CN101525641A

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

Info

Publication number: CN101525641A
Application number: CN200810053749A
Authority: CN
Inventors: 白钢; 陈宁; 杨文博; 余养盛; 刘春琴; 段静静
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2008-07-04
Filing date: 2008-07-04
Publication date: 2009-09-09

Abstract

The invention provides a method for producing L-tryptophan by a microbial enzyme method. The method comprises that: full cells of Pseudomonas putida Ts1138 and bacillus coli thalli expressing tryptophanase in high efficiency, a thallus cracking solution or immobilized enzyme are used as enzyme sources; and from a substrate DL-2-amino-delta <2>-thiazoline-4-carboxylic acid (DL-ATC), the L-tryptophan is synthesized by a two-step continuous or one-step mixing enzyme catalytic reaction. The purification method comprises that: an S8 type macroporous resin is utilized to remove the DL-2-amino- delta <2>-thiazoline-4-carboxylic acid remained in a reaction solution containing the L-tryptophan and indole; under the condition of pH of 5, an NKA-II type macroporous resin is utilized to adsorb the L-tryptophan; the interference of L-cysteine is eliminated; and finally, the obtained L-tryptophan is subjected to ethanol elution, decompression, condensation and drying to obtain the L-tryptophan. Therefore, the method provides a novel process route for the green production of the L-tryptophan. Because the production cost of the substrate DL-ATC is relatively low, the method has a wide application prospect in the field of industrialized production of the L-tryptophan.

Description

Method for producing L-tryptophan by microbial enzyme method

[ technical field ]:

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

[ background Art ] A method of:

l-tryptophan is an important essential amino acid in human and animal bodies, and has very wide application in the fields of medicines, foods, feeds and the like. In recent years, the demand for L-tryptophan is increasing at home and abroad due to rapid development of the feed industry and continuous expansion of the application of L-tryptophan in the pharmaceutical industry. However, L-tryptophan has high production difficulty and high price for a long time, and cannot be widely popularized and applied in other fields except medical application.

L-tryptophan is one of the most difficult amino acids to produce by direct fermentation, and the production method mainly comprises four methods, namely a proteolytic extraction method, a chemical synthesis method, a microbial fermentation method and an enzymatic synthesis method. Wherein, the enzymatic synthesis method utilizes the precursors of chemical synthesis as raw materials, not only gives full play to the advantages of organic synthesis technology, but also has the advantages of high product concentration, high yield, high purity, less by-products, easy refining operation and the like, and is a better method for producing L-tryptophan at low cost which is currently preferred.

The enzymatic synthesis pathway of L-tryptophan mainly involves tryptophan synthase (EC 4.2.1.20) and tryptophanase (EC 4.1.99.1), both of which catalyze the synthesis of L-tryptophan from L-serine and indole. In recent years, attention has been paid to the application of tryptophanase in L-tryptophan biosynthesis because indole has a strong inhibitory effect on tryptophanase, which has good stability against indole.

Tryptophanase normally decomposes L-tryptophan to produce pyruvate, indole and ammonia, but can effectively catalyze pyruvate, indole and ammonia to synthesize L-tryptophan under the condition of high concentration of pyruvate and ammonia. The enzyme can also catalyze L-serine or L-cysteine and indole to synthesize L-tryptophan. In the way of synthesizing L-tryptophan by catalyzing pyruvic acid, indole and ammonia by tryptophanase, the substrate indole has certain practicability because of weak inhibition effect on tryptophanase and low price of pyruvic acid, but the way is the reverse reaction of tryptophan hydrolysis, requires high substrate concentration and is difficult to grasp reaction balance. In the synthesis route of L-tryptophan using L-serine and indole as raw materials, the price of L-serine as a substrate is almost equivalent to that of L-tryptophan, and therefore, the method is not practical. In the way of synthesizing L-tryptophan by catalyzing L-cysteine and indole by utilizing tryptophanase, the production cost of the substrate L-cysteine is low, so the way has important industrial application value.

In the way of synthesizing L-tryptophan by converting L-cysteine and indole by an enzymatic method, the main difficulty of separating and purifying the product L-tryptophan is that the components of the reaction liquid are complex, the residual substances such as indole and L-cysteine are not easy to separate from the product L-tryptophan, and no mature and feasible method is reported at present.

[ summary of the invention ]:

the invention aims to solve the problems in the prior art and provide a novel method for producing L-tryptophan by a microbial enzyme method.

The invention relates to a method for preparing a tryptophanase from a substrate DL-2-amino-delta-alpha by using a Pseudomonas putida TS1138(Pseudomonas putida TS1138) cell and a whole cell of escherichia coli thallus, thallus lysate or immobilized enzyme for efficiently expressing the tryptophanase as enzyme sources²-thiazoline-4-carboxylic acid (DL-2-amino-Delta)²-thiazoline-4-carboxylic acid, DL-ATC), synthesizing L-tryptophan by two-step continuous or one-step mixed enzymatic reaction, and finally separating and purifying to obtain the product L-tryptophan.

The invention automatically separates and obtains a Pseudomonas putida TS1138 strain (Pseudomonas putida TS-1138), the strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.1920, and the L-cysteine synthase system contained in the strain can catalyze a substrate DL-ATC to synthesize L-cysteine. In addition, the invention also constructs the Escherichia coli genetic engineering strain for efficiently expressing the tryptophanase by self, namely, the Escherichia coli JM109 strain genome DNA is taken as a template, the tryptophanase gene is obtained by PCR amplification and is expressed in Escherichia coli BL21(DE3), and the activity of the tryptophanase which is recombined and expressed reaches 110 times of that of the host bacterium.

The invention respectively establishes a new method for biologically synthesizing L-tryptophan by two-step continuous or one-step mixed enzymatic reactions of L-cysteine by taking the thallus whole cells, the thallus lysate or the immobilized enzyme of the two strains as enzyme sources, and the synthetic route is shown in figure 1.

The method for producing L-tryptophan by using the microbial enzyme method provided by the invention specifically comprises the following steps:

1 st, two-step sequential enzymatic reaction

1.1, firstly, using whole cells of Pseudomonas putida TS1138 thallus or thallus lysate as enzyme source, converting substrate DL-2-amino-delta by enzyme method²-thiazoline-4-carboxylic acid to obtain a reaction solution containing L-cysteine;

1.2, taking whole cells of escherichia coli thalli for efficiently expressing tryptophanase, thalli lysate or immobilized enzyme as an enzyme source, taking the L-cysteine reaction solution and indole as substrates, and synthesizing the L-tryptophan through enzymatic conversion;

or,

2, one-step Mixed enzymatic reaction

Takes whole cells or thallus lysate of pseudomonas putida TS1138 thallus and whole cells, thallus lysate or immobilized enzyme of escherichia coli thallus for efficiently expressing tryptophanase as a mixed enzyme source, and converts a substrate DL-2-amino-delta by an enzyme method²Synthesizing L-tryptophan by using thiazoline-4-carboxylic acid and indole.

In the two-step continuous enzymatic reaction process, coenzyme pyridoxal phosphate is added simultaneously, the dosage of the coenzyme pyridoxal phosphate is 0.05g/L to 1g/L, and hydroxylamine is used as an inhibitor of L-cysteine desulfhydrase, and the final concentration of the hydroxylamine is 0.5mmol/L to 3.0 mmol/L; the dosage of TS1138 thalli is generally 50g/L to 500g/L, and the dosage of thalli lysate is generally 10g to 100g of lysate of thalli cells; the using amount of the escherichia coli thalli for efficiently expressing the tryptophanase is generally 1g/L to 100g/L, the using amount of the thalli lysate is generally 0.5g to 30g of lysate of thalli cells, and the using amount of the immobilized enzyme is generally 1g/L to 50 g/L; substrate DL-2-amino-Delta²-thiazoline-4-carboxylic acid is at a concentration of 1g/L to 15 g/L; the dosage of the indole serving as the substrate is 1g/L to 20 g/L.

The dosage of the coenzyme pyridoxal phosphate is preferably 0.15g/L, and the final concentration of the hydroxylamine is preferably 1.0 mmol/L; the dosage of TS1138 thalli is preferably 150g/L, and the dosage of thalli lysate is preferably 30g of lysate of thalli cells; the dosage of the escherichia coli thalli for efficiently expressing the tryptophanase is preferably 10g/L, the dosage of thalli lysate is preferably 3g of lysate of thalli cells, and the dosage of immobilized enzyme is preferably 15 g/L; substrate DL-2-amino-Delta²The final concentration of the thiazoline-4-carboxylic acid is preferably 6 g/L; the dosage of the indole as the substrate is preferably 4.5 g/L.

In the one-step mixed enzymatic reaction process, the coenzyme pyridoxal phosphate can be added simultaneously, and the dosage of the coenzyme pyridoxal phosphate is 0.05g/L to 1 g/L; the dosage of TS1138 is generally 50gThe dosage of the bacterial cell lysate is generally 10g to 100g of the bacterial cell lysate; the using amount of the escherichia coli thalli for efficiently expressing the tryptophanase is generally 1g/L to 100g/L, the using amount of the thalli lysate is generally 0.5g to 30g of lysate of thalli cells, and the using amount of the immobilized enzyme is generally 1g/L to 50 g/L; substrate DL-2-amino-Delta²The final concentration of the thiazoline-4-carboxylic acid is 1g/L to 15g/L, and the dosage of the indole is 1g/L to 20 g/L.

The dosage of the coenzyme pyridoxal phosphate is preferably 0.15g/L, the dosage of TS1138 thalli is preferably 200g/L, and the dosage of a thalli lysate is preferably 40g of lysate of thalli cells; the dosage of the escherichia coli thalli for efficiently expressing the tryptophanase is preferably 15g/L, the dosage of thalli lysate is preferably 5g of lysate of thalli cells, and the dosage of immobilized enzyme is preferably 20 g/L; substrate DL-2-amino-Delta²The final concentration of the thiazoline-4-carboxylic acid is preferably 6g/L, and the amount of indole is preferably 5 g/L.

The method for separating and purifying the synthesized L-tryptophan adopts S8 type macroporous resin to remove the residual DL-2-amino-delta in the reaction liquid containing the L-tryptophan²And (3) adsorbing the L-tryptophan by using NKA-II type macroporous resin under the condition that the pH value is 5, eliminating the interference of the L-cysteine, eluting by using ethanol, concentrating under reduced pressure and drying to obtain the L-tryptophan.

In the embodiment of the invention, the content of L-cysteine is determined by adopting a Gaitonde method, which comprises the following steps: accurately weighing a certain amount of L-cysteine standard substance, dissolving the L-cysteine standard substance in a certain amount of 0.05mol/L HCl to prepare a standard mother solution with a final concentration of 500mg/L, and further diluting the mother solution with distilled water to obtain a standard solution with a concentration of 10, 20. Taking 0.2mL of a certain amount of standard solution or dilution of a sample solution to be detected, adding 0.2mL of glacial acetic acid, adding 0.2mL of acidic ninhydrin reagent, reacting in a boiling water bath for 10min, immediately cooling in cold water, and finally adding 2.4mL of alcohol to make the total volume be 3 mL. After standing for 10min, the absorbance was measured at 560nm, and a standard curve was drawn by concentration and absorbance. The concentration of L-cysteine in the unknown solution was calculated from the standard curve.

In the embodiment of the invention, the content of L-tryptophan is measured by adopting a high performance liquid chromatography, and the specific method is as follows: and respectively taking 20 mu L of 50-1 and 500 mg/L-tryptophan standard solution for sample injection, carrying out continuous sample injection for 5 times, calculating the peak height, averaging, and drawing a standard curve by taking the L-tryptophan concentration (X, mg/L) as an abscissa and the peak height (Y) as an ordinate. The L-tryptophan produced by the enzymatic reaction can be accurately quantified by comparing with a standard curve after being detected by high performance liquid chromatography.

The invention has the beneficial effects that: the invention starts from a chemically synthesized substrate DL-ATC, utilizes pseudomonas putida TS1138 and whole cells of escherichia coli thalli for efficiently expressing tryptophanase, thalli lysate or immobilized enzyme as enzyme sources, and produces L-tryptophan by two-step continuous and one-step mixed enzymatic reaction respectively. The production cost of the substrate DL-ATC is low, so the method has wide application prospect in the field of industrial production of L-tryptophan.

[ description of the drawings ]

FIG. 1: a schematic route for synthesizing L-tryptophan by converting DL-ATC by an enzyme method,

FIG. 2: detecting the L-tryptophan in the reaction solution and after purification by high performance liquid chromatography,

(A) reaction solution (ultraviolet detection); (B) reaction solution (detection by evaporative light scatterer); (C) purified L-tryptophan (uv detection); (D) purified L-tryptophan (evaporative light scatterer detection); the chromatographic peaks are respectively: 1, L-tryptophan (retention time, 4.732 min); 2, indole (retention time, 5.965 min); 3, L-cysteine (retention time, 3.223 min).

The pseudomonas putida TS1138 is named after classification: pseudomonas putida, which has been deposited at "China general microbiological culture Collection center" on 17 th 1 month in 2007, with the deposition time address: the preservation number is CGMCC No.1920 in the institute of microbiology of Zhongguancun, Haizu district, Beijing. Meanwhile, the strain has been described in patent application publication No. 200710056754.9 published on 10.17.2007.

[ detailed description ] embodiments

Example 1

Cloning and expression of E.coli tryptophanase gene

According to the tryptophanase gene sequence of the Escherichia coli K12 strain, an upstream primer 5'-CCGGAATTCATGGAAAACTTTAAACATCTCC-3' and a downstream primer 5'-CCCAAGCTTTTAAACTTCTTTCAGTTTTGCGG-3' of the tryptophanase gene are designed. The genomic DNA of Escherichia coli strains DH5 alpha, JM109 and BL21 are used as templates, the tryptophanase gene is obtained by PCR amplification, and recombinant expression plasmids pET-tnaA (D), pET-tnaA (J) and pET-tnaA (B) are respectively constructed.

The three recombinant plasmids are respectively transformed into escherichia coli BL21(DE3) to construct respective tryptophanase gene engineering strains. Inoculating the recombinant strain into 4mL LB culture medium containing 100 ug/mL ampicillin, performing shake culture at 37 deg.C for 12-16 h, inoculating to 50mL fresh LB culture medium at 1% inoculum size, and culturing to OD₆₀₀When the concentration reached 0.5, IPTG was added to the mixture at a final concentration of 0.1mmol/L, and expression was induced at 37 ℃ for 2 hours. Collecting the thallus, washing with potassium phosphate buffer solution (pH 8.0), re-suspending, ultrasonic crushing for 5min to crack the thallus fully, centrifuging, collecting supernatant, and determining the activity of recombinant expression tryptophanase. The results show that the enzyme activities of the recombinant expression products of the tryptophanase genes from three different escherichia coli are similar and all reach more than 100 times of the activity of the tryptophanase of the host bacteria (table 1).

TABLE 1 tryptophanase Activity of different strains

All values are Mean ± sd of three replicates (Mean value ± s.d.).

Example 2

Preparation of Pseudomonas putida TS1138 strain enzyme source

A ring of Pseudomonas putida TS1138 was picked from the plate and inoculated into 4mL of seed medium (glucose 20g, ATC3g, corn steep liquor 5g, urea 3g, NaCl 1.5g, MnSO)₄·H₂O 0.1g，K₂HPO₄ 3g，MgSO₄·7H₂O 0.5g，FeSO₄·7H₂O0.01 g, constant volume to 1L, pH 7.5), culturing at 28 deg.C and 160r/min for 16h, transferring to 50mL enzyme production culture medium (glucose 30g, ATC 4g, corn steep liquor 1g, urea 3g, NaCl 1.5g, MnSO₄·H₂O 0.1g，K₂HPO₄ 3g，MgSO₄·7H₂O0.5g，FeSO4·7H₂O0.01 g, constant volume to 1L, pH 7.5), culturing at 28 ℃ for 16h at 160r/min, centrifuging, collecting thalli, washing the thalli for 1 to 2 times by using glycine-sodium hydroxide buffer solution (pH 8.0), weighing a certain amount of the thalli, and adding glycine-sodium hydroxide buffer solution to obtain the enzyme source cell suspension.

And (3) carrying out ultrasonic disruption on the enzyme source cells, centrifuging and collecting supernatant to obtain the thallus lysate.

Example 3

Preparation of Escherichia coli enzyme source for efficiently expressing tryptophanase

E.coli BL21(DE3) recombinant strain containing pET-tnaA (J) prepared in example 1 was inoculated into 4mL LB medium containing ampicillin 100. mu.g/mL, shake-cultured at 37 ℃ for 12-16 hours, transferred to 50mL of fresh LB medium at an inoculum size of 1%, and further cultured until OD is reached₆₀₀When the concentration reached 0.5, IPTG was added to the mixture at a final concentration of 0.1mmol/L, and expression was induced at 37 ℃ for 2 hours. Collecting thallus, washing thallus with potassium phosphate buffer (pH 8.0) for 1-2 times, weighing a certain amount of the thallus, and adding potassium phosphate buffer to obtain enzyme source cell suspensionAnd (4) liquid. And (3) breaking enzyme source cells by adopting ultrasonic waves, centrifuging and collecting supernatant to obtain the thallus lysate.

Weighing 1g of DEAE-22 cellulose, adding 40mL of 3.5% glutaraldehyde, stirring at room temperature for 5h, standing overnight, centrifuging to remove supernatant, repeatedly washing the obtained carrier with distilled water to remove residual glutaraldehyde, and performing suction filtration to obtain the cross-linked carrier. 30mL of thallus lysate with the soluble protein content of 15g/L is added into the cross-linked carrier, and the mixture is kept stand for 12 hours at 4 ℃ and stirred once every 0.5 hour. Centrifuging, discarding supernatant, repeatedly washing precipitate with distilled water to remove free lysate, and vacuum filtering to obtain immobilized enzyme.

Example 4

Takes thallus whole cell as enzyme source to synthesize L-tryptophan by two-step continuous enzymatic reaction

In a vessel having an internal volume of 500mL and equipped with a stirrer, 300mL of an enzymatic reaction solution containing 6g/L DL-ATC and 6g/L K was prepared₂HPO₄1.0mmol/L hydroxylamine and 150g/L of enzyme source cells of the TS1138 strain prepared in example 2, the pH value was adjusted to 8.0 with 5% aqueous solution of potassium hydroxide, and the mixture was placed in a thermostatic water bath at 42 ℃ and stirred for reaction for 2.5 hours.

After the reaction is finished, centrifuging for 5min at the temperature of 4 ℃ at 12,000r/min, separating solid from liquid, wherein the supernatant is a mixed solution containing L-cysteine, and detecting that the content of the L-cysteine is 4.8g/L and the molar conversion rate of the substrate DL-ATC is 96.5%.

Indole with the final concentration of 4.5g/L, coenzyme pyridoxal phosphate with the final concentration of 0.15g/L and escherichia coli enzyme source cells capable of efficiently expressing tryptophanase with the final concentration of 10g/L are respectively added into the reaction liquid containing the L-cysteine, the pH value is adjusted to 8.0 by 5% potassium hydroxide aqueous solution, and the mixture is stirred and reacted for 3 hours in a constant-temperature water bath at the temperature of 45 ℃.

After the reaction is finished, centrifuging at the temperature of 4 ℃ for 5min at 12,000r/min, separating solid from liquid, wherein the supernatant is a mixed solution containing L-tryptophan, the content of the L-tryptophan is detected to be 6.2g/L, the molar conversion rate of the substrate L-cysteine is 76.6 percent, the molar conversion rate of the indole is 79.0 percent, and the total molar conversion rate from the substrate DL-ATC to the product L-tryptophan is 73.9 percent.

The content of the L-cysteine is measured by adopting a Gaitonde method, and the specific method is as follows: accurately weighing a certain amount of L-cysteine standard substance, dissolving the L-cysteine standard substance in a certain amount of 0.05mol/L HCl to prepare a standard mother solution with a final concentration of 500mg/L, and further diluting the mother solution with distilled water to obtain a standard solution with a concentration of 10, 20. Taking 0.2mL of a certain amount of standard solution or dilution of a sample solution to be detected, adding 0.2mL of glacial acetic acid, adding 0.2mL of acidic ninhydrin reagent, reacting in a boiling water bath for 10min, immediately cooling in cold water, and finally adding 2.4mL of alcohol to make the total volume be 3 mL. After standing for 10min, the absorbance was measured at 560nm, and a standard curve was drawn by concentration and absorbance. The concentration of L-cysteine in the unknown solution was calculated from the standard curve.

The content of L-tryptophan is determined by high performance liquid chromatography, which comprises the following steps: respectively taking 20 mu L of L-tryptophan standard liquid with the concentration of 50-1,500 mg/L for sample injection, continuously carrying out sample injection for 5 times, calculating the peak height, taking the average value, and drawing a standard curve by taking the concentration (X, mg/L) of L-tryptophan as an abscissa and the peak height (Y) as an ordinate. The L-tryptophan produced by the enzymatic reaction can be accurately quantified by comparing with a standard curve after being detected by high performance liquid chromatography.

Example 5

Synthesizing L-tryptophan by using thallus lysate as enzyme source and one-step mixed enzymatic reaction

In a vessel having an internal volume of 500mL and equipped with a stirrer, 300mL of an enzymatic reaction solution containing 6g/L of DL-ATC and 0.6% of K was prepared₂HPO₄1.0mmol/L hydroxylamine, 5g/L indole, 0.15g/L pyridoxal phosphate, 25g of lysate of TS1138 strain bacteria and 5g of lysate of escherichia coli efficiently expressing tryptophanase, adjusting the pH value to 8.0 by using a 5% potassium hydroxide aqueous solution, placing the mixture in a constant-temperature water bath at 42 ℃, and stirring for reaction for 3 hours.

After the reaction is finished, centrifuging at 12,000r/min for 5min at 4 ℃, separating solid from liquid, wherein the supernatant is a mixed solution containing L-tryptophan, and detecting that the content of the L-tryptophan is 6.7g/L (the method is the same as example 4), the molar conversion rate of the substrate DL-ATC is 79.9 percent, and the molar conversion rate of the indole is 76.9 percent.

Example 6

Synthesizing L-tryptophan by using immobilized enzyme as enzyme source and through two-step continuous enzymatic reaction

In a vessel having an internal volume of 500mL and equipped with a stirrer, 300mL of an enzymatic reaction solution containing 6g/L DL-ATC and 6g/L K was prepared₂HPO₄1.0mmol/L hydroxylamine and 150g/L TS1138 strain enzyme source cell, regulating the pH value to 8.0 with 5% potassium hydroxide water solution, placing in 42 deg.C constant temperature water bath, stirring and reacting for 2.5 h.

After the reaction is finished, centrifuging at 12,000r/min for 5min at 4 ℃, separating solid from liquid, wherein the supernatant is a mixed solution containing L-cysteine, the content of the L-cysteine is detected to be 4.8g/L (the method is the same as example 4), and the molar conversion rate of the substrate DL-ATC is 96.5%.

Indole with a final concentration of 4.5g/L, pyridoxal phosphate coenzyme with a final concentration of 0.15g/L, and immobilized tryptophanase with a final concentration of 15g/L were added to the reaction solution containing L-cysteine, and the pH was adjusted to 8.0 with a 5% aqueous solution of potassium hydroxide, followed by reaction with stirring in a thermostatic water bath at 50 ℃ for 3 hours.

After the reaction, the mixture was centrifuged at 12,000r/min at 4 ℃ for 5min, the solid and liquid were separated, the supernatant was a mixture containing L-tryptophan, and the L-tryptophan content was detected to be 6.8g/L (same as in example 4), the molar conversion of the substrate L-cysteine was 84.0%, the molar conversion of indole was 86.7%, and the total molar conversion from the substrate DL-ATC to the product L-tryptophan was 81.1%.

Example 7

Separation and purification of L-tryptophan

Centrifuging the enzymatic reaction solution containing L-tryptophan at 4 ℃ for 10min at 12,000r/min to remove thallus and undissolved indole, adjusting the pH value of the supernatant to 5.0 by HCl, loading 300mL of the solution onto an S8 type macroporous resin column (40 × 2.6cm) at a flow rate of 1.5mL/min to adsorb DL-ATC and indole remaining in the solution, loading the effluent onto an NKA-II macroporous resin column (40 × 2.6cm) at a flow rate of 1.0mL/min, washing with water, eluting with 50% ethanol at an elution rate of 0.7 mL/min. Collecting the eluent, and detecting the content of the L-tryptophan in the eluent. The active fractions were combined, concentrated under reduced pressure and dried to obtain 1.652g of L-tryptophan as a white powder. The purity of the product reaches 98.3 percent of that of the standard product by high performance liquid chromatography detection (figure 2).

Claims

1. A method for producing L-tryptophan by a microbial enzyme method, which is characterized by comprising the following steps:

1 st, two-step sequential enzymatic reaction

or,

2, one-step Mixed enzymatic reaction

2. The method according to claim 1, wherein in the two-step continuous enzymatic reaction process, pyridoxal phosphate coenzyme in an amount of 0.05 to 1g/L and hydroxylamine in a final concentration of 0.5 to 3.0mmol/L are simultaneously added as an inhibitor of L-cysteine desulfhydrase; the dosage of TS1138 thalli is generally 50g/L to 500g/L, and the dosage of thalli lysate is generally 10g to 100g of lysate of thalli cells; the dosage of the colibacillus thallus for efficiently expressing the tryptophanase is generally 1g/L to 100g/L, the dosage of thallus lysate is generally 0.5g to 30g of lysate of thallus cells, and the dosage of immobilized enzyme is generally 1g/L to 50g/L of enzyme protein; substrate DL-2-amino-Delta²-thiazoline-4-carboxylic acid is at a concentration of 1g/L to 15 g/L; the dosage of the indole serving as the substrate is 1g/L to 20 g/L.

3. The process according to claim 2, wherein the amount of pyridoxal phosphate of the coenzyme used in the two successive enzymatic reactions is preferably 0.15g/L and the final concentration of hydroxylamine used is preferably 1.0 mmol/L; the dosage of TS1138 thalli is preferably 150g/L, and the dosage of thalli lysate is preferably 30g of lysate of thalli cells; the dosage of the escherichia coli thalli for efficiently expressing the tryptophanase is preferably 10g/L, the dosage of thalli lysate is preferably 3g of lysate of thalli cells, and the dosage of immobilized enzyme is preferably 15 g/L; substrate DL-2-amino-Delta²The final concentration of the thiazoline-4-carboxylic acid is preferably 6 g/L; the dosage of the indole as the substrate is preferably 4.5 g/L.

4. The process according to claim 1, wherein pyridoxal phosphate coenzyme is added simultaneously during the enzymatic reaction in one mixing step in an amount of 0.05 to 1 g/L; the dosage of TS1138 thalli is generally 50g/L to 500g/L, and the dosage of thalli lysate is generally 10g to 100g of lysate of thalli cells; the using amount of the escherichia coli thalli for efficiently expressing the tryptophanase is generally 1g/L to 100g/L, the using amount of the thalli lysate is generally 0.5g to 30g of lysate of thalli cells, and the using amount of the immobilized enzyme is generally 1g/L to 50 g/L; substrate DL-2-amino-Delta²The final concentration of the thiazoline-4-carboxylic acid is 1g/L to 15g/L, and the dosage of the indole is 1g/L to 20 g/L.

5. The process according to claim 4, wherein the enzymatic reaction with one mixing step is carried out in an amount of pyridoxal phosphate coenzyme preferably 0.15 g/L; the dosage of TS1138 thalli is preferably 200g/L, and the dosage of thalli lysate is preferably 40g of lysate of thalli cells; the dosage of the escherichia coli thalli for efficiently expressing the tryptophanase is preferably 15g/L, the dosage of thalli lysate is preferably 5g of lysate of thalli cells, and the dosage of immobilized enzyme is preferably 20 g/L; substrate DL-2-amino-Delta²The final concentration of the thiazoline-4-carboxylic acid is preferably 6g/L, and the amount of indole is preferably 5 g/L.

6. The method according to claims 1 to 5, wherein the Pseudomonas bacteria TS1138(Pseudomonas putida TS-1138) is deposited in the China general microbiological culture Collection center (CGMCCNo) with the collection number of CGMCCNo.1920.

7. The method according to claims 1 to 5, wherein the E.coli highly expressing tryptophanase is a genetically engineered strain which is highly expressed in E.coli by cloning tryptophanase gene of E.coli itself.

8. The method according to claims 1 to 5, wherein the synthesized L-tryptophan is separated and purified by removing DL-2-amino-delta remaining in the reaction solution containing L-tryptophan by using S8 type macroporous resin²And (3) adsorbing the L-tryptophan by using NKA-II type macroporous resin under the condition that the pH value is 5, eliminating the interference of the L-cysteine, eluting by using ethanol, concentrating under reduced pressure and drying to obtain the L-tryptophan.