Method 1. Hydrolysis method
Acid hydrolysis was carried out with silkworm cocoon coat as raw material, and then ion-exchanged water was used for separation and purification.
Process: Cocoon coat [acid hydrolysis]→[HCl, 110℃, 24h] hydrolyzate [deacidification, decolorization]→[732 resin, ammonia water, pH3.5-8] eluent [fractionation]→[717 resin] ] Collection solution [concentration, crystallization, purification] → [thin film evaporation] → L-serine
For acid hydrolysis, put 30kg of cocoon coat in the hydrolysis tank, add 150L of HCl with a concentration of 6mol/L, stir and heat at 110℃ for 24h, cool down to below 60℃, filter again, and wash the filter residue with pure water with five times the amount of filtrate. , the washings were combined into the filtrate to obtain an aqueous solution of about 800L.
Deacidification and deliquoring The hydrolyzate 100L is processed into H+ type 732 cation exchange resin column (150mm×2000mm polyvinyl chloride, two each with 25L of internal resin) from top to bottom with the flow rate of 100-120ml/min, and then use Pure water washes away the pigment to a clear solution without Cl-. Next, ammonia water with a concentration of 0.3 mol/L is eluted through the column from top to bottom at a flow rate of 80-100 ml/min until the amino acid flows out, and the eluate in the range of pH 3.5-8 is collected. Finally, the tyrosine was washed with 1 mol/L ammonia water, and the tyrosine was obtained by de-ammonia concentration and crystallization.
The above-mentioned eluent is obtained by fractionation and conventionally treated as 4 OH-type 717 anion exchange resin columns:
The first one is 150mm×2000mm, with 24L resin (polyvinyl chloride) inside
The second one is 150mm×1800mm, with 22L resin (polyvinyl chloride) inside
The third one is 150mm×1600mm, with 20L resin (polyvinyl chloride) inside
The fourth one is 150mm×1400mm, with 10L resin (polyvinyl chloride) inside
First, adjust the eluent to pH 7-8 with 1 mol/L NaOH solution, go to the first column at a flow rate of 120-150 ml/min, wash with pure water until the resin is saturated, and then connect the first and second columns in series , eluted with 0.1mol/L HCl, the flow rate is about 80-100ml/min, when the effluent has amino acids, it starts to collect, a total of 25 bottles (1000ml/bottle) are collected, and then the third column is connected in series, and the collection is performed when amino acids flow out. 50L, discard the 1st column. Connect the fourth column in series, and continue to elute until the effluent contains amino acids and collect until pH 2-3, about 50 bottles. Paper chromatography was performed on the pools of each group, and the pools containing serine were combined.
Concentration, crystallization and purification The above-mentioned combined solution is concentrated by thin-film evaporation until crystals appear. After cooling, about 2 times the amount of absolute ethanol is added and placed in a refrigerator overnight. The crystals are separated out, filtered, and dried to obtain L-serine. Calculated by cocoon coat, the yield is about 4%.
Method 2. Fermentation method with the addition of precursors
The metabolism of L-serine in the body is extremely fast, and it is difficult to produce it by direct fermentation. Generally, the fermentation method with the addition of precursors is generally used. The added precursors are mainly glycine, glycine trimethylidene or glyceric acid, and the industrialization of glycine as the precursor is carried out. Producers can be divided into two categories: heterooxotrophic and methylotrophic bacteria.
Heterotype strain, glycine produces L-serine
Glycine [Glycinophilic acid Corynebacterium or Nocardia butanes or Sarcinus albicans] → [fermentation] L-serine methylotrophic strain, glycine produces L-serine
Glycine [Pseudomonas or Mycobacterium or methanol-assimilating bacteria Arthrobacter sphaeroides] → [fermentation] L-serine
Method 3. Chemical synthesis method DL-serine can be synthesized by using hydroxyacetaldehyde as a raw material, and then split to obtain L-serine.
Synthetic method using glycolaldehyde as raw material
Synthetic method using diethyl bromomalonate as raw material
Synthetic method using vinyl compounds as raw materials
Method four, enzymatic method
Using the intermediate DL-2-oxooxazolidine-4-carboxylic acid (DL-OOC) in the chemical synthesis of DL-serine as raw material, using L-OOC hydrolase produced by Pseudomonas testosteroni or OOC produced from Bacillus subtilis Racemase action to produce L-serine. DL-2-oxooxazolidine-4-carboxylic acid (DL-OOC) [L-OOC hydrolase or racemase] → L-serine.