Tryptophan enzyme mutant TnaA-P2 / 3 as well as preparation method and application thereof

Abstract

The invention discloses a tryptophan enzyme mutant TnaA-P2 / 3 as well as a preparation method and an application of the tryptophan enzyme mutant TnaA-P2 / 3. The tryptophan enzyme mutant TnaA-P2 / 3 disclosed by the invention is obtained by mutating I of the 396th amino acid of wild type tryptophan enzyme TnaA-WT of which the sequence is SEQ ID NO.1 into N or L. The tryptophan enzyme mutant TnaA-P2 / 3 disclosed by the invention is a novel tryptophan enzyme mutant TnaA-P2 / 3. The tryptophanase mutant TnaA-P2 / 3 can be used for converting 6-Br-TrP into 6-Br-indole, the efficiency of converting 6-Br-Trp into 6-Br-indole is improved, the tryptophanase mutant TnaA-P2 / 3 can be used for improving the synthesis efficiency of bone snail violet, raw materials for obtaining bone snail violet are easier to obtain, and the production method is simple and easy to operate.

Description

A tryptophanase mutant TnaA-P2 / 3, 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 isoleucine (I) at position 396, with specific mutation forms including:

[0009] The mutant TnaA-P2 is formed by mutating isoleucine at position 396 to asparagine (I396N), and the amino acid sequence of the mutant TnaA-P2 is shown in SEQ ID NO.2;

[0010] The mutant TnaA-P3 is formed by mutating isoleucine at position 396 to leucine (I396L), and the amino acid sequence of the mutant TnaA-P3 is shown in SEQ ID NO.3.

[0011] 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.

[0012] 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.

[0013] 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 recombinant expression vectors PET-28a / TnaA-P2 and PET-28a / TnaA-P3.

[0014] 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.

[0015] 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:

[0016] 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;

[0017] 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.

[0018] 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.

[0019] 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).

[0020] 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.

[0021] 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

[0022] Figure 1 shows the three-dimensional modeling structure of the tryptophanase mutant.

[0023] Figure 2 shows the expression and purification results of wild-type and mutant tryptophanase. (The figure is missing from the original text.)

[0024] A represents wild-type tryptophanase (TnaA-WT);

[0025] B is a tryptophanase mutant (TnaA-P2).

[0026] C represents the tryptophanase mutant (TnaA-P3). In the SDS-PAGE protein electrophoresis diagram shown, lane M is the protein marker (14.3-97.2 kDa), lane 1 is the supernatant obtained after centrifugation following cell lysis, lane 2 is the precipitate obtained after centrifugation following cell lysis, 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.

[0027] Figure 3 shows the differential scanning fluorescence (DSF) curves of TnaA protein (wild type and mutant).

[0028] Figure 4 shows the tryptophanase activity results. (The figure is missing from the original text.)

[0029] A indicates that Trp is the substrate.

[0030] B indicates that 6-Br-Trp is used as the substrate; (C indicates the control group without enzyme, C1 indicates the positive control Indole, and C2 indicates the positive control 6-Br-Indole). Detailed Implementation

[0031] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.

[0032] 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 396 (I396N, I396L) in the sequence of SEQ ID NO:1. SEQ ID NO:1 is derived from GenBank: CAQ34053.1. The amino acid sequence of the wild-type tryptophanase TnaA-WT is as follows:

[0033] MENFKHLPEPFRIRVIEPVKRTTRAYREEAIIKSGMNPFLLDSEDVFIDLLTDSGTGAVTQSMQAAMMRGDEAYSGSRSYYALAESVKNIFGYQYTIPTHQGRGAEQIYIPVLIKKR EQEKGLDRSKMVAFSNYFFDTTQGHSQINGCTVRNVYIKEAFDTGVRYDFKGNFDLEGLERGIEEVGPNNVPYIVATITSNSAGGQPVSLANLKAMYSIAKKYDIPVVMDSARFAENA YFIKQREAEYKDWTIEQITRETYKYADMLAMSAKKDAMVPMGGLLCMKDDSFFDVYTECRTLCVVQEGFPTYGGLEGGAMERLAVGLYDGMNLDWLAYRIAQVQYLVDGLEEEIGVVCQ QAGGHAAFVDAGKLLPHIPADQFPAQALACELYKVAGIRAVEIGSFLLGRDPKTGKQLPCPAELLRLLTIPRATYTQTHMDFIIEAFKHVKENAANIKGLTFTYEPKVLRHFTAKLKEV

[0034] 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:

[0035] Table 1. English-Chinese translation and abbreviation of the amino acids involved in this application.

[0036]

[0037] As the basic template for constructing the tryptophanase mutant, the amino acid sequence of the wild-type tryptophanase enzyme TnaA-WT 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). Using error-prone PCR, a novel protein SEQ ID NO:2 or SEQ ID NO:2 was obtained by replacing isoleucine at position 396 with aspartic acid or leucine ((I396N, I396L)).

[0038] Example 1: Construction of a recombinant expression vector containing a nucleotide sequence encoding a tryptophanase mutant

[0039] Based on the amino acid sequences encoding TnaA-WT, TnaA-P2, and TnaA-P3, Hunan Aikerui Biotechnology Co., Ltd. designed histidine tags (HHHHHHHHH) at their 3' ends and synthesized the corresponding DNA molecules. These DNA molecules were then ligated to the PET-28a vector via overlap PCR to obtain recombinant expression vectors PET-28a / TnaA-WT, PET-28a / TnaA-P2, and PET-28a / TnaA-P3. 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.

[0040] Example 2: Culture and expression of tryptophanase mutants

[0041] The recombinant expression vector obtained in Example 1 was transformed into E. coli C3013I host cells. Single colonies were picked and inoculated into 20 mL of LB medium containing 50 μg / mL kanamycin. The cells were then cultured overnight at 37°C with shaking. The seed culture obtained overnight was inoculated into 200 mL of LB medium containing 50 μg / mL kanamycin at a volume ratio of 1:100. The cells were cultured at 37°C with shaking until the OD600 reached 0.6-0.8. IPTG was added to a final concentration of 0.4 mmol / L, and the cells were induced for another 5 h with shaking at 37°C. The cells were collected by centrifugation and weighed. The wet weight of the cells was recorded and stored at -80°C.

[0042] Strain ID Mutation Site SEQ ID NO Cell Wet Weight TnaA-WT Wild Type 10.91 TnaA-P2I396N2 0.85 TnaA-P3I396L3 0.82 surface

[0043] The above bacterial cell wet weight test showed that the expression levels of TnaA-WT and TnaA-P2 / P3 were basically the same, indicating that the mutation of the base did not change the expression of the protein.

[0044] Example 3: Purification of tryptophanase mutant

[0045] 1. High-pressure disruption of induced expression bacterial cells

[0046] Bacterial cells frozen at -80℃ after induction of expression were taken. Based on the wet weight of the 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℃, and the supernatant was collected into a 200 mL sterile beaker. Samples (30 μL) were taken from each step after cell lysis, and SDS-PAGE electrophoresis was performed. The results are shown in Figure 2.

[0047] Figure 2A shows the protein purity verification of TnaA-WT. Lane "M" is the protein molecular weight standard (marker), which indicates molecular weight bands of 97.2, 66.4, 44.3, 29.0, and 20.1 kDa. Lane "TnaA-WT" is the purified wild-type TnaA protein, showing only a single clear band (molecular weight of approximately 44.3 kDa), indicating that TnaA-WT has been successfully purified and has high purity.

[0048] 2. Nickel ion affinity chromatography purification

[0049] 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-P2. Lane "M" represents the molecular weight standard, and lanes 1-5 represent different purification stages or treatment groups of TnaA-P2. Lane 1 is the supernatant obtained after centrifugation of TnaA-P2 cells after cell lysis; lane 2 is the precipitate obtained after centrifugation of TnaA-P2 cells after cell lysis; lane 3 is the elution collection from TnaA-P2 nickel ion affinity chromatography; lane 4 is the supernatant obtained after centrifugation of TnaA-P2 after dialysis; and lane 5 is the precipitate obtained after centrifugation of TnaA-P2 after dialysis. Lane 4 shows the final TnaA-P2 protein, with a clear and single band, indicating that TnaA-P2 has been successfully purified to a high purity. Figure 2C shows the protein expression of TnaA-P3, with lane 4 showing the final TnaA-P2 protein, and the clear and single band, indicating that TnaA-P2 has been successfully purified to a high purity.

[0050] Example 4: Differential scanning fluorescence (DSF) detection of Tm value of tryptophanase mutant

[0051] DSF refers to the process of slowly heating a sample on a real-time PCR instrument and measuring the amount of fluorescent dye binding to proteins whose structure has changed during the heating process to evaluate the thermal stability of proteins. The higher the measured Tm value, the stronger the thermal stability of the protein.

[0052] A 20 μL reaction mixture (1X dsf buffer, 1 mg enzyme, SYPRO Orange dye) was placed in a real-time PCR instrument. The excitation and emission wavelengths were adjusted to the corresponding wavelengths of the fluorescent dye. The heating rate was set to 0.05℃ / 1s, and the temperature range was from 25℃ to 99℃. Fluorescence intensity was measured every 1℃ to 3℃. After the reaction, the Tm value of the protein sample was calculated using data processing software.

[0053] Table 2. Tm values ​​of protein samples

[0054]

[0055] The results are shown in Figure 3 and Table 2. The Tm values ​​of TnaA-WT, TnaA-P2, and TnaA-P3 are basically the same, indicating that the thermal stability of tryptophanase remains as stable as that of wild-type tryptophanase after point mutation.

[0056] Example 5: Conversion of 6-bromotryptophan to 6-bromoindole by tryptophanase

[0057] 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 mutants (TnaA-P2, TnaA-P3) 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:

[0058] (1) Incubation: Take 42ug of TnaA-WT obtained in Example 3 and TnaA-P1 and TnaA-P3 respectively and mix them with 2mL of 2mMMTrp and 2mL of 2mM 6-Br-Trp and incubate them at 30℃ for 16h. The solvent for Trp and 6-Br-Trp is glucose-PBS (0.3% w / v) solution.

[0059] (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;

[0060] (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.

[0061] (4) Detection: The obtained reaction products were analyzed by thin-layer chromatography. The group without enzyme was used as a blank control, Trp was used as the substrate and 20 mM indole as the positive control, and 6-Br-Trp was used as the substrate and 20 mM 6-Br-indole as the positive control. The developing solvent was petroleum ether:ethyl acetate (5:3). The detection results are shown in Figure 4 and Table 3:

[0062] Figure 4A 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 substrate Trp; "P2" refers to the reaction group of tryptophanase mutant (TnaA-P2) with substrate Trp; and "P3" refers to the reaction group of tryptophanase mutant (TnaA-P3) with 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. Groups "P2" and "P3" have no bands.

[0063] Figure 4B 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; "P2" refers to the reaction group of tryptophanase mutant (TnaA-P2) with the substrate 6-Br-Trp; and "P3" refers to the reaction group of tryptophanase mutant (TnaA-P3) with the substrate 6-Br-Trp. It can be observed that 6-Br-Trp itself has no band, while the WT group, "P2" and "P3" groups, and 6-Br-Indole all have bands at the same position, indicating that all can convert the substrate 6-Br-Trp to 6-Br-Indole.

[0064] The conversion efficiency of group “WT” for Trp and 6-Br-TrP is basically the same. Groups “P2” and “P3” do not convert Trp to indole, but they can convert 6-Br-TrP to 6-Br-indole. This indicates that TnaA-P2 and TnaA-P3 reduce the generation of the byproduct indole.

[0065] Table 3. Conversion capacity of tryptophanase for 6-bromo-tryptophan (6-Br-Trp)

[0066] (Note: Area refers to the region area, Mean refers to the average fluorescence intensity, and Integrated Density refers to the total fluorescence density.)

[0067] The results showed that in the 6-bromoindole related system (B):

[0068] The mean (26.708) and integrated density (26281) of TnaA-WT were significantly higher than those of C2 (6-Br-Indole, 24.876 / 24478), indicating that TnaA-WT enhanced the fluorescence signal of the system; while the mean (7.54 / 6.411) and integrated density (7419 / 6308) of TnaA-P2 and TnaA-P3 were much lower than those of TnaA-WT, indicating that these two mutants significantly weakened the fluorescence signal.

[0069] In the indole system (A):

[0070] The mean (12.495) and integrated density (12295) of TnaA-WT were lower than those of C1 (Indole, 15.856 / 15602), which is the opposite of the "enhanced fluorescence" trend of TnaA-WT in system B. This result indicates that the reaction characteristics of the tryptophan / indole system are different.

[0071] In summary, the above results reflect the differences in fluorescence response between wild-type and mutant TnaA in different indole systems. The mutant (P2 / P3) significantly reduced the fluorescence signal in the 6-bromoindole system.

[0072] In summary, TnaA-WT exhibits essentially the same conversion efficiency for Trp and 6-Br-TrP. TnaA-P2 and TnaA-P3 do not convert Trp to indole, but they can convert 6-Br-TrP to 6-Br-indole. This indicates that TnaA-P2 and TnaA-P3 reduce the conversion efficiency of Trp and decrease the generation of the byproduct indole.

[0073] Sequence list information:

[0074] DTD Version: V1_3

[0075] Filename: Cancer Hospital.xml

[0076] Software Name: WIPO Sequence

[0077] Software version: 2.1.0

[0078] Generation Date: 2025-12-18

[0079] Basic Information:

[0080] Current application / Intellectual Property Office: CN

[0081] Current application / Application date: 2025-12-18

[0082] Current application / applicant file name: 12530000431201824Q

[0083] Applicant's Name or Title: Yunnan Cancer Hospital (The Third Affiliated Hospital of Kunming Medical University)

[0084] Applicant's name or organization name / Language: zh

[0085] Applicant's Name or Title / Latin Name: Yunnan Cancer Hospital

[0086] Invention Title: A Tryptophanase Mutant TnaA-P2 / 3, Its Preparation Method and Application (zh)

[0087] Total sequence count: 3

[0088] sequence:

[0089] Serial Number (ID): 1

[0090] Length: 471

[0091] Molecular type: AA

[0092] Feature location / qualifier:

[0093] - source, 1..471

[0094] > mol_type, protein

[0095] > organism, TnaA-WT

[0096] residues:

[0097] MENFKHLPEP FRIRVIEPVK RTTRAYREEA IIKSGMNPFL LDSEDVFIDLLTDSGTGAVT 60

[0098] QSMQAAMMRG DEAYSGSRSY YALAESVKNI FGYQYTIPTH QGRGAEQIYIPVLIKKREQE 120

[0099] KGLDRSKMVA FSNYFFDTTQ GHSQINGCTV RNVYIKEAFD TGVRYDFKGNFDLEGLERGI 180

[0100] EEVGPNNVPY IVATITSNSA GGQPVSLANL KAMISIAKKY DIPVVMDSAR FAENAYFIKQ240REAEYKDWTI EQITRETYKY ADMLAMSAKK DAMVPMGGLL CMKDDSFFDV YTECRTLCVV 300

[0101] QEGFPTYGGL EGGAMERLAV GLYDGMNLDW LAYRIAQVQY LVDGLEEIGV VCQQAGGHAA 360

[0102] FVDAGKLLPH IPADQFPAQA LACELYKVAG IRAVEIGSFL LGRDPCTGKQ LPCPAELLRL 420

[0103] TIPRATYTQT HMDFIIEAFK HVKENAANIK GLTFTYEPKV LRHFTAKLKE V 471

[0104] Serial number (ID): 2

[0105] Length: 471

[0106] Molecular type: AA

[0107] Characteristic:

[0108] - source, 1..471

[0109] > mol_type, protein

[0110] > organism, TnaA-P2

[0111] residue:

[0112] MENFKHLPEP FRIRVIEPVK RTTRAYREEA IIKSGMNPFL LDSEDVFIDL LTDSGTGAVT 60

[0113] QSMQAAMMRG DEAYSGSRSY YALAESVKNI FGYQYTIPTH QGRGAEQIYI PVLIKKREQE 120

[0114] KGLDRSKMVA FSNYFFDTTQ GHSQINGCTV RNVYIKEAFD TGVRYDFKGN FDLEGLERGI 180

[0115] EEVGPNNVPY IVATITSNSA GGQPVSLANL KAMYSIAKKY DIPVVMDSAR FAENAYFIKQ 240

[0116] REAEYKDWTI EQITRETYKY ADMLAMSAKK DAMVPMGGLL CMKDDSFFDV YTECRTLCVV 300

[0117] QEGFPTYGGL EGGAMERLAV GLYDGMNLDW LAYRIAQVQY LVDGLEEIGV VCQQAGGHAA 360

[0118] FVDAGKLLPH IPADQFPAQA LACELYKVAG IRASENGSFL LGRDPKTGKQ LPCPAELLRL 420

[0119] TIPRATYTQT HMDFIIEAFK HVKENAANIK GLTFTYEPKV LRHFTAKLKE V 471

[0120] Sequence number (ID): 3

[0121] Length: 471

[0122] Molecular type: AA

[0123] Feature location / qualifier:

[0124] - source, 1..471

[0125] > mol_type, protein

[0126] > organism, TnaA-P3

[0127] Residues:

[0128] MENFKHLPEP FRIRVIEPVK RTTRAYREEA IIKSGMNPFL LDSEDVFIDL LTDSGTGAVT 60

[0129] QSMQAAMMRG DEAYSGSRSY YALAESVKNI FGYQYTIPTH QGRGAEQIYI PVLIKKREQE 120

[0130] KGLDRSKMVA FSNYFFDTTQ GHSQINGCTV RNVYIKEAFD TGVRYDFKGN FDLEGLERGI 180

[0131] EEVGPNNVPY IVATITSNSA GGQPVSLANL KAMYSIAKKY DIPVVMDSAR FAENAYFIKQ 240

[0132] REAEYKDWTI EQITRETYKY ADMLAMSAKK DAMVPMGGLL CMKDDSFFDV YTECRTLCVV 300

[0133] QEGFPTYGGL EGGAMERLAV GLYDGMNLDW LAYRIAQVQY LVDGLEEIGV VCQQAGGHAA 360

[0134] FVDAGKLLPH IPADQFPAQA LACELYKVAG IRASELGSFL LGRDPCTGKQ LPCPAELLRL 420

[0135] TIPRATYTQT HMDFIIEAFK HVKENAANIK GLTFTYEPKV LRHFTAKLKE V 471

[0136] END.

Claims

1. A tryptophanase mutant TnaA-P2 / 3, wherein the amino acid sequence of the tryptophanase parent is shown in SEQ ID NO.1, characterized in that, The tryptophanase mutants were obtained by mutating amino acid I at position 396 of the tryptophanase parent to N and L, respectively, to obtain TnaA-P2 and TnaA-P3. The amino acid sequence of TnaA-P2 is shown in SEQ ID NO.2, and the amino acid sequence of TnaA-P3 is shown in SEQ ID NO.

3.

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 as described in claim 2.

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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