L-tryptophan produced mainly by chemical synthesis of the first method and the method of manufacture of a protein hydrolyzate. With the continuous development of microbial production of tryptophan research, people began using microbial fermentation method tryptophan. Now to practical and dominant. Microbiological method can be roughly divided into microbial fermentation and enzymatic conversion method. In recent years, there was even a direct fermentation and chemical synthesis, research and production of tryptophan direct fermentation and transformation Combination. Another technology, genetic engineering, enzyme immobilization and high density cultivation on microorganisms and enzymes breeding industry has greatly promoted the industrialization process of direct fermentation and enzymatic production of tryptophan.
Protein hydrolysis
L-tryptophan produced mainly by proteolysis first and chemical synthesis method. Protein hydrolysis method is based on hair, blood meal and waste as raw silk and other protein families, by alkaline hydrolysis and enzymatic hydrolysis method for producing L-tryptophan. With the development of amino acid production technology, it is now rarely used in the production of L-tryptophan.
Chemical synthesis
Chemical synthesis method is to use production methods of organic synthesis and chemical engineering technology or a combination of amino acids was prepared. DL-tryptophan chemical synthesis method can be broadly divided into two synthesis of indole synthesis as raw material and as raw material with phenylhydrazine. Snydcr and MacDonald developed a simple synthesis of DL-tryptophan
Method, i.e. the use of indole acrylic acid and α-acetamido direct condensation in the presence of acetic acid and acetic anhydride to give N-acetyl-DL-tryptophan, the substance can be hydrolyzed in a solution of sodium hydroxide to give DL-tryptophan, the yield was 57.7%. Moe with phenylhydrazine and MacDonald reported for the synthesis of tryptophan, i.e., in the presence of sodium acetate, and acrolein diethyl acetamido malonate condensation condensate is then reacted with hydrazine to produce phenylhydrazone in phenylhydrazone BF3 refluxing aqueous H2S04 or hydrolyzed cyclized compound 3 – indolyl – methyl – acetamido – malonate, hydrolysis of this compound can be obtained decarboxylation DL-tryptophan.
The biggest advantage is that the chemical synthesis method is not limited in the variety of amino acids, prepared either natural amino acids, unnatural amino acids can be prepared in a variety of special structure. This does not mean having a value of production industry, because the amino acid is synthesized racemic DL-type, must be split in order to obtain L-amino acids can be utilized. Therefore, when produced by chemical synthesis DL-tryptophan, synthesis conditions should be considered in addition to, but also consider the use of splitting with racemic isomer D-tryptophan isomers, three are indispensable. Thus, the chemical synthesis of L-tryptophan in industrial applications are also subject to certain restrictions.
Enzymatic conversion method
Enzymatic catalytic function is the use of microorganisms to produce L-tryptophan, L-tryptophan biosynthetic enzymes are able to use chemical precursors for the synthesis of raw materials, give full play to the advantages of both organic synthesis techniques, but also has the product high concentration, high yield, high purity, less byproducts, easy purification operation, etc., is a low-cost method for the production of industrial production of tryptophan. Currently in production of L-tryptophan is widely used. These enzymes include enzymes tryptophan, tryptophan, serine racemic enzymes. According to these enzymes provide the number of microbial species can be divided into double and single bacterial enzyme bacterial enzyme types.
Dual enzyme is using two strains of bacteria are needed to provide the enzymatic reaction of tryptophan synthase (TS), serine racemase (SR), indole and DL-serine as a substrate enzymatic conversion of L-color histidine. This method can be of high activity with different enzymes required for the enzymatic conversion of tryptophan together to achieve the advantages of complementary species, improve the conversion rate of the substrate. Makiguchi like E. coli tryptophan synthase and serine racemase from Pseudomonas putida, with DL-serine and indole as a substrate, the reaction in the reaction tank 200L 24h, L-tryptophan production can be achieved 110g / L, the absorption rate of 100% indole (molar ratio, the same below), the yield of DL-serine was 91%. Using a single strain of bacteria is provided by enzymatic synthesis of the desired enzyme tryptophan Trp, tryptophan, serine enzymes and enzymatic conversion of racemic tryptophan enzymes. Won-giBang bacteria and other single enzyme tryptophan production were studied using high-Ts activity of E. coli B10 conversion indole and DL-serine, adding non-ionic surfactant Triton X-100, 37 ℃ reaction 60h, tryptophan acid yield up to 141.4g / L, for a yield of 93.2% indole, the DL-serine yield of 93.6%.
Since the substrate tryptophan indole synthesis inhibitory strong and weak inhibition of the enzyme tryptophan, so in recent years tend to be more enzymes for tryptophan L-tryptophan biosynthesis. Normally tryptophanase degrades tryptophan, L-pyruvate, indole and ammonia, but at high concentrations of pyruvate and ammonia conditions effective to pyruvate, indole and ammonia synthesizing L-tryptophan acid. The enzyme can catalyze L-serine or L-cysteine ​​and L-tryptophan indole synthesis. Nakazawa, such as indole to 20g, 30g sodium pyruvate, 50g ammonium acetate and 4gProteus rettgeri (Proteus Reye) bacterial enzyme tryptophan as a source, 37 ℃ reaction 48h could accumulate 23gL-tryptophan. Ujimaru other enzymatic tryptophan and L-serine with indole synthesis Achromabacterliquidum (Achromobacter liquid form) L-tryptophan, L-serine conversion was 82.4%, conversion of indole was 92.4%.
There are also a study of L-cysteine ​​and indole as raw enzymatic production of L-tryptophan. Wei et tryptophan levels and gene engineering strain WWW-4 L-cysteine ​​and the catalytic synthesis of L-tryptophan indole, 80mL reaction solution (L-cysteine ​​0.75g, 0.75g indole ) 37 ℃ reaction 48h, can accumulate L-tryptophan 1.18g, L-cysteine ​​conversion was 93.2% and 90.1% conversion of indole, the recovery rate of 70% of total product. In addition, the strain has also been reported using enzymatic conversion of L-tryptophan in high yield and with a high activity of pyruvate enzyme tryptophan.
The enzymatic conversion method using a high activity can be either a tryptophan synthase, tryptophanase, or the catalytic activity of a synthetic cell tryptophan synthase enzyme L tryptophan or tryptophan, or the enzyme may synthesis of L-tryptophan or the immobilized cells. After the bacteria and enzyme immobilization of enzymes with improved stability to facilitate re-use, easy to implement continuous and automated production advantages. Won-Bang et polyacrylamide fixed tryptophan synthase having a high activity of Escherichia coli B10 E. coli bacterial cells, in a continuous stirred tank reactor 50 days of continuous use, a tryptophan synthase activity The 80% maximum acidogenic 0.12gL-1h-1. There immobilization techniques using other enzymatic conversion of L-tryptophan. Eggers et al reported a use of organic lipid membrane systems utilize tryptophan enzymatic conversion of L-tryptophan. It is cyclohexane as the organic phase, the organic resin film to two separate aqueous phase and an organic phase, wherein the aqueous phase having an enzymatic reaction system, constituting the aqueous phase was back-extracted another system, using bis-tris-propane as two water maintaining the two-phase buffers the pH of the aqueous phase difference, thus affecting the substances in the reaction system constant distribution of the two aqueous phases, the aqueous phase is then exchanged two L pyruvate and the organic phase through an anion exchanger Aliquat-336 – tryptophan. Such a system is conducive to the transport of L-tryptophan in the aqueous phase was back-extracted, and help reduce the extraction of L-tryptophan and the inhibition of the enzyme tryptophan; Moreover, the organic phase may also store indole, the concentration of indole in the enzyme reaction system is lower than the level of inhibition of the enzyme. Eggers et also established a reaction system of reverse micelles of the enzymatic conversion of L-tryptophan, it is dissolved in water containing tryptophanase reverse micelles of the surfactant Brij56 composed of cyclohexane and water phase , the use of indole and serine as a substrate in the organic phase was added Aliquat-336 anion exchanger and transfer the aqueous phase to the organic phase L-tryptophan. To buffer bis-tris-propane as a two-aqueous phase, select the appropriate parameters such as moisture content and pH conditions, results within 1dm reaction volume per g of tryptophan can produce the enzyme reaction after lh acid 10g. The advantage of the above system in addition to the lipid membrane of the reaction system, but also can improve the stability of the enzyme tryptophan. So, has broad application prospects in the enzymatic conversion of L-tryptophan.
Microbial fermentation
Microbial fermentation method including direct fermentation and added precursor fermentation.
A direct fermentation
Direct fermentation is glucose, cane molasses as carbon source of cheap raw materials, the use of tryptophan-producing excellent strain under suitable fermentation conditions, the direct fermentation of tryptophan. Strain tryptophan excellent high and stable yield is central direct fermentation studies. In breeding technology, the traditional mutation breeding and abroad have done a lot of research. Shiio etc. Brevibacterium tyrosine deficient for fluorophenylalanine (4FP) as the starting strains resistant mutants, breeding 5 – fluoro-tryptophan (5-FT) resistant mutant strain No. 187, the strain can produce L-tryptophan, 8.0 g / L. Continue to be No. 187 for the parent strain breeding with anthranilate structurally similar heavy chlorine serine (AsaSer) resistant mutant A100, its acid production rate increased to lO. 3 g / L, then from A-100 breeding sulfaguanidine (SG) resistant mutant strain S-225, which further improves the ratio of acid to 19g / L. Zhang Suzhen and others to domestic nitrosoguanidine Beijing Corynebacterium AS1.299, mutants obtained CG45. This strain has 5MT, 6FT, 4MP resistance marker, and the arginine and uracil as essential growth factors, in a medium containing 12% glucose, 30 ℃ shaking for 5 days. Can accumulate tryptophan 8g / L. The method of earlier studies, but not for a long time to reach the industrial production. The main reason is the biosynthetic pathway from glucose to tryptophan is relatively long, the metabolic flux is relatively weak and requires a variety of synthetic tryptophan precursor (PRPP, glutamine, L-serine). To further improve the yield of L-tryptophan must also increase production of these precursors. Tryptophan biosynthetic pathway hand adjustment mechanism is relatively complex, in addition to the presence of multiple feedback regulation, there also exists a weakening subsystem. This makes the amino acid tryptophan, an amino acid fermentation industry to become one of the most difficult fermentation. With the application of recombinant DNA technology in microbial breeding, for the excellent tryptophan Strains provide a reliable technical guarantee. Making acid levels gradually reached the industrial production requirement. Katsumata. The recombinant plasmid with R, DAHP synthase (DS) and tryptophan synthase (TS) gene introduced into the production of L-tryptophan 43g / L glutamicum KY10-894, allowing the engineering strain L-tryptophan production reached 66g / L acid levels increased by 54%.
2 Add the precursor fermentation
This method is also known tryptophan microbial conversion method, which is the use of glucose as a carbon source, while adding the required synthesis of tryptophan precursors (e.g., anthranilic acid, indole, L-serine, etc.), the use of microbial acid synthesis enzymes in the body prior to conversion to synthesize L-tryptophan. This method is very early into the industrial production, the world’s largest manufacturer of tryptophan Japan Showa Denko company is using anthranilic acid as precursors using Hansenula (Hansenula) or Bacillus (Bacillus coli) strains to convert tryptophan production methods, Yokozcnki like to DL-5-indole – methyl hydantoin as raw materials, the use of Flavobacterium T-523 which is the decomposition of tryptophan, L-tryptophan can produce acid 7.1 g / L. Breeding the Bacillus subtilis Fukui et 5 – fluoro-tryptophan (5-FT) resistant mutant strains, containing l% soluble starch and 5% glucose medium, the continuous fed-anthranilic acid, can accumulate L-color acid 9.6g / L. Nakayarna further transform this mutant such, it has a 5-FT and 8 – azaguanine (8-AG) double resistance in medium containing 10% glucose, the continuous fed-anthranilic acid, can accumulate L-tryptophan 15.6g / L.
The inadequacies of the microbial transformation is higher when converted precursor solution concentration, the conversion rate has dropped, but can be fed through a small batches to reduce its precursor inhibition. In addition, the precursors are expensive, is not conducive to reducing costs. Therefore, some studies use fermentation to provide a low-cost precursors, combined with the advantages of other methods were tryptophan production. Hajimu MOrikota other P390 Brevibacterium flavum using direct fermentation of L-glutamic acid-β-semialdehyde (GSA) was 13.2g / L, and after appropriate dilution of the broth was added phenylhydrazine 1mol/LH2S04 solution was heated at reflux for 1 hour after GSA 48% L-tryptophan can be converted to. SMgeru oita such dual use of lipoic acid and thiamine deficient strain Enterobacter aetogene LT-94, production of pyruvic acid 30g / L glucose in the medium containing 5%, then by adding ammonium chloride and indole using bacteria color neuraminidase enzymatic conversion of L-tryptophan 16.7%.