g/ml 10% HCl). Reduction Put 175g of glutamic acid-δ-ethyl ester in a three-necked flask, add 875ml of distilled water, stir and cool to 5℃, then add 53.9g of KBH4 in portions, add it in about 1h, react for 1h at room temperature, and react at 50℃ for 3h . Cool to 0°C, add 6mol/L HCl to adjust the pH to 4, filter the filtrate to obtain the crude L-proline aqueous solution. Separation and purification Ion exchange resin-alumina column chromatography The crude L-proline aqueous solution was fed into a 732-H+ resin exchange column at a flow rate of 4ml/min (1g acid feed requires 10ml resin). First rinse with distilled water to neutrality, and then eluted with 1mol/L ammonia water, and collect the eluent containing the L-proline segment (controlled by silica gel G thin layer chromatography). The eluent was concentrated under reduced pressure to dryness, and then dissolved in a small amount of water, and then passed into a neutral alumina chromatographic column, and then eluted with a 60% ethanol aqueous solution (still controlled by silica gel G thin layer chromatography). The collected eluate was concentrated under reduced pressure to dryness, and then washed several times with absolute ethanol. After a little cooling, anhydrous ether was added, and the crystals were collected by cooling and filtration, and dried in vacuum to obtain L-proline. The melting point is 220–222℃ (decomposition), and the yield is about 28%. [α] 24D-82.4 (C=1g/ml, H2O). Pentachlorophenol precipitation and desorption separation method for salt formation. Place the crude proline aqueous solution in a reaction flask, add pentachlorophenol ethanol solution (0.111mol/70ml ethanol) dropwise when heated to 50℃, and keep it warm and stir for 5h, then let it cool To 0℃, filter the crystals, wash with a small amount of ice water, drain, and dry to obtain a double salt with a melting point of 240–242℃ and a precipitation rate of 95%. Analyze 38.4g of double salt, put it into a three-necked flask, add 200ml of distilled water, 20ml of ammonia, stir at room temperature for 8h, cool to 0℃, filter to take the filtrate, concentrate the filtrate under reduced pressure, add 100ml of distilled water, filter to take the filtrate, add activated carbon for decolorization . Extract with ether, separate the water layer, continue to concentrate to dryness, decolorize with absolute ethanol several times, then add a small amount of absolute ethanol to moisten, add 2 times the amount of anhydrous ether, cool to crystallize, filter the crystals, and vacuum dry to obtain L-Pro Acid product. Scale-up production process esterification Put 15kg of L-glutamic acid and 100L of absolute ethanol into a 200L reaction tank, cool to 0℃, add 8.1L of concentrated H2SO4 dropwise under stirring, keep 0℃, stir for 1h, and then keep at 25℃ After stirring the reaction for 1h, add triethylamine to make the pH 8.0–8.5. After stirring for 1 hour, a white precipitate appeared. Cool to 5°C, filter the precipitate, wash with 50L of 95% ethanol, and dry the precipitate in vacuum at 50°C to obtain L-glutamic acid-δ-ethyl ester. Reduction Put the obtained L-glutamic acid-δ-ethyl ester into a 100L reaction tank, add 70L of water, stir and cool to 5℃, add 4.3kg KBH4 in 1h, heat and keep at 200℃, stir for 1h, and then increase the temperature Stir the reaction at 50°C for 3–4h, cool to 0°C, adjust the pH to 4.0 with 6mol/L HCl, and filter the filtrate to obtain a crude L-proline 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℃ and react for 5h, then cool to 0℃ The crystals are separated out, filtered to take the crystals, and drained to obtain the double salt. Analyze and refine the double salt into a 100L reaction tank, add 20L of 3% ammonia water, stir at room temperature for 7–8 hours, then cool to 0℃ and filter, wash the precipitate with a small amount of ice water, drain it, and combine the washing liquid and the filtrate, and then reduce Concentrate to dryness, stir to dissolve with 10L deionized water, filter to take the filtrate, and add 0.5% activated carbon, heat at 70℃ and stir to decolorize for 1h, filter to take the filtrate, let it cool to 0℃, add an equal volume of ether to extract, separate the water layer , Concentrate under reduced pressure to dryness, add 10L of absolute ethanol for dehydration 3 times, drain off, add 2L of absolute ethanol to the precipitate and stir evenly, then add 10L of ether, cool to 0℃, filter to collect the precipitate, vacuum ether, and dry at 80℃ , Get the finished product of L-proline.
It is obtained by ion exchange resin column chromatography after acid hydrolysis with gelatin as raw material.
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