Method for immobilizing tryptophanase and application of tryptophanase

Abstract

The invention discloses a method for immobilizing tryptophanase and application of the tryptophanase, and the method comprises the following steps: S1, adding dilute acetic acid into a reaction container filled with chitosan, stirring and dissolving, adding FeCl3 6H2O and FeCl2 4H2O, stirring, then adding ammonia water under vigorous stirring, continuously stirring until the reaction is complete, after the reaction is completed, cooling, carrying out magnetic separation, and washing to obtain a chitosan solution; the chitosan magnetic nano material is obtained; and S2, dissolving the chitosan magnetic nano material in deionized water, then adding tryptophanase to prepare a solution with the concentration of 0.8-1.2 mg / mL, carrying out electrostatic adsorption, then adding a glutaraldehyde solution to carry out crosslinking, reacting for a certain time, and carrying out magnetic adsorption separation and washing to obtain the tryptophanase-chitosan immobilized enzyme. According to the invention, chitosan modification is carried out on the magnetic nanoparticles, so that the prepared chitosan magnetic nano material can be easily and rapidly separated from a reaction system in a magnetic field, and the catalytic activity and stability of enzyme are greatly improved.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme immobilization, and in particular to a method for immobilizing tryptophanase and application thereof. Background Art

[0002] L-tryptophan plays an important role in the growth, development and metabolism of humans and animals, and is therefore widely used in the fields of medicine, cosmetics, food and feed. At present, the main methods for preparing L-tryptophan are fermentation, chemical synthesis and enzymatic conversion. The fermentation method can be divided into direct fermentation and precursor addition fermentation. The direct microbial fermentation method uses cheap raw materials such as glucose and sugarcane molasses as carbon sources and uses L-tryptophan production strains for fermentation production. However, the synthesis pathway is long, the metabolic flow is weak, and a variety of precursors are required. If the accumulation of L-tryptophan is to be further increased, the metabolic flow of these precursors must be enhanced; on the other hand, the regulatory mechanism in the biosynthetic pathway of L-tryptophan is studied, but this method cannot meet the requirements of industrial production for a long time. The chemical synthesis method mainly includes two categories: directly using indole as a raw material and generating indole rings during the synthesis process. The reaction conditions of this method are complex, the cycle is long, and the pollution is serious, which is not suitable for industrial production. The enzymatic method can synthesize L-tryptophan from indole and L-serine under the action of tryptophan synthase, which has the advantages of high accumulation of the final product, short reaction cycle and easy separation and purification. It is a cheap and effective method for producing L-tryptophan.

[0003] However, from an industrial perspective, free enzymes also have many disadvantages, such as poor reusability, short catalytic life, low operational stability to temperature, pH and certain environmental factors, and difficulty in recycling and reuse, which seriously hinder their large-scale application at the industrial level. In order to solve this technical problem, it is necessary to study the properties of immobilized enzymes. Enzyme immobilization is a powerful means to overcome this limitation, however, efficient and stable immobilization remains to be studied. Magnetic nanoparticles have the advantages of small particle size, stable performance, strong superparamagnetism, and simple separation, and have the prerequisites to become an excellent carrier for lipase immobilization.

[0004] For example, a Chinese patent (CN1224703C) discloses a method for preparing magnetic microsphere immobilized cellulase, wherein magnetic agarose microspheres are used as carriers to immobilize industrial cellulase onto the microspheres by physical adsorption, and magnetic microsphere immobilized cellulase with magnetic responsiveness, high protein loading, high specific activity, and high activity recovery rate is prepared. In this patent, industrial cellulase can be repeatedly used, thereby improving the utilization rate of cellulase and reducing production costs. However, the enzyme is mainly immobilized by physical adsorption, and there is a problem of unsatisfactory immobilization effect. Therefore, how to prepare an immobilized tryptophanase with higher safety, stability, and reusability is a problem that needs to be solved urgently. Summary of the invention

[0005] The main purpose of the present invention is to provide a method for fixing tryptophanase and its application, aiming to solve the problem that the effect of fixing tryptophanase in the prior art is poor, resulting in low yield of converted tryptophan.

[0006] To achieve the above object, the present invention provides a method for fixing tryptophanase, comprising the following steps:

[0007] S1, add dilute acetic acid to the reaction vessel containing chitosan, stir and dissolve, add FeCl36H2O and FeCl24H2O, stir, then add ammonia water under vigorous stirring, continue stirring until the reaction is complete, after the reaction is completed, cool, perform magnetic separation, wash, and obtain chitosan magnetic nanomaterials;

[0008] S2, dissolving the chitosan magnetic nanomaterial in deionized water, then adding tryptophanase to prepare a solution with a concentration of 0.8-1.2 mg / mL, subjecting it to electrostatic adsorption, and then adding glutaraldehyde solution for cross-linking. After a certain reaction time, magnetic adsorption separation and washing are performed to obtain tryptophanase-chitosan immobilized enzyme.

[0009] Preferably, in step S1, the usage ratio of chitosan, FeCl3 6H2O, FeCl2 4H2O and aqueous ammonia is (90-110 mg): (8-12 mg): (8-12 mg): (8-12 mL).

[0010] Preferably, in steps S1 and S2, deionized water is used for washing.

[0011] Preferably, in step S2, the volume ratio of the deionized water to the glutaraldehyde solution is (90-110 mL): (8-12 μL).

[0012] The present invention also provides an immobilized tryptophanase prepared by the method as described above.

[0013] The present invention further provides an industrial application of the immobilized tryptophanase as described above in converting L-tryptophan.

[0014] Preferably, the application comprises the following steps: preparing a certain concentration of serine and 2-methylindole in a reaction container, adding the coenzyme pyridoxal phosphate, and then adding an immobilized enzyme, stirring in a reaction tank, reacting for a period of time, and obtaining converted L-tryptophan.

[0015] Preferably, the concentration of serine is 8-12 g / L, the concentration of 2-methylindole is 0.8-1.2 g / L, the concentration of coenzyme pyridoxal phosphate is 8-12 mg / L, the concentration of immobilized enzyme is 80-120 mg / L, and the pH value of the reaction system is 7-9.

[0016] Preferably, the concentration of serine is 10 g / L, the concentration of 2-methylindole is 1 g / L, the concentration of coenzyme pyridoxal phosphate is 10 mg / L, the concentration of immobilized enzyme is 100 mg / L, and the pH value of the reaction system is 8.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] (1) The method for immobilizing tryptophanase provided by the present invention is to modify magnetic nanoparticles with chitosan to prepare chitosan magnetic nanomaterials, which can effectively solve the defects of enzyme immobilization with pure magnetic nanomaterials. The chitosan magnetic nanomaterials are used as carriers to immobilize tryptophanase, which not only retains the original advantages of the enzyme, but also gives full play to the special role of the chitosan magnetic nanomaterials. The tryptophanase can be easily and quickly separated from the reaction system in a magnetic field, thereby greatly improving the catalytic activity and stability of the enzyme.

[0019] (2) The chitosan used in the present invention is a biodegradable polymer with good biocompatibility and adsorption properties. It contains a large number of active groups, such as -NH2 and -OH. At the same time, it further improves the stability of the immobilized tryptophanase by cross-linking reaction with glutaraldehyde. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is a diagram of enzyme expression at different bacterial concentrations in Example 1 of the present invention;

[0022] Figure 2 The activity diagram of tryptophanase-chitosan immobilized enzyme at different pH values ​​in Example 5 of the present invention;

[0023] Figure 3 The activity diagram of tryptophanase-chitosan immobilized enzyme at different temperatures in Example 5 of the present invention;

[0024] Figure 4 The activity diagram of tryptophanase-chitosan immobilized enzyme at different times of use in Example 5 of the present invention;

[0025] Figure 5 This is a graph showing the tryptophan conversion amount of the tryptophanase-chitosan immobilized enzyme in Example 6 of the present invention.

[0026] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0027] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be clearly and completely described below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0028] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0029] Example 1 Activation and induction of enzyme production by enzyme engineering strains

[0030] (1) Culture medium preparation

[0031] Luria-Bertani (LB) liquid medium: 1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1% (w / v) NaCl, double distilled water, mix thoroughly, adjust pH to 7.0, dispense into 100 mL portions, and sterilize in an autoclave at 121°C for 20 min. LB solid medium: add 1.5% (w / v) agar powder to LB liquid medium.

[0032] (2) Inducing enzyme production

[0033] The tryptophanase engineered strain (carrying plasmid pET-28-Tryptophanase-2×His), Tryptophanase sequence see SEQ1 (amino acid sequence of tryptophanase see SEQ2), was streaked onto a LB solid medium containing kanamycin (10μg / mL) and cultured at 37°C for 8h. A single colony was picked and inoculated into LB activation medium to obtain an activated strain. The strain was aspirated into LB liquid medium containing kanamycin (10μg / mL) at a 1% (v / v) inoculation amount and cultured in a 37°C constant temperature shaker until OD600 reached 0.4, 0.6, 0.8 and 1. Then IPTG was added at a final concentration of 0.001 mol / L at 25°C to induce enzyme production for 24h, and the bacteria were collected by centrifugation at 5000r / min for 10min. The enzyme expression level at different bacterial concentrations was detected by SDS-PAGE electrophoresis to obtain Figure 1 ,Depend on Figure 1 It was found that when the bacterial concentration OD600 = 0.8, the enzyme expression reached the maximum and remained stable after 24 hours.

[0034]

[0035] Protein sequence SEQ2: MENFKHLPEPFRIRVIEPVKRTTRAYREEAIIKSGMNPFLLDSEDVFIDLLTDSGTGAVTQSMQAAMMRGDEAYSGSRSYYALAESVKNIFGYQYTIPTHQGRG AEQIYIPVLIKKREQEKGLDRSKMVAFSNYFFDTTQGHSQINGCTVRNVYIKEAFDTGVRYDFKGNFDLEGLERGKEVGPNNVPYIVATITSNSAGGQPVSLANLKAMYSIAKKYDIPVVMDSARFAENAYFIKQREAEYKDWTIEQITRETYKYADMLAMSAKKDAMVPMGGLLCMKDDSFF DVYTECRTLCVVQEGFPTYGGLEGGAMERLAVGLYDGMNLDWLAYRIAQVQYLVDGLEEEIGVVCQQAGGHAAFVDAGKLLPHIPADQFPAQALACELYKVAGIRAVEIGSFLLGRDPKTGKQLPCPAELLRLTIPRATYTQTHMDFIIEAFKHVKENAANIKGLTFTYEPKVLRHFTAKLKEV

[0036] Example 2 Isolation and purification of enzyme

[0037] The tryptophanase in this experiment has a 2× 6-histidine (His) tag at the end and is tagged with Ni 2+ Column affinity chromatography purification, the specific steps are as follows: (1) Resuspend the bacteria with resuspension buffer and use an ultrasonic cell crusher (150W, ultrasonic 5s, stop 5s) in an ice bath to break for 20min. Then centrifuge (8000r / min, 4℃) for 10min to collect the supernatant, remove impurities through a 0.22μm water filter membrane, and obtain a crude enzyme solution. (2) During purification, first use the loading buffer to balance the HisTrap HP 1mL Ni 2+ The column was filled with 5 column volumes, and then the crude enzyme solution was loaded at a loading flow rate of 1 mL / min. After loading, 5% elution buffer was pumped in to wash away impurities (10 column volumes), and then 38% elution buffer was pumped in to elute the target protein. The salt ions in the sample were removed by a gel column, and the target protein was collected in a 5 mL centrifuge tube.

[0038] Example 3 Enzyme immobilization

[0039] Tryptophanase-chitosan immobilized enzyme: After dissolving 100 mg chitosan, add 10 mg FeCl36H2O and 10 mg FeCl2 4H2O, stir for 30 minutes, then add 10 ml ammonia water under vigorous stirring, continue stirring until the reaction is complete, after the reaction is completed, cool the reaction system to room temperature, obtain chitosan magnetic nanomaterials by magnetic separation, and immediately wash with deionized water; the chitosan magnetic nanomaterials are dissolved in 100 mL deionized water, and tryptophanase is added to 100 mL of chitosan magnetic nanomaterials to prepare a solution with a concentration of 1 mg / mL for electrostatic adsorption; then add 10 uL of glutaraldehyde solution for cross-linking, after a certain reaction time, separate the prepared tryptophanase-nanocatalyst by magnetic adsorption, and wash with deionized water, and finally obtain tryptophanase-chitosan immobilized enzyme.

[0040] Tryptophanase-hyaluronic acid immobilized enzyme: Weigh 100 mg of hyaluronic acid and dissolve it in distilled water. Under magnetic stirring, add crosslinking agents carbodiimide and N-hydroxysuccinimide at a molar ratio of 1:1 to activate the carboxyl group. After stirring at room temperature, add a certain amount of 10 mg of histidine to react for 24 hours. Then put the reaction solution into a dialysis bag and dialyze with distilled water for 72 hours. Ultrasonicate with an ultrasonic crusher for 5-10 minutes, and use 1% mannitol as a freeze-drying protective agent to obtain hyaluronic acid nanoparticle frozen material. Add tryptophanase to 100 mL of hyaluronic acid nanomaterial to prepare a solution with a concentration of 1 mg / mL for electrostatic adsorption. Then add 1 mg of triethylamine and let it stand for 24 hours. Put the solution into a dialysis bag and dialyze with distilled water for 72 hours. Finally, tryptophanase-hyaluronic acid immobilized enzyme is obtained.

[0041] Tryptophanase-silica microsphere immobilized enzyme: 1g of hollow silica microspheres with sulfonic acid groups was dispersed in 90mL of water, and 30mL of ethylene glycol, 1g of potassium nitrate, and 5g of hexamethylenetetramine (HMTA) were added in sequence to prepare a mixed solution A. This mixed solution A was placed in a 250mL flask and dispersed evenly by ultrasound; 0.01g of ferric chloride hexahydrate and 0.05g of magnesium nitrate hexahydrate were dissolved in 100mL of water to prepare solution B; the two solutions were mixed for 30min under nitrogen protection and continuous mechanical stirring at 200r / min, and the system was heated to 90℃ for reaction for 3-5h; heating was stopped and stirring was continued until cooling. Wash with deionized water until neutral, and silica microspheres were obtained by magnetic separation. Tryptophanase was added to 100mL of silica microspheres to prepare a solution with a concentration of 1mg / mL for electrostatic adsorption. Then it was washed with PBS 3 times. Finally, tryptophanase-silica microsphere immobilized enzyme was obtained.

[0042] Detection of immobilization rate: The immobilization rate of the enzyme was determined according to the Bradford assay. The specific operation is as follows: Dissolve 10.0 mg of Coomassie Brilliant Blue G-250 in 5 mL of 95% ethanol (C2H5OH). Next, carefully add 12 mL of 85% phosphoric acid (H3PO4) while stirring. Then, make up to 100 mL with deionized water. Mix 100 μL of supernatant and 5 mL of Coomassie Brilliant Blue G-250 solution completely and incubate at room temperature for 5 minutes. Then, determine its absorbance at 595 nm by ultraviolet spectrophotometry. Use tryptophan enzyme solution as the standard curve and use the standard curve equation to calculate the mass of the enzyme in the supernatant. Finally, calculate the immobilization rate of the enzyme according to the following formula:

[0043]

[0044] The calculation results are shown in Table 1 below.

[0045] Table 1 Solidification rate results

[0046] Immobilization method Solid loading rate Tryptophanase-chitosan immobilized enzyme 73.5％ Tryptophanase-hyaluronan immobilized enzyme 71.2％ Tryptophanase-silica microspheres immobilized enzyme 55.8％

[0047] According to the results in Table 1, the immobilization rates of tryptophanase-chitosan immobilized enzyme and tryptophanase-hyaluronic acid immobilized enzyme were both higher than 70%, with no significant difference (P<0.05), and were both higher than 55.8% of tryptophanase-silica microsphere immobilized enzyme.

[0048] Example 4 Study on the stability of immobilized enzyme

[0049] (1) Study on the effect of pH on the stability of immobilized enzyme

[0050] 1 mg of tryptophanase-chitosan immobilized enzyme, tryptophanase-hyaluronic acid immobilized enzyme and tryptophanase-silica microsphere immobilized enzyme were placed in 2 mL of PBS solution and stirred for 12 hours at pH 6.0, pH 6.5, pH 7.0, pH 7.5 and pH 8.0, respectively. The Bradford assay was used to detect the amount of enzyme initially added and the amount of enzyme in the supernatant after centrifugation at 10000 rpm / min for 10 min. The tryptophan protein content in the supernatant was also calculated, and the immobilization rate was obtained as shown in Table 2.

[0051] Table 2 Immobilization rate at different pH

[0052]

[0053] From the results in Table 2, it can be found that the immobilization rate of tryptophanase-chitosan immobilized enzyme is 91.5%-98.5% in the range of pH 6.0-pH 8.0, and it is least affected by pH.

[0054] The immobilization rate of tryptophanase-hyaluronic acid immobilized enzyme was 77.3%-95.2% in the range of pH 6.0-pH 8.0, and it was most affected by pH.

[0055] The immobilization rate of tryptophanase-silica microspheres is 88.1%-92.2% in the pH range of 6.0-8.0, and is less affected by pH.

[0056] (2) Effect of temperature on the stability of immobilized enzyme

[0057] 1 mg of tryptophanase-chitosan immobilized enzyme, tryptophanase-hyaluronic acid immobilized enzyme and tryptophanase-silica microsphere immobilized enzyme were placed in 2 mL of PBS solution and stirred for 12 h at 20 ° C, 30 ° C, 40 ° C, 50 ° C and 60 ° C. The Bradford assay was used to detect the amount of enzyme initially added and the amount of enzyme in the supernatant after centrifugation at 10000 rpm / min for 10 min. The tryptophan protein content in the supernatant was also determined, and the immobilization rate was calculated to obtain Table 3.

[0058] Table 3 Solid loading rate at different temperatures

[0059]

[0060]

[0061] From the results in Table 3, it can be found that the immobilization rate of tryptophanase-chitosan immobilized enzyme is 88.1%-98.5% in the range of 20°C-60°C, and it is least affected by temperature.

[0062] The immobilization rate of tryptophanase-hyaluronic acid immobilized enzyme was 80.0%-97.1% in the range of 20℃-60℃, and it was most affected by temperature.

[0063] The immobilization rate of tryptophanase-silica microspheres is 89.8%-96.6% within the range of 20°C-60°C, and is less affected by temperature.

[0064] Example 5 Determination of the activity of tryptophanase-chitosan immobilized enzyme

[0065] Tryptophanase activity test: In a 13mm×100mm test tube, add 0.3ml of 60mmol / L L-serine, 0.04ml of 0.2mmol / L indole, 0.16ml of 80mmol / L potassium phosphate buffer (pH8), 0.1ml of 10mg of pyridoxal phosphate, 0.4ml of enzyme extract, and then add heavy distilled water to a final volume of 1.0ml, and keep warm in a 35℃ water bath for 1h. Then add 0.2ml of 5% NaOH to stop the reaction, centrifuge for 1h, and filter the supernatant through a 0.22μm filter membrane to determine the tryptophan content. Enzyme activity definition: The amount of enzyme required to generate 1μmol of L-tryptophan in 1min is one enzyme activity unit (U).

[0066] (1) Determination of the optimal pH value of tryptophanase-chitosan immobilized enzyme: In this experiment, the reaction was carried out at 35°C in substrate buffers with different pH values ​​(6, 7, 8, 9, 10) for 1 h, and the supernatant was filtered through a 0.22 μm filter membrane to determine the enzyme activity. Figure 2 .Depend on Figure 2 It was found that the optimal enzyme activity was 245U at pH 8.

[0067] (2) Determination of the optimal temperature of tryptophanase-chitosan immobilized enzyme: In this experiment, the reaction solution of pH 8 was reacted at different temperatures (25, 35, 45, 55°C) for 1 h, centrifuged, and the supernatant was filtered through a 0.22 μm filter membrane to determine the enzyme activity. Figure 3 .Depend on Figure 3 It was found that the optimal enzyme activity was 210U at 35℃.

[0068] (3) Determination of the maximum number of times tryptophanase-chitosan immobilized enzyme was used: In this experiment, the pH 5 reaction solution was reacted at 35°C for 1 hour, centrifuged, and the supernatant was filtered through a 0.22 μm filter membrane to determine the enzyme activity. The above experiment was repeated 5 times, 10 times, 15 times, 20 times, 25 times, and 30 times to determine the enzyme activity. Figure 4 .Depend on Figure 4 It was found that there was no significant change in enzyme activity from 240U to 265U after 1-20 times, and the enzyme activity decreased to 200U after 25 times, and to 180U after 30 times.

[0069] Example 6 Industrial application of tryptophanase-chitosan immobilized enzyme

[0070] In a 50L reaction system, 10g / L-serine and 1g / L 2-methylindole were prepared, and 10mg / L coenzyme pyridoxal phosphate was added, and then 100mg / L immobilized enzyme was added. The mixture was stirred in a reaction tank and reacted at 40°C for 12 hours. The reaction formula is shown below. During this period, the supernatant was collected and the tryptophan content was detected. Figure 5 .Depend on Figure 5It was found that the synthesis of tryptophan basically reached the maximum value of 12g / L after 10 hours, and the reaction was completed. The immobilized enzyme reacted continuously for 20 times and prepared 12kg of tryptophan for industrial application.

[0071]

[0072] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A method for immobilizing tryptophanase, characterized in that: The following steps are involved: S1. Add dilute acetic acid to a reaction vessel containing chitosan, stir to dissolve, add FeCl36H2O and FeCl24H2O, stir, then add aqueous ammonia under vigorous stirring, continue stirring until the reaction is complete, after the reaction is completed, cool, perform magnetic separation, and wash to obtain chitosan magnetic nanomaterials; S2, dissolving the chitosan magnetic nanomaterial in deionized water, then adding tryptophanase to prepare a solution with a concentration of 0.8-1.2 mg / mL, subjecting it to electrostatic adsorption, and then adding glutaraldehyde solution for cross-linking. After a certain reaction time, magnetic adsorption separation and washing are performed to obtain tryptophanase-chitosan immobilized enzyme.

2. The method for immobilizing tryptophanase according to claim 1, characterized in that: In step S1, the usage ratio of chitosan, FeCl3 6H2O, FeCl2 4H2O and ammonia water is (90-110 mg): (8-12 mg): (8-12 mg): (8-12 mL).

3. The method for immobilizing tryptophanase according to claim 1, characterized in that: In steps S1 and S2, deionized water is used for washing.

4. The method for immobilizing tryptophanase according to claim 1, characterized in that: In step S2, the volume ratio of the deionized water to the glutaraldehyde solution is (90-110 mL): (8-12 μL).

5. An immobilized tryptophanase prepared by the method according to any one of claims 1 to 4.

6. An industrial application of the immobilized tryptophanase as claimed in claim 5 in converting L-tryptophan.

7. The use according to claim 6, characterized in that The method comprises the following steps: a certain concentration of serine and 2-methylindole are prepared in a reaction container, and coenzyme pyridoxal phosphate is added, and then an immobilized enzyme is added, and the mixture is stirred in a reaction tank and reacted for a period of time to obtain converted L-tryptophan.

8. The use according to claim 7, characterized in that The concentration of serine is 8-12 g / L, the concentration of 2-methylindole is 0.8-1.2 g / L, the concentration of coenzyme pyridoxal phosphate is 8-12 mg / L, the concentration of immobilized enzyme is 80-120 mg / L, and the pH value of the reaction system is 7-9.

9. The use according to claim 8, characterized in that The concentration of serine is 10 g / L, the concentration of 2-methylindole is 1 g / L, the concentration of coenzyme pyridoxal phosphate is 10 mg / L, the concentration of immobilized enzyme is 100 mg / L, and the pH value of the reaction system is 8.

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