CN101525641A - Method for producing L-tryptophan by microbial enzyme method - Google Patents

Abstract

A method for microbial enzyme production of L-tryptophan. The present invention uses Pseudomonas putida TS1138 (Pseudomonas putida TS1138) and whole cells of Escherichia coli cells highly expressing tryptophanase, cell lysates or immobilized enzymes as enzyme sources, from the substrate DL-2-amino- Δ ² -thiazoline-4-carboxylic acid (DL-2-amino-Δ ² -thiazoline-4-carboxylic acid, DL-ATC) is converted into L-color through two-step continuous or one-step mixed enzymatic reaction The purification method is to use S8 type macroporous resin to remove the residual DL-2-amino-Δ ² -thiazoline-4-carboxylic acid and indole in the reaction solution containing L-tryptophan, and then at a pH of Under the condition of 5, use NKA-II type macroporous resin to adsorb L-tryptophan, eliminate the interference of L-cysteine, and finally elute with ethanol, concentrate and dry under reduced pressure to obtain L-tryptophan, thus providing A new green production process route of L-tryptophan. Because the production cost of the substrate DL-ATC is relatively low, the invention has broad application prospects in the field of industrial production of L-tryptophan.

Description

A kind of method that microorganism enzymatically produces L-tryptophan

【Technical field】：

The invention belongs to the technical field of amino acid production, and relates to a method for producing L-tryptophan by transforming a substrate precursor through microbial enzymatic method.

【Background technique】:

L-tryptophan is an important essential amino acid in humans and animals, and it has a very wide range of uses in the fields of medicine, food and feed. In recent years, due to the rapid development of the feed industry and the continuous expansion of the use of L-tryptophan in the pharmaceutical industry, the demand for L-tryptophan at home and abroad is increasing day by day. However, due to the high difficulty and high price in the production of L-tryptophan for a long time, except for medical use, L-tryptophan has not been widely used in other fields.

L-tryptophan is one of the most difficult amino acids to produce by direct fermentation. Its production methods mainly include proteolytic extraction, chemical synthesis, microbial fermentation and enzymatic synthesis. Among them, the enzymatic synthesis method uses chemically synthesized precursors as raw materials, which not only fully utilizes the advantages of organic synthesis technology, but also has the advantages of high product concentration, high yield, high purity, few by-products and easy refining operations. A good way to produce L-tryptophan cheaply and respected at present.

The enzymatic synthesis of L-tryptophan mainly involves tryptophan synthase (EC 4.2.1.20) and tryptophanase (EC 4.1.99.1), both of which can catalyze the synthesis of L-chrome from L-serine and indole acid. Since indole has a strong inhibitory effect on tryptophan synthase, and tryptophanase has good stability to indole, people have paid more attention to the role of tryptophanase in L-tryptophan biosynthesis in recent years. Applications.

Tryptophanase normally decomposes L-tryptophan to generate pyruvate, indole and ammonia, but it can also effectively catalyze the synthesis of L-tryptophan from pyruvate, indole and ammonia under conditions of high concentrations of pyruvate and ammonia acid. The enzyme can also catalyze the synthesis of L-tryptophan from L-serine or L-cysteine and indole. In the pathway of tryptophanase catalyzing the synthesis of L-tryptophan from pyruvate, indole and ammonia, the substrate indole has a weak inhibitory effect on tryptophanase and the price of pyruvate is not high, so it has certain practicability , but this pathway is the reverse reaction of tryptophan hydrolysis, which requires a higher substrate concentration, and the reaction balance is not easy to grasp. In the route of synthesizing L-tryptophan from L-serine and indole as raw materials, since the price of the substrate L-serine is almost equivalent to that of L-tryptophan, it is not practical. In the way of using tryptophanase to catalyze L-cysteine and indole to synthesize L-tryptophan, since the production cost of the substrate L-cysteine is relatively low, this way has important industrial application value.

In the way of enzymatic conversion of L-cysteine and indole to synthesize L-tryptophan, the main difficulty in separating and purifying the product L-tryptophan is that the composition of the reaction solution is relatively complex, and the residual indole and L- Substances such as cysteine and the product L-tryptophan are not easy to separate, and no mature and feasible method has been reported yet.

【Invention content】：

The purpose of the present invention is to solve the above-mentioned problems existing in the prior art, and provide a new method for microbial enzymatic production of L-tryptophan.

The present invention relates to the use of Pseudomonas putida TS1138 (Pseudomonas putida TS1138) cells and Escherichia coli whole cells expressing tryptophanase efficiently, cell lysates or immobilized enzymes as enzyme sources, from the substrate DL-2- Starting from amino-Δ ² -thiazoline-4-carboxylic acid (DL-2-amino-Δ ² -thiazoline-4-carboxylic acid, DL-ATC), the L- Tryptophan, and the final separation and purification process to obtain the product L-tryptophan.

The present invention isolates and obtains a Pseudomonas putida TS1138 bacterial strain (Pseudomonas putida TS-1138) by itself, and the bacterial strain is preserved in the "Common Microorganism Center of the China Microbiological Culture Collection Management Committee", and the preservation number is CGMCC No.1920. The contained L-cysteine synthetase system can catalyze the substrate DL-ATC to synthesize L-cysteine. In addition, the present invention has also constructed an Escherichia coli genetically engineered strain that expresses tryptophanase efficiently, that is, using the Escherichia coli JM109 strain genome DNA as a template, PCR amplification to obtain the tryptophanase gene, and using it in Escherichia coli BL21 ( DE3) for expression, the tryptophanase activity of the recombinant expression reached 110 times that of the host bacterium.

Using the whole cells of the bacteria of the above two strains, the lysate of the bacteria or the immobilized enzyme as the enzyme source, the present invention respectively establishes the enzymatic reaction biosynthesis of L-color through two-step continuous or one-step mixing of L-cysteine A new method for amino acid, the synthetic route is shown in Figure 1.

The method for microbial enzymatic production of L-tryptophan provided by the invention specifically includes:

1. Two-step continuous enzymatic reaction

1.1. First, use the whole cell or lysate of Pseudomonas putida TS1138 cells as the enzyme source, and enzymatically convert the substrate DL-2-amino- ^Δ2 -thiazoline-4-carboxylic acid to obtain L- Cysteine reaction solution;

1.2. Then use the whole cell of E. coli thallus, lysate or immobilized enzyme of E. coli thallus highly expressing tryptophanase as the enzyme source, and use the above-mentioned L-cysteine reaction solution and indole as the substrate. Enzymatic conversion to synthesize L-tryptophan;

or,

2. One-step mixed enzymatic reaction

Using the whole cell or cell lysate of Pseudomonas putida TS1138 and the whole cell, cell lysate or immobilized enzyme of Escherichia coli highly expressing tryptophanase as the mixed enzyme source, the enzymatic transformation substrate L-tryptophan can be synthesized from DL-2-amino-Δ ² -thiazoline-4-carboxylic acid and indole.

In the described two-step continuous enzymatic reaction process, add coenzyme pyridoxal phosphate and hydroxylamine as the inhibitor of L-cysteine desulfhydrylase simultaneously, the consumption of described coenzyme pyridoxal phosphate is 0.05g/ L to 1g/L, the final concentration of hydroxylamine is 0.5mmol/L to 3.0mmol/L; the amount of TS1138 cells is generally 50g/L to 500g/L, and the amount of cell lysate is generally 10g to 100g cells lysate; the amount of E. coli cells that express tryptophanase is generally 1g/L to 100g/L, the amount of cell lysate is generally 0.5g to 30g cell lysate, and the amount of immobilized enzyme is generally 1g/L to 50g/L; the concentration of the substrate DL-2-amino- ^Δ2 -thiazoline-4-carboxylic acid is 1g/L to 15g/L; the dosage of the substrate indole is 1g/L to 20g/L .

The consumption of described coenzyme pyridoxal phosphate is preferably 0.15g/L, the final concentration of hydroxylamine used is preferably 1.0mmol/L; the dosage of TS1138 cells is preferably 150g/L, and the dosage of cell lysate is preferably 30g cells The lysate of the lysate; the Escherichia coli thalline dosage of high-efficiency expression tryptophanase is preferably 10g/L, the lysate of thalline lysate dosage is preferably the lysate of 3g bacterial cells, and the dosage of immobilized enzyme is preferably 15g/L; substrate DL The final concentration of -2-amino- ^{Δ 2} -thiazoline-4-carboxylic acid is preferably 6 g/L; the dosage of substrate indole is preferably 4.5 g/L.

In the one-step mixed enzymatic reaction process, the coenzyme pyridoxal phosphate can be added at the same time, and its dosage is 0.05g/L to 1g/L; the dosage of TS1138 cells is generally 50g/L to 500g/L, and the cells are lysed The amount of solution is generally 10g to 100g of cell lysate; the amount of E. coli cells that express tryptophanase is generally 1g/L to 100g/L, and the amount of cell lysate is generally 0.5g to 30g of cells lysate, the amount of immobilized enzyme is generally 1g/L to 50g/L; the final concentration of the substrate DL-2-amino-Δ ² -thiazoline-4-carboxylic acid is 1g/L to 15g/L, indole The dosage is 1g/L to 20g/L.

The dosage of coenzyme pyridoxal phosphate is preferably 0.15g/L, the dosage of TS1138 cells is preferably 200g/L, and the dosage of cell lysate is preferably the lysate of 40g cells; the dosage of Escherichia coli cells highly expressing tryptophanase Preferably 15g/L, the lysate dosage of thalline lysate is preferably the lysate of 5g thalline cells, and the ^dosage of immobilized enzyme is preferably 20g/L; The final concentration is preferably 6g/L, and the amount of indole is preferably 5g/L.

The separation and purification method of L-tryptophan synthesized by the present invention is to use S8 type macroporous resin to remove DL-2-amino-Δ ² -thiazoline-4-carboxylic acid remaining in the reaction solution containing L-tryptophan and indole, and then use NKA-II type macroporous resin to adsorb L-tryptophan under the condition of pH 5 to eliminate the interference of L-cysteine, and finally elute with ethanol, concentrate and dry under reduced pressure to obtain L - Tryptophan.

In the embodiment of the present invention, the Gaitonde method is used to measure the content of L-cysteine, and the specific method is as follows: Accurately weigh a certain amount of L-cysteine standard substance, dissolve it in a certain amount of 0.05mol/L HCl, Prepare a standard mother solution with a final concentration of 500 mg/L, and further dilute the above mother solution with distilled water to 10, 20, ..., 100 mg/L standard solutions. Take a certain amount of standard solution or 0.2mL of the diluent of the sample solution to be tested, add 0.2mL of glacial acetic acid, then add 0.2mL of acidic ninhydrin reagent, react in a boiling water bath for 10min, then immediately cool in cold water, and finally add 2.4 mL of alcohol to make a total volume of 3 mL. After standing for 10min, measure its absorbance at 560nm, and draw a standard curve with concentration and absorbance. The L-cysteine concentration in the unknown solution was calculated from this standard curve.

In the embodiment of the present invention, high performance liquid chromatography is used to determine the content of L-tryptophan, and the specific method is as follows: 20 μL of L-tryptophan standard solution with a concentration of 50 to 1,500 mg/L is respectively injected into the sample, continuously Samples were injected 5 times, peak heights were calculated and averaged, and a standard curve was drawn with L-tryptophan concentration (X, mg/L) as the abscissa and peak height (Y) as the ordinate. The L-tryptophan produced by the enzymatic reaction can be accurately quantified after being detected by high performance liquid chromatography and compared with the standard curve.

Beneficial effects of the present invention: The present invention starts from the chemically synthesized substrate DL-ATC, and utilizes the whole cells of Pseudomonas putida TS1138 and Escherichia coli cells highly expressing tryptophanase, cell lysates or immobilized enzymes As an enzyme source, L-tryptophan is produced through two-step continuous and one-step mixed enzymatic reactions. Because the production cost of the substrate DL-ATC is relatively low, the present invention has broad application prospects in the field of industrial production of L-tryptophan.

【Description of drawings】

Figure 1: Schematic diagram of the route of enzymatic conversion of DL-ATC to L-tryptophan,

Figure 2: High performance liquid chromatography detection of L-tryptophan in the reaction solution and after purification,

(A), reaction solution (ultraviolet detection); (B), reaction solution (evaporative light scattering detection); (C), purified L-tryptophan (ultraviolet detection); (D), purified L- Tryptophan (detected by evaporative light diffuser); chromatographic peaks are: 1, L-tryptophan (retention time, 4.732min); 2, indole (retention time, 5.965min); 3, L-cysteine Amino acid (retention time, 3.223min).

The Pseudomonas putida TS1138 described in the present invention, classified and named: Pseudomonas putida, has been preserved in the "Common Microbiology Center of China Microbiological Culture Collection Management Committee" on January 17, 2007, and the address at the time of preservation: Zhongguancun, Haidian District, Beijing In the Institute of Microbiology, Chinese Academy of Sciences, the preservation number is CGMCC No.1920. Meanwhile, the bacterial strain has been recorded in the patent application document No. 200710056754.9 published on October 17, 2007.

【Detailed ways】

Example 1

Cloning and Expression of Escherichia coli Tryptophanase Gene

According to the tryptophanase gene sequence of Escherichia coli K12 strain, the upstream primer 5'-CCGGAATTCATGGAAAACTTTAAACATCTCC-3' and the downstream primer 5'-CCCAAGCTTTTAAACTTCTTTCAGTTTTGCGG-3' of the tryptophanase gene were designed. Using the genomic DNA of Escherichia coli DH5α, JM109 and BL21 strains as templates, the tryptophanase gene was amplified by PCR, and the recombinant expression plasmids pET-tnaA(D), pET-tnaA(J) and pET-tnaA( B).

The three recombinant plasmids were respectively transformed into Escherichia coli BL21(DE3), and respective tryptophanase genetically engineered strains were constructed. Inoculate the recombinant strain in 4 mL of LB medium containing 100 μg/mL of ampicillin, culture with shaking at 37°C for 12-16 hours, then transfer to 50 mL of fresh LB medium with 1% inoculum amount, continue to cultivate until the _OD600 reaches 0.5, add The final concentration of IPTG was 0.1mmol/L, and the expression was induced at 37°C for 2 hours. The collected cells were washed with potassium phosphate buffer (pH 8.0) and then resuspended, ultrasonically disrupted for 5 minutes to fully lyse the cells, and the supernatant was collected after centrifugation to measure the activity of the recombinantly expressed tryptophanase. The results showed that the tryptophanase gene recombination expression products derived from three different strains of Escherichia coli had similar enzyme activities, all reaching more than 100 times the tryptophanase activity of the host bacteria itself (Table 1).

Table 1 Tryptophanase activity of different bacterial strains

All values are mean ± standard deviation (Mean value ± S.D.) of three parallel experiments.

Example 2

Preparation of Enzyme Source of Pseudomonas putida TS1138 Strain

Pick a ring of Pseudomonas putida TS1138 from the plate and insert it into 4mL seed medium (glucose 20g, ATC 3g, corn steep liquor 5g, urea 3g, NaCl 1.5g, MnSO ₄ ·H ₂ O 0.1g, K ₂ HPO ₄ 3g, MgSO ₄ 7H ₂ O 0.5g, FeSO ₄ 7H ₂ O 0.01g, constant volume to 1L, pH 7.5), culture at 160r/min at 28°C for 16h, transfer to 50mL enzyme-producing culture with 1% inoculum Base (glucose 30g, ATC 4g, corn steep liquor 1g, urea 3g, NaCl 1.5g, MnSO ₄ ·H ₂ O 0.1g, K ₂ HPO ₄ 3g, MgSO ₄ 7H ₂ O 0.5g, FeSO4·7H ₂ O 0.01 g, set the volume to 1L, pH 7.5), incubate at 160r/min at 28°C for 16 hours, centrifuge, collect the bacteria, wash the bacteria 1 to 2 times with glycine-sodium hydroxide buffer (pH 8.0), weigh a certain amount The above bacterium was added to glycine-sodium hydroxide buffer to prepare the enzyme source cell suspension.

The enzyme source cells are ultrasonically disrupted, and the supernatant is collected after centrifugation to obtain the cell lysate.

Example 3

Preparation of Escherichia coli Enzyme Source Highly Expressing Tryptophanase

Inoculate the E.coli BL21(DE3) recombinant strain containing pET-tnaA(J) prepared in Example 1 in 4 mL of LB medium containing 100 μg/mL of ampicillin, and shake at 37°C for 12 to 16 hours, then add 1% The inoculum was transferred to 50 mL of fresh LB medium, and the culture was continued until the OD ₆₀₀ reached 0.5. IPTG was added at a final concentration of 0.1 mmol/L, and the expression was induced at 37°C for 2 hours. The collected cells were washed with potassium phosphate buffer solution (pH 8.0) for 1 to 2 times, and a certain amount of the above cells was weighed, and potassium phosphate buffer solution was added to obtain the enzyme source cell suspension. The enzyme source cells are disrupted by ultrasonic waves, and the supernatant is collected after centrifugation to obtain the cell lysate.

Weigh 1g of DEAE-22 cellulose, add 40mL of 3.5% glutaraldehyde, stir at room temperature for 5h, let it stand overnight, centrifuge to remove the supernatant, wash the obtained carrier repeatedly with distilled water to remove residual glutaraldehyde, and filter That is, the cross-linked carrier is obtained. Add 30 mL of cell lysate with a soluble protein content of 15 g/L to the cross-linked carrier, let stand at 4°C for 12 h, and stir once every 0.5 h. Centrifuge, discard the supernatant, wash the precipitate with distilled water repeatedly to remove free lysate, and obtain the immobilized enzyme after suction filtration.

Example 4

Using the whole cell of the bacteria as the enzyme source, L-tryptophan is synthesized through two-step continuous enzymatic reactions

In a container with an inner volume of 500mL and equipped with a stirrer, prepare 300mL of enzymatic reaction solution, including 6g/L DL-ATC, 6g/L K ₂ HPO ₄ , 1.0mmol/L hydroxylamine and 150g/L of Example 2 The TS1138 strain enzyme source cells prepared in , were adjusted to pH 8.0 with 5% potassium hydroxide aqueous solution, placed in a constant temperature water bath at 42° C., and stirred for 2.5 hours.

After the reaction is completed, centrifuge at 12,000r/min for 5min at 4°C to separate the solid from the liquid. The supernatant is a mixture containing L-cysteine, and the L-cysteine content is 4.8g/L. , the molar conversion of the substrate DL-ATC was 96.5%.

In the above-mentioned reaction solution containing L-cysteine, add indole with a final concentration of 4.5g/L, coenzyme pyridoxal phosphate of 0.15g/L and Escherichia coli with high expression of tryptophanase at 10g/L For the enzyme source cells, the pH value was adjusted to 8.0 with 5% aqueous potassium hydroxide solution, and stirred and reacted for 3 hours in a constant temperature water bath at 45°C.

After the reaction is completed, centrifuge at 12,000r/min for 5min at 4°C to separate the solid from the liquid. The supernatant is a mixture containing L-tryptophan. The content of L-tryptophan in it is detected to be 6.2g/L. The molar conversion rate of product L-cysteine was 76.6%, the molar conversion rate of indole was 79.0%, and the total molar conversion rate from substrate DL-ATC to product L-tryptophan was 73.9%.

The determination of the above-mentioned L-cysteine content adopts the Gaitonde method, and the specific method is as follows: Accurately weigh a certain amount of L-cysteine standard substance, dissolve it in a certain amount of 0.05mol/L HCl, and prepare a final concentration of 500mg/L The standard mother solution of the above-mentioned mother solution is further diluted with distilled water to be 10, 20,..., 100mg/L standard solution respectively. Take a certain amount of standard solution or 0.2mL of the diluent of the sample solution to be tested, add 0.2mL of glacial acetic acid, then add 0.2mL of acidic ninhydrin reagent, react in a boiling water bath for 10min, then immediately cool in cold water, and finally add 2.4 mL of alcohol to make a total volume of 3 mL. After standing for 10min, measure its absorbance at 560nm, and draw a standard curve with concentration and absorbance. The L-cysteine concentration in the unknown solution was calculated from this standard curve.

The determination of L-tryptophan content adopts high-performance liquid chromatography, and the specific method is as follows: take 20 μL of L-tryptophan standard solution with a concentration of 50-1,500 mg/L and inject 5 times continuously, calculate the peak height and calculate the peak height. Take the average value, and draw a standard curve with the L-tryptophan concentration (X, mg/L) as the abscissa and the peak height (Y) as the ordinate. The L-tryptophan produced by the enzymatic reaction can be accurately quantified after being detected by high performance liquid chromatography and compared with the standard curve.

Example 5

Synthesis of L-tryptophan through a one-step mixed enzymatic reaction using bacterial cell lysate as an enzyme source

In a container with an inner volume of 500mL and equipped with a stirrer, prepare 300mL of enzymatic reaction solution, containing 6g/L DL-ATC, 0.6% K ₂ HPO ₄ , 1.0mmol/L hydroxylamine, 5g/L indole, 0.15 g/L pyridoxal phosphate, the lysate of 25g TS1138 strain thalline and 5g lysate of Escherichia coli expressing tryptophanase efficiently, adjust the pH value to 8.0 with 5% potassium hydroxide aqueous solution, place in 42 ℃ constant temperature water bath, stirred for 3h.

After the reaction was completed, it was centrifuged at 12,000r/min for 5min at 4°C to separate the solid from the liquid. The supernatant was a mixture containing L-tryptophan, and the content of L-tryptophan was detected to be 6.7g/L (method Same example 4), the molar conversion rate of substrate DL-ATC is 79.9%, and the molar conversion rate of indole is 76.9%.

Example 6

Synthesis of L-Tryptophan by Two-step Continuous Enzymatic Reaction Using Immobilized Enzyme as Enzyme Source

In a container with an inner volume of 500mL and equipped with a stirrer, prepare 300mL of enzymatic reaction solution, containing 6g/L DL-ATC, 6g/L K ₂ HPO ₄ , 1.0mmol/L hydroxylamine and 150g/L TS1138 strain enzyme The source cells were adjusted to pH 8.0 with 5% potassium hydroxide aqueous solution, placed in a constant temperature water bath at 42°C, and stirred for 2.5 hours.

After the reaction is completed, centrifuge at 12,000r/min for 5min at 4°C to separate the solid from the liquid. The supernatant is a mixture containing L-cysteine, and the L-cysteine content is 4.8g/L. (Method is the same as example 4), the molar conversion rate of substrate DL-ATC is 96.5%.

In the above-mentioned reaction solution containing L-cysteine, add the indole that final concentration is 4.5g/L, the coenzyme pyridoxal phosphate of 0.15g/L and the immobilized tryptophanase of 15g/L respectively, use 5 % potassium hydroxide aqueous solution to adjust the pH value to 8.0, and stirred and reacted for 3 hours in a constant temperature water bath at 50°C.

After the reaction was completed, it was centrifuged at 12,000r/min for 5min at 4°C to separate the solid from the liquid. The supernatant was a mixture containing L-tryptophan, and the content of L-tryptophan was detected to be 6.8g/L (method Same example 4), the molar conversion rate of substrate L-cysteine is 84.0%, the molar conversion rate of indole is 86.7%, and the total molar conversion rate from substrate DL-ATC to product L-tryptophan is 81.1%.

Example 7

Separation and Purification of L-Tryptophan

Centrifuge the enzymatic reaction solution containing L-tryptophan at 12,000r/min for 10min at 4°C to remove bacteria and undissolved indole, adjust the pH value of the supernatant to 5.0 with HCl, take 300mL of the above solution and load S8 type macroporous resin column (40×2.6cm), flow rate 1.5mL/min, to absorb DL-ATC and indole remaining in the solution, and then load the effluent on NKA-II macroporous resin column (40×2.6cm ) at a flow rate of 1.0 mL/min, fully washed with water and then eluted with 50% ethanol at a rate of 0.7 mL/min. Collect the eluate, and detect the content of L-tryptophan in the eluate. The active eluted fractions were combined, concentrated and dried under reduced pressure to obtain 1.652 g of L-tryptophan white powder. Detected by high performance liquid chromatography (Figure 2), the product purity reached 98.3% of the standard product.

Claims (8)

1. A method for microbial enzymatic production of L-tryptophan, characterized in that the method comprises: 1. Two-step continuous enzymatic reaction 1.1. First, use the whole cell or lysate of Pseudomonas putida TS1138 cells as the enzyme source, and enzymatically convert the substrate DL-2-amino- ^Δ2 -thiazoline-4-carboxylic acid to obtain L- Cysteine reaction solution; 1.2. Then use the whole cell of E. coli thallus, lysate or immobilized enzyme of E. coli thallus highly expressing tryptophanase as the enzyme source, and use the above-mentioned L-cysteine reaction solution and indole as the substrate. Enzymatic conversion to synthesize L-tryptophan; or, 2. One-step mixed enzymatic reaction Using the whole cell or cell lysate of Pseudomonas putida TS1138 and the whole cell, cell lysate or immobilized enzyme of Escherichia coli highly expressing tryptophanase as the mixed enzyme source, the enzymatic transformation substrate L-tryptophan can be synthesized from DL-2-amino-Δ ² -thiazoline-4-carboxylic acid and indole. 2. The method according to claim 1, characterized in that, in the two-step continuous enzymatic reaction process, coenzyme pyridoxal phosphate and hydroxylamine are added simultaneously as the inhibitor of L-cysteine desulfhydrylase. agent, the dosage of the coenzyme pyridoxal phosphate is 0.05g/L to 1g/L, the final concentration of the hydroxylamine is 0.5mmol/L to 3.0mmol/L; the dosage of TS1138 cells is generally 50g/L to 500g/L L, the amount of cell lysate is generally 10g to 100g of cell lysate; the amount of Escherichia coli cells that express tryptophanase is generally 1g/L to 100g/L, and the amount of cell lysate is generally 0.5g To the lysate of 30g bacterial cells, the amount of immobilized enzyme is generally 1g/L to 50g/L enzyme protein; the concentration of substrate DL-2-amino- ^Δ2 -thiazoline-4-carboxylic acid is 1g/L to 15g/L; the dosage of substrate indole is 1g/L to 20g/L. 3. The method according to claim 2, characterized in that, in the two-step continuous enzymatic reaction process, the dosage of said coenzyme pyridoxal phosphate is preferably 0.15g/L, and the final concentration of hydroxylamine used is preferably 1.0mmol /L; the dosage of TS1138 cells is preferably 150g/L, and the dosage of cell lysate is preferably 30g of cell lysate; the dosage of E. The dosage is preferably the lysate of 3g bacterial cells, and the dosage of immobilized enzyme is preferably 15g/L; the final concentration of the substrate DL-2-amino- ^Δ2 -thiazoline-4-carboxylic acid is preferably 6g/L; The dosage of indole is preferably 4.5g/L. 4. The method according to claim 1, characterized in that during the one-step mixed enzymatic reaction process, the coenzyme pyridoxal phosphate is added at the same time, and the dosage is 0.05g/L to 1g/L; the dosage of TS1138 cells is generally 50g/L to 500g/L, the amount of cell lysate is generally 10g to 100g of cell lysate; the amount of E. The amount of solution is generally 0.5g to 30g of lysate of bacterial cells, and the amount of immobilized enzyme is generally 1g/L to 50g/L; the final concentration of substrate DL-2-amino-Δ ² -thiazoline-4-carboxylic acid It is 1g/L to 15g/L, and the dosage of indole is 1g/L to 20g/L. 5. The method according to claim 4, characterized in that, in the enzymatic reaction process of one-step mixing, the dosage of coenzyme pyridoxal phosphate is preferably 0.15g/L; the dosage of TS1138 cells is preferably 200g/L, The amount of thalline lysate is preferably the lysate of 40g thalline cells; the amount of Escherichia coli thalline that highly expresses tryptophanase is preferably 15g/L, and the amount of thalline lysate is preferably the lysate of 5g thalline cells, immobilized The dosage of enzyme is preferably 20g/L; the final concentration of substrate DL-2-amino-Δ ² -thiazoline-4-carboxylic acid is preferably 6g/L, and the dosage of indole is preferably 5g/L. 6. The method according to claims 1 to 5, characterized in that the Pseudomonas TS1138 (Pseudomonas putida TS-1138) is preserved in the "General Microorganism Center of China Committee for the Collection of Microbial Cultures", and the preservation number is CGMCCNo .1920. 7. The method according to claims 1 to 5, characterized in that the high-efficiency expression of tryptophanase in the Escherichia coli is obtained by cloning the tryptophanase gene of Escherichia coli itself and expressing it in Escherichia coli Engineering strains. 8. The method according to claims 1 to 5, characterized in that the separation and purification method of the synthesized L-tryptophan is to use S8 type macroporous resin to remove the residual DL in the reaction solution containing L-tryptophan -2-amino- ^Δ2 -thiazoline-4-carboxylic acid and indole, and then use NKA-II type macroporous resin to adsorb L-tryptophan under the condition of pH 5 to exclude the loss of L-cysteine interference, and finally eluted with ethanol, concentrated and dried under reduced pressure to obtain L-tryptophan.

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