Tryptophan enzyme mutant TnaA-P1 as well as preparation method and application thereof

Abstract

The invention discloses a tryptophan enzyme mutant TnaA-P1 as well as a preparation method and an application of the tryptophan enzyme mutant TnaA-P1. The tryptophan enzyme mutant TnaA-P1 disclosed by the invention is obtained by mutating H of the 463rd amino acid of wild type tryptophan enzyme TnaA-WT of which the sequence is SEQ ID NO.1 into S. The tryptophanase mutant TnaA-P1 can be used for converting 6-Br-TrP into 6-Br-indole, so that the efficiency of converting 6-Br-TrP into 6-Br-indole is improved, the tryptophanase mutant TnaA-P1 can be used for improving the synthesis efficiency of bone snail violet, the raw materials of bone snail violet are easier to obtain, and the production method is simple and easy to operate.

Description

A tryptophanase mutant TnaA-P1, its preparation method and application Technical Field

[0001] This application relates to the field of tryptophanase mutation technology, and in particular to a tryptophanase mutant and its application. Background Technology

[0002] Tryptophanase (TnaA) is a class of pyridoxal phosphate-dependent lysins widely found in microorganisms such as *Escherichia coli* and *Klebsiella pneumoniae*. Its core function is to catalyze the breakdown of L-tryptophan into indole, pyruvate, and ammonia, playing a crucial role in the microbial tryptophan metabolic pathway. It is also a highly promising industrial enzyme in the field of biosynthesis. In recent years, with the development of biocatalysis technology, tryptophanase has been gradually applied to the green synthesis of indole compounds, demonstrating significant value, especially in the precursor preparation of precious dyes such as Tyrian Purple and Indigo.

[0003] Tyre purple, also known as royal purple, is a historically legendary natural dye. Its natural source is specific marine gastropods such as *Bolinus brandaris* and *Hexaplex trunculus*. Due to the difficulty in obtaining natural raw materials, high fishing costs, and the complexity and extremely low yield of traditional extraction processes, tyre purple has long been a scarce commodity, making it difficult to meet the demands of large-scale applications. Modern research shows that 6-bromoindole (6-Br-indole) is the core precursor in the biosynthesis of tyre purple, and 6-bromotryptophan (6-Br-Trp), as a direct precursor of 6-bromoindole, is crucial for the efficient and targeted conversion of tyre purple into a biosynthetic product.

[0004] Currently, several patents disclose related technologies for tryptophanase mutants, aiming to optimize their catalytic performance. For example, CN120485164A discloses a tryptophanase mutant that improves the catalytic efficiency for L-tryptophan by modifying the amino acid site of the active site, but it is mainly used for the general synthesis of indole and is not designed for the specific conversion of 6-bromo-tryptophan; CN110904086A discloses another tryptophanase mutant that focuses on optimizing the enzyme's thermal stability and pH adaptability, but its catalytic substrate is still mainly natural L-tryptophan, and its activity for the conversion of halotryptophan is limited; CN119506260A discloses a tryptophanase mutant for the production of indole, which reduces production costs by increasing enzyme expression and stability, but it also does not solve the problem of insufficient specificity for halotryptophan conversion.

[0005] In existing technologies, wild-type tryptophanases and their disclosed mutants generally suffer from a key drawback: poor catalytic specificity for 6-bromo-tryptophan. While converting 6-bromo-tryptophan to 6-bromoindole, they readily catalyze the conversion of L-tryptophan, which may be present in the system, into indole, leading to severe interference from byproducts (CN110904086A; CN119506260A). This non-specific catalysis not only reduces the purity of the 6-bromoindole product and increases the difficulty of subsequent separation and purification, but also wastes raw materials, severely limiting its application in the synthesis of high-purity products such as spiroviolet. Furthermore, while some mutants show improvements in catalytic efficiency or stability, they do not address the specific conversion requirements of halogenated substrates, failing to meet the core demands of "high efficiency, high purity, and low byproducts" in industrial production.

[0006] Therefore, developing a tryptophanase mutant with high specificity and high conversion efficiency for 6-bromo-tryptophan, which significantly reduces the formation of the byproduct indole while maintaining enzyme stability, is of great significance for breaking through the bottleneck of large-scale production of spirulina violet precursors and promoting the green biosynthesis of natural dyes. It can also provide a technical reference for the targeted synthesis of other halogenated indole compounds. Summary of the Invention

[0007] To address the shortcomings of existing wild-type tryptophanases in catalyzing the conversion of 6-Br-Trp to 6-bromoindole, which suffers from insufficient specificity and high indole content as a byproduct, this invention provides a tryptophanase mutant. This mutant significantly improves the conversion specificity to 6-Br-Trp while maintaining thermal stability, and reduces the generation of indole byproduct. Furthermore, this invention provides a preparation method and applications for this mutant, enabling the efficient and high-purity production of 6-bromoindole. The specific technical solution is as follows:

[0008] The first objective of this invention is to modify the wild-type tryptophanase shown in SEQ ID NO.1 using site-directed mutagenesis, targeting histidine (L) at position 463, with specific mutation forms including:

[0009] The mutant TnaA-P1 is created by mutating histidine at position 463 to serine (H463S), and the amino acid sequence of the mutant TnaA-P1 is shown in SEQ ID NO.2.

[0010] The mutants retain the thermostability of wild-type tryptophanase while significantly improving the conversion specificity to 6-Br-Trp. They essentially do not catalyze the conversion of L-tryptophan (Trp) to indole, effectively reducing interference from byproducts.

[0011] A second objective of this invention is to provide a nucleic acid molecule encoding the above-mentioned tryptophanase mutant, wherein the nucleic acid molecule encodes the tryptophanase mutant of claim 1, and has a sequence encoding the histidine tag HHHHHHHH fused to the 3' end of the nucleic acid molecule, which facilitates subsequent purification.

[0012] A third objective of this invention is to provide a recombinant vector comprising the aforementioned nucleic acid molecule, wherein the recombinant vector is a PET-28a vector, and the nucleic acid molecule encoding the aforementioned tryptophanase mutant is ligated to the PET-28a vector via overlap PCR to construct the recombinant expression vector PET-28a / TnaA-P1.

[0013] The fourth objective of this invention is to provide a recombinant bacterium, wherein the recombinant bacterium is transformed into host cells E. coli C3013I by the above-mentioned recombinant vector and screened in LB medium containing 50 μg / mL kanamycin to obtain a recombinant bacterium capable of stably expressing a tryptophanase mutant.

[0014] Another object of the present invention is to provide a method for preparing a tryptophanase mutant, the method comprising the steps of recombinant bacterial culture, induced expression, bacterial cell disruption and purification, as detailed below:

[0015] Recombinant bacterial culture: Single colonies of recombinant bacteria were picked and inoculated into LB medium containing 50 μg / mL kanamycin. The culture was incubated overnight at 37°C with shaking at 200 rpm to obtain a seed culture. The seed culture was then inoculated into fresh LB medium at a 1:100 volume ratio and cultured until OD (Organic Demand). 600 =0.6~0.8;

[0016] Induction of expression: IPTG was added to a final concentration of 0.4 mmol / L, and the cells were induced by shaking at 37°C for 5 h. The cells were then collected by centrifugation.

[0017] Cell disruption: Resuspend each gram of cells in 3 mL of lysis buffer (10 mM PBS, pH 7.0), autoclave at 800 bar for three cycles, centrifuge at 4°C and 12000 rpm for 30 min, and collect the supernatant.

[0018] Purification: The supernatant was purified by nickel ion affinity chromatography, equilibrated with buffer A, and eluted with a gradient of buffer B. The target protein was collected and dialyzed, and finally stored in 10 mM PBS and 10% glycerol (pH 7.0).

[0019] The final objective of the invention is to provide a method for producing 6-bromoindole: a purified tryptophanase mutant is incubated with 2 mM 6-Br-Trp (solvent is 0.3% w / v glucose-PBS solution) at 30°C for 16 h. After incubation, an equal volume of ethyl acetate is added for extraction for 15 min. After standing and layering, the upper organic phase is collected and concentrated by rotary evaporation to obtain the 6-bromoindole product. The purity of the product is verified by thin-layer chromatography.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: the tryptophanase mutant described in the present invention is as stable as the wild-type tryptophanase; the tryptophanase mutant improves the efficiency of converting 6-Br-Trp to 6-Br indole compared with the wild-type tryptophanase, reduces the production of the byproduct indole, and improves the production efficiency of spirulina violet. Attached Figure Description

[0021] Figure 1 shows the three-dimensional modeling structure of the tryptophanase mutant.

[0022] Figure 2 shows the expression and purification results of wild-type and mutant tryptophanase; where:

[0023] A represents wild-type tryptophanase (TnaA-WT).

[0024] B represents the tryptophanase mutant (TnaA-P1); in the SDS-PAGE protein electrophoresis diagram shown, lane M is the protein marker (14.3-97.2kDa), lane 1 is the supernatant obtained after centrifugation following cell disruption, lane 2 is the precipitate obtained after centrifugation following cell disruption, lane 3 is the elution collection buffer from nickel ion affinity chromatography, lane 4 is the supernatant obtained after centrifugation following dialysis, and lane 5 is the precipitate obtained after centrifugation following dialysis.

[0025] Figure 3 shows the tryptophanase activity results; where:

[0026] A indicates that Trp is the substrate;

[0027] B indicates that 6-Br-Trp was used as the substrate; in the results shown in the figure; (where C represents the control group without enzyme, C1 represents the positive control Indole, and C2 represents the positive control 6-Br-Indole.) Detailed Implementation

[0028] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.

[0029] The tryptophanase mutant constructed in this invention is a mutant of the wild-type tryptophanase TnaA-WT with the amino acid sequence shown in SEQ ID NO:1. It is a new protein formed by replacing the amino acid at position 463 (H463S) in the sequence of SEQ ID NO:1, where SEQ ID NO:1 is derived from GenBank: CAQ34053.1. The amino acid sequence of the wild-type tryptophanase TnaA-WT is as follows:

[0030] MENFKHLPEPFRIRVIEPVKRTTRAYREEAIIKSGMNPFLLDSEDVFIDLLTDSGTGAVTQSMQAAMMRGDEAYSGSRSYYALAESVKNIFGYQYTIPTHQGRGAEQIYIPVLIKKR EQEKGLDRSKMVAFSNYFFDTTQGHSQINGCTVRNVYIKEAFDTGVRYDFKGNFDLEGLERGIEEVGPNNVPYIVATITSNSAGGQPVSLANLKAMYSIAKKYDIPVVMDSARFAENA YFIKQREAEYKDWTIEQITRETYKYADMLAMSAKKDAMVPMGGLLCMKDDSFFDVYTECRTLCVVQEGFPTYGGLEGGAMERLAVGLYDGMNLDWLAYRIAQVQYLVDGLEEEIGVVCQ QAGGHAAFVDAGKLLPHIPADQFPAQALACELYKVAGIRAVEIGSFLLGRDPKTGKQLPCPAELLRLLTIPRATYTQTHMDFIIEAFKHVKENAANIKGLTFTYEPKVLRHFTAKLKEV

[0031] For ease of description, the amino acid abbreviations for proteins can use either three letters or single letters, as is well known to those skilled in the art. These abbreviations are listed in the table below:

[0032] Table 1. English-Chinese translation and abbreviation of the amino acids involved in this application.

[0033]

[0034] The amino acid sequence of the wild-type tryptophanase enzyme TnaA-WT, serving as the basic template for constructing the tryptophanase mutant, is shown in SEQ ID NO:1. To obtain a novel functional enzyme capable of catalyzing the synthesis of D-tryptophan from D-serine and indole, this invention involves point mutation of the gene sequence of the wild-type tryptophanase enzyme TnaA-WT (SEQ ID NO:1). A new protein, SEQ ID NO:2, was obtained by replacing histidine at amino acid position 463 with serine (H463S) using error-prone PCR.

[0035] Example 1: Construction of a recombinant expression vector containing a nucleotide sequence encoding a tryptophanase mutant

[0036] Based on the amino acid sequences of TnaA-WT and TnaA-P1, Hunan Aikerui Biotechnology Co., Ltd. designed a histidine tag (HHHHHHHHH) at the 3' end and synthesized the corresponding DNA molecules. These DNA molecules were then ligated to the PET-28a vector via overlap PCR to obtain the recombinant expression vectors PET-28a / TnaA-WT and PET-28a / TnaA-P1. Further, PCR amplification was performed using these recombinant expression vectors as templates. The PCR products were then purified and recovered using a SteadyPure PCR reaction purification kit (Hunan Aikerui Biotechnology Co., Ltd., catalog number AG21003). The recovered products were transformed into DH5α competent cells, and positive clones were screened for sequencing verification. The results were consistent with expectations.

[0037] Example 2: Culture and expression of tryptophanase mutants

[0038] The recombinant expression vectors obtained in Example 1 were transformed into E. coli C3013I host cells. Single colonies were picked and inoculated into 20 mL of LB medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C with shaking. The seed culture from the overnight culture was then inoculated into 200 mL of LB medium containing 50 μg / mL kanamycin at a volume ratio of 1:100, and cultured at 37°C with shaking until 0D. 600 The concentration was 0.6-0.8; IPTG was added to a final concentration of 0.4 mmol / L, and the cells were induced by shaking at 37°C for 5 hours; the cells were collected by centrifugation and weighed, and the wet weight of the cells was recorded and stored at -80°C.

[0039]

[0040] The above bacterial cell wet weight test showed that the expression levels of TnaA-WT and TnaA-P1 were basically the same, indicating that the mutation of the base did not change the expression of the protein.

[0041] Example 3: Purification of tryptophanase mutant

[0042] 1. High-pressure disruption of induced expression bacterial cells

[0043] After induction of expression, bacterial cells were frozen at -80°C. Based on the wet weight of the bacterial cells recorded in Example 2, 3 mL of lysis buffer (10 mmol / L PBS pH 7.0) was added per gram of bacterial cells to resuspend the cells. The cells were then lysed using a high-pressure homogenizer at 800 bar for three cycles. The lysed cells were centrifuged at 12000 rpm for 30 min at 4°C, and the supernatant was transferred to a 200 mL sterile beaker. Samples were taken from each step of the process starting after cell disruption, with 30 μL of each sample. SDS-PAGE electrophoresis analysis was performed, and the results are shown in Figure 2A, verifying the protein purity of TnaA-WT. Lane "M" represents the protein molecular weight marker, indicating molecular weight bands of 97.2, 66.4, 44.3, 29.0, and 20.1 kDa. Lane "TnaA-WT" represents the purified wild-type TnaA protein, showing only a single clear band (molecular weight approximately 44.3 kDa), indicating that TnaA-WT has been successfully purified with high purity.

[0044] 2. Nickel ion affinity chromatography purification

[0045] The selected chromatography column was a Ni-NTA Purose 6 Fast Flow (purchased from Jiaxing Qianchun Biotechnology Co., Ltd.). The binding buffer was Buffer A: 50 mmol / L potassium phosphate, 300 mM NaCl, 20 mmol / L imidazole, pH 7.0; the elution buffer was Buffer B: 50 mmol / L potassium phosphate, 300 mM NaCl, 700 mmol / L imidazole, pH 7.0. The supernatant from step 1 was loaded onto the chromatography column equilibrated with Buffer A. After sample loading, the column was first rinsed with buffer A, followed by gradient elution from 0% to 100% with buffer B. The eluted fractions were then analyzed by SDS-PAGE protein electrophoresis. The eluent was collected based on the results and dialyzed back into buffer A. The dialysate was collected for later use. The results are shown in Figure 2B, which illustrates the protein expression of TnaA-P1. Lane "M" represents the molecular weight standard, and lanes 1-5 represent different purification stages or treatment groups of TnaA-P1. Lane 1 is the supernatant obtained after centrifugation following TnaA-P1 cell lysis; lane 2 is the precipitate obtained after centrifugation following TnaA-P1 cell lysis; lane 3 is the elution collection from TnaA-P1 nickel ion affinity chromatography; lane 4 is the supernatant obtained after centrifugation following TnaA-P1 dialysis; and lane 5 is the precipitate obtained after centrifugation following TnaA-P1 dialysis. Lane 4 shows the final TnaA-P1 protein, with a clear and single band, indicating that TnaA-P1 has been successfully purified to a high degree of purity.

[0046] Example 4: Conversion of 6-bromotryptophan to 6-bromoindole by tryptophanase

[0047] The tryptophanase mutant obtained in Example 3 was used to evaluate its conversion ability to 6-bromo-tryptophan (6-Br-Trp). Wild-type tryptophanase (TnaA-WT) and the mutant (TnaA-P1) were incubated with the substrates tryptophan and 6-Br-Trp, respectively, for a certain period. After the reaction, the incubation system was extracted and concentrated, and the products were analyzed by thin-layer chromatography. The specific operating steps are as follows:

[0048] (1) Incubation: Take 42ug of TnaA-WT and TnaA-P1 obtained in Example 3, and mix them with 2mL of 2mM Trp and 2mL of 2mM 6-Br-Trp respectively and incubate at 30℃ for 16h. The solvent for Trp and 6-Br-Trp is glucose-PBS (0.3% w / v) solution.

[0049] (2) Extraction: Add 2 ml of ethyl acetate to the above incubated solution, mix by inversion for 15 min, let stand for 1 h and then take the upper layer solution;

[0050] (3) Concentration: The obtained upper layer solution is evaporated to dryness in a rotary evaporator, and then redissolved with 100 μL of ethyl acetate. The liquid after redissolution is the product of tryptophanase reaction.

[0051] (4) Detection: The obtained reaction products were subjected to thin-layer chromatography analysis. The group without enzyme was used as a blank control, Trp was used as the substrate positive control and 20mM indole was used as the substrate positive control and 6-Br-Trp was used as the substrate positive control and 20mM 6-Br-indole was used as the developing solvent. The developing solvent was petroleum ether: ethyl acetate (5:3).

[0052] The results are shown in Figure 3. Figure 3A shows the detection results using Trp as the reaction substrate. "C" represents the group without tryptophanase, containing only Trp; "C1" represents the reaction product indole; "WT" refers to the reaction group of wild-type tryptophanase (TnaA-WT) with the substrate Trp; and "P1" refers to the reaction group of tryptophanase mutant (TnaA-P1) with the substrate Trp. It can be observed that Trp itself has no band, while the WT group and indole both have bands at the same position, indicating that TnaA-WT converts the substrate Trp to indole. The "P1" group has no band.

[0053] Figure 3B shows the detection results using 6-Br-Trp as the reaction substrate. "C" represents the group without tryptophanase, containing only 6-Br-Trp; "C2" represents the reaction product 6-bromoindole (6-Br-Indole); "WT" refers to the reaction group of wild-type tryptophanase (TnaA-WT) with the substrate 6-Br-Trp; and "P1" refers to the reaction group of tryptophanase mutant (TnaA-P1) with the substrate 6-Br-Trp. It can be observed that 6-Br-Trp itself has no band, while the WT group, the "P1" group, and 6-Br-Indole all show bands at the same position, indicating that both can convert the substrate 6-Br-Trp to 6-Br-Indole.

[0054] The conversion efficiency of the “WT” group for Trp and 6-Br-TrP is basically the same, while the “P1” group does not convert Trp to indole, but it can convert 6-Br-TrP to 6-Br-indole. This indicates that TnaA-P1 reduces the generation of the byproduct indole.

[0055] The detection results are shown in Figure 3 and Table 3. TnaA-WT showed essentially the same conversion efficiency for Trp and 6-Br-TrP. TnaA-P1 did not convert Trp to indole, but it could convert 6-Br-TrP to 6-Br-indole. This indicates that TnaA-P1 reduced the generation of the byproduct indole.

[0056] Table 3. Conversion capacity of tryptophanase for 6-bromo-tryptophan (6-Br-Trp)

[0057]

[0058] In summary, TnaA-WT exhibits essentially the same conversion efficiency for Trp and 6-Br-TrP, while TnaA-P1 does not convert Trp to indole, but it can convert 6-Br-TrP to 6-Br-indole. This indicates that TnaA-P1 reduces the conversion efficiency of Trp and decreases the generation of the byproduct Indole.

[0059] Sequence list information:

[0060] DTD Version: V1_3

[0061] Filename: A tryptophanase mutant TnaA-P1, its preparation method and application.xml

[0062] Software Name: WIPO Sequence

[0063] Software version: 2.1.0

[0064] Generation Date: 2025-12-30

[0065] Basic Information:

[0066] Current application / applicant file name: 12530000431201824Q

[0067] Applicant's Name or Organization: Yunnan Cancer Hospital (The Third Affiliated Hospital of Kunming Medical University)

[0068] Applicant's name or organization name / Language: zh

[0069] Applicant's name or title / Latin name: 12530000431201824Q

[0070] Invention Title: A Tryptophanase Mutant TnaA-P1, Its Preparation Method and Application (zh)

[0071] Total sequence count: 2

[0072] sequence:

[0073] Serial Number (ID): 1

[0074] Length: 471

[0075] Molecular type: AA

[0076] Feature location / qualifier:

[0077] - source, 1..471

[0078] > mol_type, protein

[0079] > organism, Tryptophanase

[0080] Residues:

[0081] MENFKHLPEP FRIRVIEPVK RTTRAYREEA IIKSGMNPFL LDSEDVFIDL LTDSGTGAVT 60

[0082] QSMQAAMMRG DEAYSGSRSY YALAESVKNI FGYQYTIPTH QGRGAEQIYI PVLIKKREQE 120

[0083] KGLDRSKMVA FSNYFFDTTQ GHSQINGCTV RNVYIKEAFD TGVRYDFKGN FDLEGLERGI 180

[0084] EEVGPNNVPY IVATITSNSA GGQPVSLANL KAMYSIAKKY DIPVVMDSAR FAENAYFIKQ 240

[0085] REAEYKDWTI EQITRETYKY ADMLAMSAKK DAMVPMGGLL CMKDDSFFDV YTECRTLCVV 300

[0086] QEGFPTYGGL EGGAMERLAV GLYDGMNLDW LAYRIAQVQY LVDGLEEIGV VCQQAGGHAA 360

[0087] FVDAGKLLPH IPADQFPAQA LACELYKVAG IRAVEIGSFL LGRDPKTGKQ LPCPAELLRL 420

[0088] TIPRATYTQT HMDFIIEAFK HVKENAANIK GLTFTYEPKV LRHFTAKLKE V 471

[0089] Sequence number (ID): 2

[0090] Length: 471

[0091] Molecular type: AA

[0092] Feature Location / Qualifier:

[0093] - source, 1..471

[0094] > mol_type, protein

[0095] > organism, synthetic construct

[0096] Residues:

[0097] MENFKHLPEP FRIRVIEPVK RTTRAYREEA IIKSGMNPFL LDSEDVFIDL LTDSGTGAVT 60

[0098] QSMQAAMMRG DEAYSGSRSY YALAESVKNI FGYQYTIPTH QGRGAEQIYI PVLIKKREQE 120

[0099] KGLDRSKMVA FSNYFFDTTQ GHSQINGCTV RNVYIKEAFD TGVRYDFKGN FDLEGLERGI 180

[0100] EEVGPNNVPY IVATITSNSA GGQPVSLANL KAMYSIAKKY DIPVVMDSAR FAENAYFIKQ 240

[0101] REAEYKDWTI EQITRETYKY ADMLAMSAKK DAMVPMGGLL CMKDDSFFDV YTECRTLCVV 300

[0102] QEGFPTYGGL EGGAMERLAV GLYDGMNLDW LAYRIAQVQY LVDGLEEIGV VCQQAGGHAA 360

[0103] FVDAGKLLPH IPADQFPAQA LACELYKVAG IRAVEIGSFL LGRDPKTGKQ LPCPAELLRL 420

[0104] TIPRATYTQT HMDFIIEAFK HVKENAANIK GLTFTYEPKV LRSFTAKLKE V 471

[0105] END.

Claims

1. A tryptophanase mutant TnaA-P1, wherein the amino acid sequence of the tryptophanase parent is shown in SEQ ID NO.1, characterized in that, The tryptophanase mutant was obtained by mutating amino acid H to S at position 463 of the tryptophanase parent to obtain TnaA-P1, and the amino acid sequence of TnaA-P1 is shown in SEQ ID NO.

2.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule is encoding the tryptophanase mutant as described in claim 1.

3. A recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid molecule of claim 3.

4. The recombinant vector according to claim 3, characterized in that, The recombinant vector is the PET-28a vector, and the nucleic acid molecule has a sequence encoding the histidine tag HHHHHHHH fused to its 3' end.

5. A recombinant bacterium, characterized in that, The recombinant bacteria were obtained by transforming host cells E. coli C3013I with the recombinant vector as described in claim 3 or 4.

6. A method for constructing a recombinant bacterium according to claim 5, characterized in that, Includes the following steps: S1, Based on the amino acid sequence of SEQ ID NO.1, obtain the nucleic acid molecule encoding the tryptophanase mutant of claim 1 through site-directed mutagenesis, and link it with the PET-28a vector to construct a recombinant expression vector; S2, transform the recombinant expression vector into DH5α competent cells, screen positive single clones and sequence them for verification; S3, extract the verified recombinant expression vector, transform it into host cell E. coli C3013I, and obtain recombinant bacteria.

7. The use of the tryptophan mutant according to claim 1, the nucleic acid molecule according to claim 2, or the recombinant bacteria according to any one of claims 4-5 in the preparation of 6-Br-indole.

8. A method for preparing 6-Br-indole, characterized in that, The tryptophanase mutant as described in claim 1 was added to a reaction system containing 6-Br-Trp to catalyze the production of 6-Br-indole.

9. The method for preparing 6-Br-indole according to claim 8, characterized in that, The concentration of 6-Br-Trp in the reaction system was 2 mM, the solvent was 0.3% w / v glucose-PBS solution, and the incubation conditions were 30℃ for 16 h. After incubation, 6-Br-indole was obtained by extraction with ethyl acetate and concentration.

10. The application of 6-Br-indole obtained by the preparation method of 6-Br-indole according to claim 8 or 9 in the preparation of spirochetal purple pigment.

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