Method for synthesizing L-7-methyltryptophan by enzymic method

Abstract

The invention relates to the technical field of medicines, and discloses a method for synthesizing L-7-methyltryptophan by an enzymic method, tryptophan synthetase is used as a biocatalyst, 7-methylindole and L-serine are used as substrates, the enzymic method synthesis of the L-7-methyltryptophan is realized through a one-step catalytic reaction, and after the reaction is finished, the L-7-methyltryptophan is obtained. A reaction solution is subjected to a series of separation and purification methods such as membrane filtration, decoloration and concentration, and a finished product of L-7-methyltryptophan is obtained. The method is mild in process reaction condition, high in substrate conversion rate, free of byproduct generation, high in chiral selectivity, environmentally friendly and safe, compared with a traditional chemical synthesis method, the yield is high, the cost is low, and the separation and purification process is easy to operate, short in production period, high in yield and suitable for large-scale industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a method for synthesizing amino acids, and in particular to a method for synthesizing L-7-methyltryptophan by an enzymatic method. Background Art

[0002] Amino acids, as important structural units and starting materials of life substances in organisms, participate in the synthesis of natural products and some complex compounds. Compared with natural amino acids, non-natural amino acids, as the source of natural amino acid derivatives, can be used not only in biosensors, protein probes, enzyme engineering and other fields due to their unique functional groups and chemical properties such as fluorescence, photosensitivity, metal chelation, etc., but also as chiral building blocks in drug precursors, viral vaccines, antibody-drug conjugates and other medical fields, such as 5-aminolevulinic acid, an important precursor of heme, porphyrin, chlorophyll, and vitamin B12, L-2,3-diaminopropionic acid, a key substance for the synthesis of various antibiotics, and β-alanine, a pharmaceutical chemical for the synthesis of basalazine and disodium pamidronate. Tryptophan derivatives also play an important role and can be used as building blocks in the biosynthesis of natural products, such as 5-hydroxytryptophan, 7-chloro-tryptophan, 1-methyltryptophan, L-2-methyl-tryptophan and 7-methyl-tryptophan. 7-Methyl-tryptophan, CAS No 17332-70-6, is a key precursor for the biosynthesis of many non-ribosomal peptide antibiotics and plays an important role in the synthesis of highly effective antibacterial agents and their analogs. Therefore, L-7-methyltryptophan, as an important pharmaceutical intermediate, has received extensive attention in the pharmaceutical field.

[0003] At present, there are few publicly available methods for synthesizing L-7-methyltryptophan, and the main synthetic route is chemical method. Chou-Hsiungchen et al. (Organic & Biomolecular Chemistry, 2014, 12, 9764-9768) used 7-methylindole as raw material, first reacted with phosphorus oxychloride to obtain 7-methylindole-3-carboxylic acid, then reacted with strong base n-butyl lithium to obtain 7-methylindole-3-acetic acid, and then amide condensation, oxidation, palladium carbon catalysis, and hydrolysis reaction to obtain the target product L-7-methyltryptophan.

[0004] This approach has the following disadvantages:

[0005] 1. The use of phosphorus reagents increases the difficulty of post-treatment and makes it difficult to purify the target product.

[0006] 2. The use conditions of the strong base n-butyl lithium are harsh. The reaction needs to be carried out at a low temperature of -78°C in anhydrous and oxygen-free environment, which increases the requirements for equipment and increases production costs.

[0007] 3. A lot of chemical wastewater is generated during the reaction, such as incompletely reacted phosphorus oxychloride, which is difficult to treat.

[0008] 4. The chiral selectivity of 7-methyltryptophan prepared by this method is not high, the reaction steps are too many, and the overall yield is not high.

[0009] Xu Yanjun and others publicly invented a method for synthesizing L-7-methyltryptophan in 2020, which includes four chemical reactions. First, the reaction is carried out under the protection of an inert gas (nitrogen) with an organic solvent, acetic anhydride, as the reaction solvent, and acetylglycine and 7-methylindole-3-carboxaldehyde are reacted at 80-120°C in anhydrous sodium acetate for 2-5 hours. After the reaction, the reaction solution is filtered, washed with water, and dried to obtain the product (1) 4-(7-methyl-1'H-indol-3'-ylmethylene)-2-methyl-5(4H)oxazolone. Secondly, in acetonitrile or tetrahydrofuran solvent, the above product (1) is reacted with water at 80-100°C for 1-5 hours. After the reaction, the reaction solution is cooled to room temperature and methyl tert-butyl ether is added, stirred and filtered, the filter cake is taken, washed with methyl tert-butyl ether, and dried to obtain the product (2) 7-methyl-3-indolyl-2-acetamidoallylic acid. Then, the alcohol solvent methanol is used as the reaction solvent, and hydrogen and the above product (2) are reacted at room temperature for 1-5 hours under the catalysis of Pd / c. After the reaction is completed, Pd / c is removed by filtration, and the filtrate is concentrated under reduced pressure and dried to obtain the product (3) 7-methyl-N-acetyltryptophan. Finally, the above product (3) is reacted in a hydrochloric acid aqueous solution at 70-150°C for 2-5 hours to be hydrolyzed. After the reaction is completed, the reaction liquid is cooled to room temperature, and the pH value of the reaction liquid is adjusted to pH=7-8 with a saturated sodium carbonate aqueous solution, and a white solid is precipitated. The solid is stirred, filtered, and the filter cake is taken and dried to obtain the product L-7-methyltryptophan. The total yield of the synthesis method is 52.6% and the purity is 98%.

[0010] This approach has the following disadvantages:

[0011] 1. This process has harsh reaction conditions, such as high temperature, nitrogen protection, hydrogenation and other steps; the reaction route is long, and separation and purification are difficult; the total yield is not high, the production cost is high, and it is not conducive to industrialization.

[0012] 2. A large number of solvents are used in the synthesis process, such as methyl tert-butyl ether, tetrahydrofuran, and methanol; acid and alkali are used in the extraction process to introduce waste salt. This increases environmental pressure and affects production capacity.

[0013] 3. The reaction solvent, acetic anhydride, is a second-class precursor chemical and a state-controlled chemical. It is flammable and corrosive. Its vapor can form an explosive mixture with air, which can cause combustion and explosion when exposed to open flames and high heat. The reaction temperature is extremely high, up to 120°C. Therefore, the reaction is extremely dangerous.

[0014] 4. This process contains multiple carcinogenic reagents such as tetrahydrofuran, methyl tert-butyl ether, etc., which are not friendly to the health of process workers.

[0015] Therefore, it is necessary to develop an alternative to chemical synthesis, which is mild, efficient, economical, safe, environmentally friendly, low-cost, simple to operate, and easy to industrialize to prepare L-7-methyltryptophan, which is of great significance to the promotion and application of L-7-methyltryptophan in the fields of medicine and so on. Summary of the invention

[0016] The present invention aims at the technical defects of the existing chemical synthesis technology of L-7-methyltryptophan, such as complicated process, unsatisfactory yield, high cost and great environmental pressure, and provides a L-7-methyltryptophan synthase synthesis method with simple process, low cost, suitability for large-scale industrial production and low environmental pressure.

[0017] To achieve the above object, the technical solution provided by the present invention is: a method for synthesizing L-7-methyltryptophan by enzymatic method, comprising the following steps: a. adding L-serine, 7-methylindole, pyridoxal 5-phosphate, water and tryptophan synthase to the reaction system to react and obtain crude L-7-methyltryptophan, wherein the reaction temperature is 25 to 40° C., the reaction pH is 6.5 to 7.5, and the feed mass ratio of L-serine to 7-methylindole is 1:1.3 to 1.5; b. After the reaction is completed, the temperature is lowered and then purified to obtain a purified product; c. decolorizing the purified product to obtain an aqueous solution of L-7-methyltryptophan; d. The L-7-methyltryptophan aqueous solution is concentrated and crystallized to obtain the finished L-7-methyltryptophan.

[0018] The present invention uses a tryptophan synthase as a biocatalyst, 7-methylindole and L-serine as substrates, and catalyzes the synthesis of L-7-methyltryptophan through a one-step reaction. The enzymatic synthesis of L-7-methyltryptophan reaction formula is as follows: The L-7-methyltryptophan conversion process and product extraction process provided by the invention have short flow, no special requirements on equipment, small equipment investment and stable process; the raw materials are easily available, the production cost is low, the conversion rate is high, the finished product yield is high, the product quality is good, the environmental pressure is small, and it is conducive to large-scale industrial production.

[0019] Since the catalyst used in the present invention is tryptophan synthase, which is a biological catalyst, the reaction conditions are mild. If the reaction temperature is too high or too low, the enzyme activity will be affected, thereby affecting the reaction rate. Therefore, the reaction temperature needs to be controlled within the suitable temperature of the enzyme. Meanwhile, if the pH value of the reaction is too high or too low, the enzyme activity will be affected, thereby affecting the enzyme reaction rate. Therefore, the pH value during the reaction needs to be controlled within a neutral state to ensure the enzyme activity and achieve the required purity and yield. Since in the above reaction formula, the reaction ratio of L-tryptophan to 7-methylindole is 1:1, a slightly excessive amount of one substrate is required to achieve complete reaction. Since the raw material of 7-methylindole is relatively cheap, a slightly excessive amount of 7-methylindole is used to ensure complete reaction of L-tryptophan, thereby maximizing the economic benefit. After multiple experiments, it was found that when the feed ratio of L-tryptophan to 7-methylindole is 1:1.3-1.5, the benefit is optimal.

[0020] Preferably, the reaction is considered complete when the conversion of L-7-methyltryptophan is ≥ 95%.

[0021] Further, the preparation process of tryptophan synthetase is: The tryptophan synthase (NP_415777.1, amino acid sequence as shown in SEQ ID NO: 1: MTTLLNPYFGEFGGMYVPQILMPALRQLEEAFVSAQKD) screened from the enzyme library of this experiment was used. PEFQAQFNDLLKNYAGRPTALT KCQNITAGTNTTLYLKREDLLHGGAHKTNQVLGQALLAKRMGKTEIIAETGAGQHGVASA LASALLGLKCRIYMGAKDVERQSPNVFRMRLMGAEVIPVHSGSATLKDACNEALRDWSGS YETAHYMLGTAAGPHPYPTIVREFQRMIGEETKAQILEREGRLPDAVIACVGGGSNAIGM FADFINETNVGLIGVEPGGHGIETGEHGAPLKHGRVGIYFGMKAPMMQTEDGQIEESYSI SAGLDFPSVGPQHAYLNSTGRADYVSITDDEALEAFKTLCLHEGIIPALESSHALAHALKMMRENPDKEQLLVVNLSGRGDKDIFTVHDILKARGEI) was inoculated into a culture medium for culture to obtain a culture solution 1; When the culture medium OD 600=0.4-0.6, inoculate into new culture medium and continue culturing to obtain culture fluid 2; When OD 600 =0.8-1.0, add IPTG for induction to obtain crude tryptophan synthase; The bacteria were collected by low-temperature centrifugation to obtain tryptophan synthase.

[0022] The tryptophan synthase used in the present invention can be obtained from recombinant Escherichia coli containing the target gene of tryptophan synthase, and the fermentation process is simple, the operability is strong, the cost of fermentation raw materials is low, and it is easy to scale up production.

[0023] Furthermore, in the preparation process of tryptophan synthetase: The culture medium is TB liquid culture medium containing 40-60 μg / L kanamycin or LB liquid culture medium containing 40-60 μg / L kanamycin, the culture conditions are 35-40°C, 200-250 rpm, the induction conditions are 25-30°C, 150-200 rpm, the IPTG concentration is 0.18-0.22 mM, and the bacterial collection temperature is 2-8°C.

[0024] Preferably, the culture medium is TB liquid culture medium containing 50 μg / L kanamycin.

[0025] Preferably, the culture conditions are 37°C and 220 rpm.

[0026] Preferably, the induction conditions are 28°C and 180 rpm.

[0027] Preferably, the concentration of IPTG is 0.2 mM.

[0028] Furthermore, the reaction time in step a is 8 to 36 hours, and the feeding amount of pyridoxal 5-phosphate is 0.2 to 0.5 g / L.

[0029] Preferably, the reaction time is 16 h.

[0030] Preferably, the feeding amount of pyridoxal-5-phosphate is 0.4 g / L.

[0031] Preferably, the reaction process can be mechanically stirred at a stirring speed of 200 to 500 r / min.

[0032] Preferably, the stirring speed is selected to be 350 r / min.

[0033] Alternatively, the tryptophan synthase in step a can be selected from crude enzyme liquid, wet bacteria or enzyme powder.

[0034] Preferably, wet cells of tryptophan synthase are used.

[0035] Preferably, the amount of wet bacteria used is 10 g / L.

[0036] Furthermore, in step b, the cooling temperature is 10-25°C; and the purification is divided into two steps, first filtration and then ultrafiltration.

[0037] Furthermore, the filtering method is centrifugal separation or ceramic membrane separation, and the molecular weight cutoff of the ultrafiltration membrane used in ultrafiltration is 1000 to 5000 Daltons.

[0038] Optionally, if the volume of the conversion liquid is small, centrifugal separation is selected; if the volume of the conversion liquid is large, ceramic membrane separation is selected.

[0039] Preferably, the ultrafiltration membrane core selects a membrane cutoff of 2000 Daltons.

[0040] Furthermore, in step c, the decolorizing agent is activated carbon, the decolorizing temperature is 50-80° C., and the decolorizing time is 30-60 min.

[0041] Preferably, the decolorization temperature is 75°C.

[0042] Preferably, the decolorization time is selected to be 30 min.

[0043] Furthermore, the amount of activated carbon used is 1 to 2.5%.

[0044] Furthermore, the concentration method in step d is nanofiltration membrane concentration or reduced pressure concentration, and the crystallization system is a solvent system or a water system.

[0045] Preferably, the concentration method is reduced pressure concentration.

[0046] Preferably, the crystallization system is an aqueous system.

[0047] Furthermore, the temperature of the reduced pressure concentration is 75 to 85°C.

[0048] Preferably, the reduced pressure concentration temperature is selected to be 80°C.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] 1. The present invention uses L-serine and 7-methylindole as substrates and utilizes the specificity of the enzyme to synthesize L-7-methyltryptophan in a one-step reaction, which has high chiral selectivity, simple steps and convenient operation.

[0051] 2. The conversion process of the present invention has a mild reaction, is environmentally friendly and safe.

[0052] 3. The process conversion rate of the present invention is high, the extraction and purification process of the product is simple, the steps are few, the total yield is high, and the cost is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1HPLC spectrum of the finished L-7-methyltryptophan in the embodiment of the present invention; Figure 2 The HPLC chromatogram data of the L-7-methyltryptophan product in Example 1 of the present invention. DETAILED DESCRIPTION

[0054] The present invention will be further described below in conjunction with the embodiments.

[0055] Example 1

[0056] (1) Preparation of tryptophan synthetase: Inoculate 100 mL of LB liquid medium with 100 uL of the recombinant E. coli containing the target gene of tryptophan synthase stored in a glycerol tube and culture at 37°C and 250 rpm until the OD reaches 600 = 0.5; transfer to new LB liquid medium with an inoculum volume of 0.2 vol% and continue to culture. 600 =0.8, add IPTG with a final concentration of 0.2 mM and induce for 16 h at 28°C and 180 rpm; after the induction, collect the bacteria by centrifugation at 4°C and 6500 r / min for 15 min to obtain tryptophan synthase.

[0057] The components of the LB liquid culture medium are: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, pH=7.0, and kanamycin 50 μg / L.

[0058] (2) Enzyme catalyzed reaction: 10 g of L-serine, 13 g of 7-methylindole, 0.4 g of PLP and 1000 g of pure water were added to a reaction container respectively and stirred evenly, and then the pH was adjusted to pH=7, and then the reaction system was heated. When the reaction temperature rose to 30°C, 10 g of wet bacteria was added, and the reaction was kept warm under stirring conditions of 350 r / min; after 16 hours, the reaction solution was taken for high performance liquid chromatography (HPLC) detection, and the L-7-methyltryptophan conversion rate was 96%.

[0059] (3) Separation and purification: After cooling the conversion solution to 20°C, the enzyme and insoluble matter were removed by centrifugation at 6500 rpm for 15 min, and 986 g of the centrifugal supernatant was collected. The centrifugal supernatant was transferred to an ultrafiltration membrane with a molecular weight cutoff of 1000 Daltons for filtration. After filtration, the ultrafiltration membrane concentrate was washed with 1000 g of pure water to obtain 1784 g of ultrafiltration membrane filtrate and 196 g of ultrafiltration membrane concentrate.

[0060] (4) Decolorization: The above 1784g ultrafiltration filtrate was heated to 75°C, 1.5wt% activated carbon was added and decolorized on a decolorization shaker for 30 minutes. When the solution was colorless and transparent, the activated carbon was removed by suction filtration with a 0.22 micron microporous filter membrane to obtain 1752g 7-methyltryptophan aqueous solution.

[0061] (5) Concentration and crystallization: 1752g of L-7-methyltryptophan aqueous solution was concentrated under reduced pressure, and the evaporation temperature was first raised to 60°C during the process of reduced pressure concentration. When the solution was concentrated to 30g, the solution color became colorless and transparent, and no solid was precipitated when cooled, the evaporation temperature was raised to 80°C and continued to concentrate; when the solution quality remained unchanged, the solution color became golden yellow, the texture was viscous, and a large amount of solid was precipitated after cooling, the concentration was stopped, and solid-liquid separation was performed by suction filtration. The separated solid was the L-7-methyltryptophan finished product, a total of 18g. The obtained L-7-methyltryptophan finished product was tested by HPLC, and the HPLC spectrum was as follows Figure 1 As shown, the HPLC spectrum data of L-7-methyltryptophan finished product is as follows Figure 2 shown.

[0062] Comparative Example 1

[0063] The difference between this comparative example and Example 1 is that in step (2), the reaction temperature is 10° C., and the rest of the preparation process is the same as that of Example 1. The specific process is as follows: (1) Preparation of tryptophan synthetase: Inoculate 100 mL of LB liquid medium with 100 uL of the recombinant E. coli containing the target gene of tryptophan synthase stored in a glycerol tube and culture at 37°C and 250 rpm until the OD reaches 600 = 0.5; transfer to new LB liquid medium with an inoculum volume of 0.2 vol% and continue to culture. 600 =0.8, add IPTG with a final concentration of 0.2 mM and induce for 16 h at 28°C and 180 rpm; after the induction, collect the bacteria by centrifugation at 4°C and 6500 r / min for 15 min to obtain tryptophan synthase.

[0064] The components of the LB liquid culture medium are: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, pH=7.0, and kanamycin 50 μg / L.

[0065] (2) Enzyme catalyzed reaction: 10 g of L-serine, 13 g of 7-methylindole, 0.4 g of PLP and 1000 g of pure water were added to the reaction container respectively and stirred evenly, and then the pH was adjusted to pH = 7. The reaction system was then temperature-controlled. When the reaction temperature reached 10°C, 10 g of wet bacteria was added and the reaction was kept warm at 350 r / min stirring conditions. After 16 hours, the reaction solution was taken for high performance liquid chromatography (HPLC) detection.

[0066] (3) Separation and purification: The conversion solution was centrifuged at 6500 rpm for 15 min to remove enzymes and insolubles, and 986 g of the centrifugal supernatant was collected. The centrifugal supernatant was transferred to an ultrafiltration membrane with a molecular weight cutoff of 1000 Daltons for filtration, and after filtration, the ultrafiltration membrane concentrate was washed with 1000 g of pure water to obtain ultrafiltration membrane filtrate and ultrafiltration membrane concentrate.

[0067] (4) Decolorization: The ultrafiltration filtrate was heated to 75°C, 1.5 wt% activated carbon was added and decolorized on a decolorization shaker for 30 minutes. When the solution was colorless and transparent, the activated carbon was removed by suction filtration with a 0.22 μm microporous filter membrane to obtain a 7-methyltryptophan aqueous solution.

[0068] (5) Concentration and crystallization: The L-7-methyltryptophan aqueous solution is concentrated under reduced pressure, and the evaporation temperature is first raised to 60°C during the process of reduced pressure concentration. When the solution is concentrated to 30g, the solution color becomes colorless and transparent, and no solid is precipitated after cooling, the evaporation temperature is raised to 80°C and the concentration is continued; when the solution quality remains unchanged, the solution color becomes golden yellow, the texture is viscous, and a large amount of solid is precipitated after cooling, the concentration is stopped, and solid-liquid separation is performed by suction filtration, and the separated solid is the L-7-methyltryptophan finished product.

[0069] In this comparative example, the HPLC purity of the L-7-methyltryptophan product obtained in step (5) is 90%, and the total yield is 48%.

[0070] Comparative Example 2

[0071] The difference between this comparative example and Example 1 is that in step (2), the reaction temperature is 50° C., and the rest of the preparation process is the same as that of Example 1. The specific process is as follows: (1) Preparation of tryptophan synthetase: Inoculate 100 mL of LB liquid medium with 100 uL of the recombinant E. coli containing the target gene of tryptophan synthase stored in a glycerol tube and culture at 37°C and 250 rpm until the OD reaches 600 = 0.5; transfer to new LB liquid medium with an inoculum volume of 0.2 vol% and continue to culture. 600=0.8, add IPTG with a final concentration of 0.2 mM and induce for 16 h at 28°C and 180 rpm; after the induction, collect the bacteria by centrifugation at 4°C and 6500 r / min for 15 min to obtain tryptophan synthase.

[0072] The components of the LB liquid culture medium are: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, pH=7.0, and kanamycin 50 μg / L.

[0073] (2) Enzyme catalyzed reaction: 10 g of L-serine, 13 g of 7-methylindole, 0.4 g of PLP and 1000 g of pure water were added to the reaction container respectively and stirred evenly, and then the pH was adjusted to pH = 7, and then the reaction system was heated. When the reaction temperature rose to 50°C, 10 g of wet bacteria was added, and the reaction was kept warm under stirring conditions of 350 r / min; after 16 hours, the reaction solution was taken for high performance liquid chromatography (HPLC) detection.

[0074] (3) Separation and purification: After the conversion solution was cooled to 20°C, the enzyme and insoluble matter were removed by centrifugation at 6500rpm for 15min, and the supernatant was collected. The supernatant was transferred to an ultrafiltration membrane with a molecular weight cutoff of 1000 Daltons for filtration. After the filtration was completed, the ultrafiltration membrane concentrate was washed with 1000g of pure water to obtain ultrafiltration membrane filtrate and ultrafiltration membrane concentrate.

[0075] (4) Decolorization: The ultrafiltration filtrate was heated to 75°C, 1.5 wt% activated carbon was added and decolorized on a decolorization shaker for 30 minutes. When the solution was colorless and transparent, the activated carbon was removed by suction filtration with a 0.22 μm microporous filter membrane to obtain a 7-methyltryptophan aqueous solution.

[0076] (5) Concentration and crystallization: The L-7-methyltryptophan aqueous solution is concentrated under reduced pressure, and the evaporation temperature is first raised to 60°C during the process of reduced pressure concentration. When the solution is concentrated to 30g, the solution color becomes colorless and transparent, and no solid is precipitated after cooling, the evaporation temperature is raised to 80°C and the concentration is continued; when the solution quality remains unchanged, the solution color becomes golden yellow, the texture is viscous, and a large amount of solid is precipitated after cooling, the concentration is stopped, and solid-liquid separation is performed by suction filtration, and the separated solid is the L-7-methyltryptophan finished product.

[0077] In this comparative example, the HPLC purity of the L-7-methyltryptophan product obtained in step (5) is 86%, and the total yield is 63%.

[0078] Table 2 Effect of temperature on L-7-methyltryptophan From the data in Table 2, it can be seen that when the reaction temperature is too low or too high, the purity and total yield of the final L-7-methyltryptophan are reduced. The reason is presumably that when the temperature is too high or too low, the activity of tryptophan synthase decreases, which reduces the enzyme reaction rate and affects the production capacity and purity.

[0079] Comparative Example 3

[0080] The difference between this comparative example and Example 1 is that in step (2), 10 g of L-serine, 20 g of 7-methylindole, 0.4 g of PLP and 1000 g of purified water are added to the reaction system respectively, and the rest of the preparation process is the same as that of Example 1. The specific process is as follows: (1) Preparation of tryptophan synthetase: Inoculate 100 mL of LB liquid medium with 100 uL of the recombinant E. coli containing the target gene of tryptophan synthase stored in a glycerol tube and culture at 37°C and 250 rpm until the OD reaches 600 = 0.5; transfer to new LB liquid medium with an inoculum volume of 0.2 vol% and continue to culture. 600 =0.8, add IPTG with a final concentration of 0.2 mM and induce for 16 h at 28°C and 180 rpm; after the induction, collect the bacteria by centrifugation at 4°C and 6500 r / min for 15 min to obtain tryptophan synthase.

[0081] The components of the LB liquid culture medium are: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, pH=7.0, and kanamycin 50 μg / L.

[0082] (2) Enzyme catalyzed reaction: 10 g of L-serine, 20 g of 7-methylindole, 0.4 g of PLP and 1000 g of pure water were added to the reaction container respectively and stirred evenly, and the pH was adjusted to pH = 7. The reaction system was then heated. When the reaction temperature reached 30°C, 10 g of wet bacteria was added and the reaction was kept warm at 350 r / min stirring conditions. After 16 hours, the reaction solution was taken for high performance liquid chromatography (HPLC) detection.

[0083] (3) Separation and purification: After the conversion solution was cooled to 20°C, it was centrifuged at 6500rpm for 15min to remove the enzyme and insoluble matter, and the centrifuged supernatant was transferred to an ultrafiltration membrane with a membrane cutoff molecular weight of 1000 Daltons for filtration. After the filtration was completed, the ultrafiltration membrane concentrate was washed with 1000g of pure water to obtain ultrafiltration membrane filtrate and ultrafiltration membrane concentrate.

[0084] (4) Decolorization: The ultrafiltration filtrate was heated to 75°C, 1.5 wt% activated carbon was added and decolorized on a decolorization shaker for 30 minutes. When the solution was colorless and transparent, the activated carbon was removed by suction filtration with a 0.22 μm microporous filter membrane to obtain a 7-methyltryptophan aqueous solution.

[0085] (5) Concentration and crystallization: The L-7-methyltryptophan aqueous solution is concentrated under reduced pressure, and the evaporation temperature is first raised to 60°C during the process of reduced pressure concentration. When the solution is concentrated to 30g, the solution color becomes colorless and transparent, and no solid is precipitated after cooling, the evaporation temperature is raised to 80°C and the concentration is continued; when the solution quality remains unchanged, the solution color becomes golden yellow, the texture is viscous, and a large amount of solid is precipitated after cooling, the concentration is stopped, and solid-liquid separation is performed by suction filtration, and the separated solid is the L-7-methyltryptophan finished product.

[0086] In this comparative example, the HPLC purity of the L-7-methyltryptophan product obtained in step (5) is 72%, and the total yield is 59%.

[0087] Comparative Example 4

[0088] The difference between this comparative example and Example 1 is that in step (1), 10 g of L-serine, 6 g of 7-methylindole, 0.4 g of PLP and 1000 g of purified water are added to the reaction system respectively, and the rest of the preparation process is the same as that of Example 1. The specific process is as follows: (1) Preparation of tryptophan synthetase: Inoculate 100 mL of LB liquid medium with 100 uL of the recombinant E. coli containing the target gene of tryptophan synthase stored in a glycerol tube and culture at 37°C and 250 rpm until the OD reaches 600 = 0.5; transfer to new LB liquid medium with an inoculum volume of 0.2 vol% and continue to culture. 600 =0.8, add IPTG with a final concentration of 0.2 mM and induce for 16 h at 28°C and 180 rpm; after the induction, collect the bacteria by centrifugation at 4°C and 6500 r / min for 15 min to obtain tryptophan synthase.

[0089] The components of the LB liquid culture medium are: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, pH=7.0, and kanamycin 50 μg / L.

[0090] (2) Enzyme catalyzed reaction: 10 g of L-serine, 6 g of 7-methylindole, 0.4 g of PLP and 1000 g of pure water were added to the reaction container respectively and stirred evenly, and the pH was adjusted to pH = 7. The reaction system was then heated. When the reaction temperature reached 30°C, 10 g of wet bacteria was added and the reaction was kept warm at 350 r / min stirring conditions. After 16 hours, the reaction solution was taken for high performance liquid chromatography (HPLC) detection.

[0091] (3) Separation and purification: After the conversion solution was cooled to 20°C, the enzyme and insoluble matter were removed by centrifugation at 6500rpm for 15min, and the supernatant was collected. The supernatant was transferred to an ultrafiltration membrane with a molecular weight cutoff of 1000 Daltons for filtration. After the filtration was completed, the ultrafiltration membrane concentrate was washed with 1000g of pure water to obtain ultrafiltration membrane filtrate and ultrafiltration membrane concentrate.

[0092] (4) Decolorization: The ultrafiltration filtrate was heated to 75°C, 1.5 wt% activated carbon was added and decolorized on a decolorization shaker for 30 minutes. When the solution was colorless and transparent, the activated carbon was removed by suction filtration with a 0.22 μm microporous filter membrane to obtain a 7-methyltryptophan aqueous solution.

[0093] (5) Concentration and crystallization: The L-7-methyltryptophan aqueous solution is concentrated under reduced pressure, and the evaporation temperature is first raised to 60°C during the process of reduced pressure concentration. When the solution is concentrated to 30g, the solution color becomes colorless and transparent, and no solid is precipitated after cooling, the evaporation temperature is raised to 80°C and the concentration is continued; when the solution quality remains unchanged, the solution color becomes golden yellow, the texture is viscous, and a large amount of solid is precipitated after cooling, the concentration is stopped, and solid-liquid separation is performed by suction filtration, and the separated solid is the L-7-methyltryptophan finished product.

[0094] In this comparative example, the HPLC purity of the L-7-methyltryptophan product obtained in step (5) is 88%, and the total yield is 47%.

[0095] Table 3 Effect of the feeding amount of 7-methyl indole on L-7-methyl tryptophan It can be seen from the data in Table 3 that the purity and total yield of the product L-7-methyltryptophan will decrease when the feeding amount of 7-methylindole is too much or too little. The reason is presumed to be that when the feeding amount of 7-methylindole is too much, 7-methylindole will be excessive and accumulate with the product L-7-methyltryptophan, and the yield and purity caused by the difficulty in separation will decrease; when the feeding amount of 7-methylindole is too little, it will lead to the L-serine process and accumulate with L-7-methyltryptophan. At the same time, due to the high price of L-serine, the economic benefit is low.

[0096] Comparative Example 5

[0097] The difference between this comparative example and Example 1 is that in step (2), the pH is adjusted to pH=5, and the rest of the preparation process is the same as that of Example 1. The specific process is as follows: (1) Preparation of tryptophan synthetase: Inoculate 100 mL of LB liquid medium with 100 uL of the recombinant E. coli containing the target gene of tryptophan synthase stored in a glycerol tube and culture at 37°C and 250 rpm until the OD reaches 600 = 0.5; transfer to new LB liquid medium with an inoculum volume of 0.2 vol% and continue to culture. 600 =0.8, add IPTG with a final concentration of 0.2 mM and induce for 16 h at 28°C and 180 rpm; after the induction, collect the bacteria by centrifugation at 4°C and 6500 r / min for 15 min to obtain tryptophan synthase.

[0098] The components of the LB liquid culture medium are: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, pH=7.0, and kanamycin 50 μg / L.

[0099] (2) Enzyme catalyzed reaction: 10 g of L-serine, 13 g of 7-methylindole, 0.4 g of PLP and 1000 g of pure water were added to the reaction container respectively and stirred evenly, and then the pH was adjusted to pH = 5, and then the reaction system was heated. When the reaction temperature rose to 30°C, 10 g of wet bacteria was added, and the reaction was kept warm under stirring conditions of 350 r / min; after 16 hours, the reaction solution was taken for high performance liquid chromatography (HPLC) detection.

[0100] (3) Separation and purification: After the conversion solution was cooled to 20°C, the enzyme and insoluble matter were removed by centrifugation at 6500rpm for 15min, and the supernatant was collected. The supernatant was transferred to an ultrafiltration membrane with a molecular weight cutoff of 1000 Daltons for filtration. After the filtration was completed, the ultrafiltration membrane concentrate was washed with 1000g of pure water to obtain ultrafiltration membrane filtrate and ultrafiltration membrane concentrate.

[0101] (4) Decolorization: The ultrafiltration filtrate was heated to 75°C, 1.5 wt% activated carbon was added and decolorized on a decolorization shaker for 30 minutes. When the solution was colorless and transparent, the activated carbon was removed by suction filtration with a 0.22 μm microporous filter membrane to obtain a 7-methyltryptophan aqueous solution.

[0102] (5) Concentration and crystallization: The L-7-methyltryptophan aqueous solution is concentrated under reduced pressure, and the evaporation temperature is first raised to 60°C during the process of reduced pressure concentration. When the solution is concentrated to 30g, the solution color becomes colorless and transparent, and no solid is precipitated after cooling, the evaporation temperature is raised to 80°C and the concentration is continued; when the solution quality remains unchanged, the solution color becomes golden yellow, the texture is viscous, and a large amount of solid is precipitated after cooling, the concentration is stopped, and solid-liquid separation is performed by suction filtration, and the separated solid is the L-7-methyltryptophan finished product.

[0103] In this comparative example, the HPLC purity of the L-7-methyltryptophan product obtained in step (5) is 85%, and the total yield is 45%. The product purity and total yield are significantly lower than those in Example 1, indicating that when the reaction conditions are too acidic, the enzyme reaction rate will be significantly reduced, and the product purity and production capacity will be reduced.

[0104] Comparative Example 6

[0105] The difference between this comparative example and Example 1 is that in step (2), the pH is adjusted to pH=6.2, and the rest of the preparation process is the same as that of Example 1. The specific process is as follows: (1) Preparation of tryptophan synthetase: Inoculate 100 mL of LB liquid medium with 100 uL of the recombinant E. coli containing the target gene of tryptophan synthase stored in a glycerol tube and culture at 37°C and 250 rpm until the OD reaches 600 = 0.5; transfer to new LB liquid medium with an inoculum volume of 0.2 vol% and continue to culture. 600 =0.8, add IPTG with a final concentration of 0.2 mM and induce for 16 h at 28°C and 180 rpm; after the induction, collect the bacteria by centrifugation at 4°C and 6500 r / min for 15 min to obtain tryptophan synthase.

[0106] The components of the LB liquid culture medium are: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, pH=7.0, and kanamycin 50 μg / L.

[0107] (2) Enzyme catalyzed reaction: 10 g of L-serine, 13 g of 7-methylindole, 0.4 g of PLP and 1000 g of pure water were added to the reaction container respectively and stirred evenly, and the pH was adjusted to pH = 6.2. Then, the reaction system was heated. When the reaction temperature rose to 30°C, 10 g of wet bacteria was added, and the reaction was kept warm under stirring conditions of 350 r / min; after 16 hours, the reaction solution was taken for high performance liquid chromatography (HPLC) detection.

[0108] (3) Separation and purification: After the conversion solution was cooled to 20°C, the enzyme and insoluble matter were removed by centrifugation at 6500rpm for 15min, and the supernatant was collected. The supernatant was transferred to an ultrafiltration membrane with a molecular weight cutoff of 1000 Daltons for filtration. After the filtration was completed, the ultrafiltration membrane concentrate was washed with 1000g of pure water to obtain ultrafiltration membrane filtrate and ultrafiltration membrane concentrate.

[0109] (4) Decolorization: The ultrafiltration filtrate was heated to 75°C, 1.5 wt% activated carbon was added and decolorized on a decolorization shaker for 30 minutes. When the solution was colorless and transparent, the activated carbon was removed by suction filtration with a 0.22 μm microporous filter membrane to obtain a 7-methyltryptophan aqueous solution.

[0110] (5) Concentration and crystallization: The L-7-methyltryptophan aqueous solution is concentrated under reduced pressure, and the evaporation temperature is first raised to 60°C during the process of reduced pressure concentration. When the solution is concentrated to 30g, the solution color becomes colorless and transparent, and no solid is precipitated after cooling, the evaporation temperature is raised to 80°C and the concentration is continued; when the solution quality remains unchanged, the solution color becomes golden yellow, the texture is viscous, and a large amount of solid is precipitated after cooling, the concentration is stopped, and solid-liquid separation is performed by suction filtration, and the separated solid is the L-7-methyltryptophan finished product.

[0111] In this comparative example, the HPLC purity of the L-7-methyltryptophan product obtained in step (5) is 87%, and the total yield is 68%. The product purity and total yield are significantly lower than those in Example 1, indicating that when the reaction conditions are too acidic, the enzyme reaction rate will be significantly reduced, and the product purity and production capacity will be reduced.

[0112] Comparative Example 7

[0113] The difference between this comparative example and Example 1 is that in step (2), the pH is adjusted to pH=7.8, and the rest of the preparation process is the same as that of Example 1. The specific process is as follows: (1) Preparation of tryptophan synthetase: Inoculate 100 mL of LB liquid medium with 100 uL of the recombinant E. coli containing the target gene of tryptophan synthase stored in a glycerol tube and culture at 37°C and 250 rpm until the OD reaches 600 = 0.5; transfer to new LB liquid medium with an inoculum volume of 0.2 vol% and continue to culture. 600 =0.8, add IPTG with a final concentration of 0.2 mM and induce for 16 h at 28°C and 180 rpm; after the induction, collect the bacteria by centrifugation at 4°C and 6500 r / min for 15 min to obtain tryptophan synthase.

[0114] The components of the LB liquid culture medium are: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, pH=7.0, and kanamycin 50 μg / L.

[0115] (2) Enzyme catalyzed reaction: 10 g of L-serine, 13 g of 7-methylindole, 0.4 g of PLP and 1000 g of pure water were added to the reaction container respectively and stirred evenly, and the pH was adjusted to pH = 7.8. Then, the reaction system was heated. When the reaction temperature rose to 30°C, 10 g of wet bacteria was added, and the reaction was kept warm under stirring conditions of 350 r / min; after 16 hours, the reaction solution was taken for high performance liquid chromatography (HPLC) detection.

[0116] (3) Separation and purification: After the conversion solution was cooled to 20°C, the enzyme and insoluble matter were removed by centrifugation at 6500rpm for 15min, and the supernatant was collected. The supernatant was transferred to an ultrafiltration membrane with a molecular weight cutoff of 1000 Daltons for filtration. After the filtration was completed, the ultrafiltration membrane concentrate was washed with 1000g of pure water to obtain ultrafiltration membrane filtrate and ultrafiltration membrane concentrate.

[0117] (4) Decolorization: The ultrafiltration filtrate was heated to 75°C, 1.5 wt% activated carbon was added and decolorized on a decolorization shaker for 30 minutes. When the solution was colorless and transparent, the activated carbon was removed by suction filtration with a 0.22 μm microporous filter membrane to obtain a 7-methyltryptophan aqueous solution.

[0118] (5) Concentration and crystallization: The L-7-methyltryptophan aqueous solution is concentrated under reduced pressure, and the evaporation temperature is first raised to 60°C during the process of reduced pressure concentration. When the solution is concentrated to 30g, the solution color becomes colorless and transparent, and no solid is precipitated after cooling, the evaporation temperature is raised to 80°C and the concentration is continued; when the solution quality remains unchanged, the solution color becomes golden yellow, the texture is viscous, and a large amount of solid is precipitated after cooling, the concentration is stopped, and solid-liquid separation is performed by suction filtration, and the separated solid is the L-7-methyltryptophan finished product.

[0119] In this comparative example, the HPLC purity of the L-7-methyltryptophan product obtained in step (5) is 89%, and the total yield is 73%. The product purity and total yield are significantly lower than those in Example 1, indicating that when the reaction conditions are too alkaline, the enzyme reaction rate will be significantly reduced, and the product purity and production capacity will be reduced.

[0120] Comparative Example 8

[0121] The difference between this comparative example and Example 1 is that in step (2), the pH is adjusted to pH=9, and the rest of the preparation process is the same as that of Example 1. The specific process is as follows: (1) Preparation of tryptophan synthetase: Inoculate 100 mL of LB liquid medium with 100 uL of the recombinant E. coli containing the target gene of tryptophan synthase stored in a glycerol tube and culture at 37°C and 250 rpm until the OD reaches 600 = 0.5; transfer to new LB liquid medium with an inoculum volume of 0.2 vol% and continue to culture. 600 =0.8, add IPTG with a final concentration of 0.2 mM and induce for 16 h at 28°C and 180 rpm; after the induction, collect the bacteria by centrifugation at 4°C and 6500 r / min for 15 min to obtain tryptophan synthase.

[0122] The components of the LB liquid culture medium are: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, pH=7.0, and kanamycin 50 μg / L.

[0123] (2) Enzyme catalyzed reaction: 10 g of L-serine, 13 g of 7-methylindole, 0.4 g of PLP and 1000 g of pure water were added to the reaction container respectively and stirred evenly, and the pH was adjusted to pH = 9. The reaction system was then heated. When the reaction temperature reached 30°C, 10 g of wet bacteria was added and the reaction was kept warm at 350 r / min stirring conditions. After 16 hours, the reaction solution was taken for high performance liquid chromatography (HPLC) detection.

[0124] (3) Separation and purification: After the conversion solution was cooled to 20°C, the enzyme and insoluble matter were removed by centrifugation at 6500rpm for 15min, and the supernatant was collected. The supernatant was transferred to an ultrafiltration membrane with a molecular weight cutoff of 1000 Daltons for filtration. After the filtration was completed, the ultrafiltration membrane concentrate was washed with 1000g of pure water to obtain ultrafiltration membrane filtrate and ultrafiltration membrane concentrate.

[0125] (4) Decolorization: The ultrafiltration filtrate was heated to 75°C, 1.5 wt% activated carbon was added and decolorized on a decolorization shaker for 30 minutes. When the solution was colorless and transparent, the activated carbon was removed by suction filtration with a 0.22 μm microporous filter membrane to obtain a 7-methyltryptophan aqueous solution.

[0126] (5) Concentration and crystallization: The L-7-methyltryptophan aqueous solution is concentrated under reduced pressure, and the evaporation temperature is first raised to 60°C during the process of reduced pressure concentration. When the solution is concentrated to 30g, the solution color becomes colorless and transparent, and no solid is precipitated after cooling, the evaporation temperature is raised to 80°C and the concentration is continued; when the solution quality remains unchanged, the solution color becomes golden yellow, the texture is viscous, and a large amount of solid is precipitated after cooling, the concentration is stopped, and solid-liquid separation is performed by suction filtration, and the separated solid is the L-7-methyltryptophan finished product.

[0127] In this comparative example, the HPLC purity of the L-7-methyltryptophan product obtained in step (5) is 80%, and the total yield is 52%. The product purity and total yield are significantly lower than those in Example 1, indicating that when the reaction conditions are too alkaline, the enzyme reaction rate will be significantly reduced, and the product purity and production capacity will be reduced.

[0128] Table 4 Effect of pH on L-7-methyltryptophan According to the data in Table 4, when the reaction pH is too acidic or too alkaline, the enzyme reaction rate will be significantly reduced, affecting the purity and total yield of the product L-7-methyltryptophan. The reason is speculated to be that too high or too low pH value will lead to a decrease in the activity of the main reaction catalyst tryptophan synthase, slowing down the reaction temperature and reducing the total yield of the final product. At the same time, the purity of the product is low due to incomplete reaction.

[0129] Example 2

[0130] (1) Preparation of tryptophan synthetase: Inoculate 100 mL of TB liquid medium with 100 uL of recombinant E. coli containing the target gene of tryptophan synthase stored in a glycerol tube and culture at 37 °C and 250 rpm until the OD reaches 600 = 0.5; transfer to new TB liquid medium with an inoculum volume of 0.2 vol% and continue to culture. 600 =0.8, add IPTG with a final concentration of 0.2mM and induce for 16h at 28℃, 180rpm; after the induction, collect the bacteria by centrifugation at 4℃, 6500r / min for 15min, dilute the collected wet bacteria with water to a final concentration of 20%, and then break the cells with a high-pressure homogenizer at low temperature. The obtained cell lysate is used as a donor of tryptophan synthase for the following reaction.

[0131] Among them, the components of TB liquid culture medium are: peptone 12g / L, yeast powder 24g / L, potassium dihydrogen phosphate 2.31g / L, dipotassium hydrogen phosphate 12.54g / L, glycerol 4g / L, kanamycin 50μg / L.

[0132] (2) Enzyme catalyzed reaction: 50 g of L-serine, 65 g of 7-methylindole, 2 g of PLP and 5000 g of purified water were added to a reaction kettle and stirred evenly. After adjusting the pH to pH=7, the reaction system was heated. When the reaction temperature reached 30°C, 250 g of the cell lysate obtained in step (1) was added, and the mixture was stirred at 200 r / min and kept warm for reaction. After 20 hours, the reaction solution was taken for high performance liquid chromatography (HPLC) detection, and the conversion rate of L-7-methyltryptophan was 94%.

[0133] (3) Separation and purification: After the inside of the reactor was cooled to 20°C, the conversion liquid was transferred to a plate and frame for filtration, and the cell lysate and bacterial residue were preliminarily removed to obtain 4980g of plate and frame filtrate. The impurities in the plate and frame filtrate and the unreacted substrate were filtered again using a 30nm ceramic membrane. After the filtration was completed, the ceramic membrane concentrate was washed with 2000g of pure water to obtain 6682g of ceramic membrane filtrate and 302g of ceramic membrane concentrate. The ceramic membrane filtrate was transferred to an ultrafiltration membrane with a membrane retention molecular weight of 2000 Daltons for filtration. After the filtration was completed, the ultrafiltration membrane concentrate was washed with 2000g of pure water to finally obtain 8500g of ultrafiltration membrane filtrate and 180g of ultrafiltration membrane concentrate.

[0134] (4) Decolorization: The above 8500 g ultrafiltration filtrate was heated to 75° C., 1.5 wt % activated carbon was added and stirred at 60 r / min for 60 min. When the solution became colorless and transparent, the activated carbon was removed by centrifugation using a plate centrifuge to obtain 8.35 kg of 7-methyltryptophan aqueous solution.

[0135] (5) Concentration and crystallization: 8350g of L-7-methyltryptophan aqueous solution was concentrated by nanofiltration membrane until the concentrated solution reached 300g. When a large amount of solid was precipitated after the concentrated solution was cooled, the concentration was stopped. The solid-liquid separation was performed by suction filtration. The separated solid was 85g of L-7-methyltryptophan product. The HPLC purity of the L-7-methyltryptophan product was 96% and the total yield was 79%.

[0136] Example 3

[0137] (1) Preparation of tryptophan synthetase: Inoculate 100 mL of TB liquid medium with 100 uL of recombinant E. coli containing the target gene of tryptophan synthase stored in a glycerol tube and culture at 37 °C and 250 rpm until the OD reaches 600 = 0.5; transfer to new TB liquid medium with an inoculum volume of 0.2 vol% and continue to culture. 600=1.0, add IPTG with a final concentration of 0.2mM and induce for 16h at 28℃ and 180rpm; after the induction, centrifuge at 4℃ and 6500r / min for 15min to collect the bacteria, dilute the collected wet bacteria with PBS to a final concentration of 20%, and then perform ultrasonic cell disruption at low temperature. Centrifuge the cell disruption solution at 4℃ and 6500r / min for 15min to remove the precipitate and collect the supernatant. The supernatant is freeze-dried to obtain a powder, which is the tryptophan synthase enzyme powder.

[0138] The components of TB liquid culture medium are: peptone 12g / L, yeast powder 24g / L, potassium dihydrogen phosphate 2.31g / L, dipotassium hydrogen phosphate 12.54g / L, glycerol 4g / L, kanamycin 50μg / L.

[0139] (2) Enzyme catalyzed reaction: 5 g of L-serine, 6.5 g of 7-methylindole, 0.2 g of PLP and 500 g of purified water were added to a three-necked flask, and the pH was adjusted to pH=7, and stirring was started and the temperature was increased. When the reaction temperature reached 35°C, 2.5 g of enzyme powder was added, and the reaction was kept warm at 250 r / min stirring condition. After 12 hours, the reaction solution was taken for high performance liquid chromatography (HPLC) detection, and the conversion rate of L-7-methyltryptophan was 96%.

[0140] (3) Separation and purification: The conversion solution was cooled to 20°C in an ice bath and centrifuged at 6500 rpm for 15 min to remove the enzyme and insoluble matter. 496 g of the centrifugal supernatant was collected and transferred to an ultrafiltration membrane with a membrane cutoff molecular weight of 2000 Daltons for filtration. After completion, the ultrafiltration membrane concentrate was washed with 1000 g of pure water to obtain 1456 g of ultrafiltration membrane filtrate and 37 g of ultrafiltration membrane concentrate.

[0141] (4) Decolorization: The above 1456 g ultrafiltration filtrate was heated to 75° C., 1.5 wt % activated carbon was added and decolorized on a decolorization shaker for 30 min. When the solution was colorless and transparent, the activated carbon was removed by suction filtration with a 0.22 μm microporous filter membrane to obtain 1432 g 7-methyltryptophan aqueous solution.

[0142] (5) Concentration and crystallization: 1752g of L-7-methyltryptophan aqueous solution was concentrated under reduced pressure, and the evaporation temperature was first raised to 60°C during the process of reduced pressure concentration. When the solution was concentrated to 40g, the solution color became colorless and transparent, and no solid was precipitated after cooling, the evaporation temperature was raised to 80°C and the concentration was continued; when the solution quality remained unchanged, the solution color became golden yellow, the texture was viscous, and a large amount of solid was precipitated after adding 100g of anhydrous ethanol and cooling, the concentration was stopped, and solid-liquid separation was performed by suction filtration. The separated solid was obtained as the L-7-methyltryptophan finished product, a total of 8.6g, the HPLC purity of the L-7-methyltryptophan finished product was 98%, and the total yield was 80%.

[0143] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0144] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for enzymatically synthesizing L-7-methyltryptophan, characterized in that: There are the following steps: a. adding L-serine, 7-methylindole, pyridoxal 5-phosphate, water and tryptophan synthase to a reaction system to react and obtain crude L-7-methyltryptophan, wherein the reaction temperature is 25 to 40° C., the reaction pH is 6.5 to 7.5, and the feed mass ratio of L-serine to 7-methylindole is 1:1.3 to 1.5; b. After the reaction is completed, the temperature is lowered and then purified to obtain a purified product; c. decolorizing the purified product to obtain an aqueous solution of L-7-methyltryptophan; d. The L-7-methyltryptophan aqueous solution is concentrated and crystallized to obtain the finished L-7-methyltryptophan.

2. The method for enzymatically synthesizing L-7-methyltryptophan according to claim 1, wherein: The amino acid sequence of the tryptophan synthase described in step a is shown in SEQ ID NO:

1.

3. A method for enzymatically synthesizing L-7-methyltryptophan according to claim 1 or 2, characterized in that: The preparation process of the tryptophan synthetase described in step a is: Inoculating the recombinant Escherichia coli containing the target gene of tryptophan synthase into the culture medium for cultivation to obtain culture solution 1; When the culture medium OD 600 =0.4-0.6, inoculate into new culture medium and continue culturing to obtain culture medium 2; When OD 600 =0.8~1.0, add IPTG for induction to obtain crude tryptophan synthase; The bacterial cells were collected by centrifugation to obtain tryptophan synthase.

4. A method for enzymatically synthesizing L-7-methyltryptophan according to claim 3, characterized in that: The culture medium is TB liquid medium containing 40-60 µg / L kanamycin or LB liquid medium containing 40-60 µg / L kanamycin; The culture conditions were 35-40°C and 200-250 rpm; The inoculation volume is 0.15~0.25vol%; The induction conditions were 25-30°C and 150-200 rpm; The concentration of IPTG is 0.18~0.22mM; The collection temperature of the bacteria is 2~8℃.

5. The method for enzymatically synthesizing L-7-methyltryptophan according to claim 1, wherein: In step a, The reaction time is 8~36 hours; The dosage of pyridoxal 5-phosphate is 0.2~0.5g / L.

6. A method for enzymatically synthesizing L-7-methyltryptophan according to claim 1 or 5, characterized in that: In step a, the tryptophan synthase is a crude enzyme solution, wet bacteria or enzyme powder.

7. The method for enzymatically synthesizing L-7-methyltryptophan according to claim 1, characterized in that: In step b, The cooling temperature is 10~25℃; The purification was divided into two steps, filtration followed by ultrafiltration.

8. The method for enzymatically synthesizing L-7-methyltryptophan according to claim 7, wherein: The filtration method is centrifugal separation or ceramic membrane separation; The molecular weight cutoff of the ultrafiltration membrane used in ultrafiltration is 1000~5000 Daltons.

9. The method for enzymatically synthesizing L-7-methyltryptophan according to claim 1, wherein: Step c middle The decolorizing agent is activated carbon, and the amount of activated carbon used is 1~2.5wt%; The decolorization temperature is 50~80℃; The decolorization time is 30~60min.

10. The method for enzymatically synthesizing L-7-methyltryptophan according to claim 1, characterized in that: In step d The concentration method is nanofiltration membrane concentration or reduced pressure concentration; The crystallization system is a solvent system / water system.

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