A mutant of l-tryptophan oxidase with improved thermal stability
By mutating the amino acid sequence and constructing genetic engineering for L-tryptophan oxidase, the problem of decreased enzyme activity under neutral to alkaline conditions was solved, resulting in a highly stable and highly catalytically active L-tryptophan oxidase mutant suitable for industrial production and biological detection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SUZHOU ZHIYUAN CHUANGLIAN BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-02-09
- Publication Date
- 2026-06-02
AI Technical Summary
Existing L-tryptophan oxidases have shortcomings in terms of catalytic activity, substrate specificity, and stability. In particular, their enzyme activity decreases significantly under neutral to alkaline conditions, making it difficult to meet the needs of industrial reaction systems.
By performing specific mutations on the amino acid sequence of L-tryptophan oxidase, including single-point mutations and combinatorial mutations, and combining them with genetic engineering techniques to express the mutant in Escherichia coli, a thermostability-enhanced L-tryptophan oxidase was constructed. Bioinformatics and crystallography methods were then used to screen for thermostability-related mutation sites.
The thermostability of L-tryptophan oxidase was improved. The half-life of single-point mutants and combined mutants at 50℃ was significantly prolonged. In particular, the thermostability of combined mutants was significantly enhanced, with a half-life of about 8 times that of wild type.
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Abstract
Description
Technical Field
[0001] This invention relates to an L-tryptophan oxidase mutant with improved thermal stability, belonging to the field of biotechnology. Background Technology
[0002] L-Tryptophan oxidase is a key enzyme that specifically catalyzes the oxidative deamination of L-tryptophan to indolepyruvate, and it has significant applications in biosynthesis, pharmaceutical intermediate preparation, and biosensor development. In recent years, with the rapid development of metabolic engineering, enzyme catalysis processes, and in vitro diagnostic technologies, the demand for efficient, stable, and substrate-specific L-tryptophan oxidases has been increasing.
[0003] Currently, the research and development of this enzyme mainly relies on natural enzyme resources derived from microorganisms. For example, L-tryptophan oxidase encoding genes are obtained by screening, cloning, and heterologously expressing them from certain bacteria or fungi, thereby enabling enzyme production. Previous studies have reported L-tryptophan oxidase genes from various microbial genera (such as Streptomyces and Bacillus), and recombinant expression has been achieved in host systems such as Escherichia coli. These natural enzymes meet some application needs to a certain extent, but still have significant limitations: First, natural enzymes generally have low catalytic activity, especially under neutral to alkaline conditions, which significantly reduces their activity and limits their applicability in industrial reaction systems. Second, they lack substrate specificity, and some homologous enzymes are prone to cross-reactions with structural analogs such as phenylalanine and tyrosine, resulting in low product purity. Third, enzymes have poor stability and low tolerance to temperature, pH, and organic solvents, making them difficult to use for extended periods under non-aqueous phases or harsh reaction conditions. In addition, the low expression levels and complex purification processes of natural enzymes also restrict their large-scale preparation and economical application.
[0004] Although protein engineering techniques such as rational design and directed evolution have been attempted to improve enzyme performance, the structure-function relationship of the enzyme has not been fully elucidated, and the construction and screening strategies for mutant libraries are inefficient. At present, there is still a lack of L-tryptophan oxidase mutants that combine high activity, high specificity, and high stability.
[0005] Therefore, there is an urgent need in the field to develop a novel L-tryptophan oxidase that, while retaining high substrate specificity, possesses significantly enhanced catalytic efficiency, thermal stability, and pH adaptability, thereby meeting the application requirements of industrial production and high-precision biological detection. This invention is proposed precisely to address the aforementioned technical deficiencies. Summary of the Invention
[0006] To address the shortcomings of the existing technology, this invention provides a thermostable L-tryptophan oxidase mutant and a method for constructing a genetically engineered bacterium of the L-tryptophan oxidase mutant, aiming to solve the technical problem of how to improve the thermostability of existing L-tryptophan oxidases.
[0007] The first technical solution provided by this invention is an L-tryptophan oxidase mutant, wherein the L-tryptophan oxidase mutant is as follows (a1) or (a2): (a1) A derived protein having the same function as the amino acid sequence shown in SEQ ID NO.2 by substituting, deleting, or adding one or more amino acids; (a2) A derived protein having at least 90% homology to the amino acid sequence shown in SEQ ID NO.2 by substitution, deletion or addition of one or more amino acids.
[0008] In some embodiments, the L-tryptophan oxidase mutant amino acid sequence, relative to the parent L-tryptophan oxidase with an amino acid sequence as shown in SEQ ID NO.2, has lysine at position 155, tyrosine at position 242, aspartic acid at position 378, and / or lysine at position 438.
[0009] In some embodiments, the L-tryptophan oxidase mutant is a single-point mutant at any one of the single-point mutation sites P155K, S242Y, T378D, or P438K in the amino acid sequence shown in SEQ ID NO.2.
[0010] In some embodiments, the thermostable L-tryptophan oxidase mutant is a combination mutant of combined mutation sites in P155K / S242Y, P155K / T378D, P155K / P438K, S242Y / T378D, S242Y / P438K, T378D / P438K, P155K / S242Y / T378D, P155K / S242Y / P438K, P155K / T378D / P438K, S242Y / T378D / P438K, and P155K / S242Y / T378D / P438K.
[0011] The second technical solution provided by the present invention is a gene encoding the L-tryptophan oxidase mutant described in the first technical solution.
[0012] The third technical solution provided by the present invention is a recombinant plasmid containing the gene described in the second technical solution.
[0013] In some embodiments, the recombinant plasmid is expressed using the pET28a plasmid as an expression vector.
[0014] The fourth technical solution provided by the present invention is to express the L-tryptophan oxidase mutant described in the first technical solution, or to carry the gene described in the second technical solution, or to transform recombinant cells with the recombinant plasmid described in the third technical solution.
[0015] In some embodiments, the recombinant cells use Escherichia coli as a host.
[0016] The fifth technical solution provided by this invention is a method for improving the thermostability of L-tryptophan oxidase, wherein the method involves performing at least one of the following mutations on the L-tryptophan oxidase parent with the amino acid sequence shown in SEQ ID NO.2: (1) The phenylalanine at position 155 is mutated to lysine; (2) Serine at position 242 is mutated to tyrosine; (3) Threonine at position 378 is mutated to aspartic acid; (4) Phenylalanine at position 438 is mutated to lysine.
[0017] The present invention also provides a method for constructing an L-tryptophan oxidase mutant with improved thermal stability as described above, comprising the following steps: By searching the Pfam and NCBI databases for the amino acid sequence shown in SEQ ID NO.2, removing duplicate sequences, and selecting amino acid sequences with a similarity greater than 30% to the amino acid sequence shown in SEQ ID NO.2, multiple sequence alignment was performed using Clustalx 1.83 software. The remaining amino acid sequences were then compiled into a FASTA file and uploaded to the Consensus Maker v2.0.0 server. After modifying the settings as needed, the online software will generate a consensus sequence that can be edited later. The three-dimensional structure of the protein shown in SEQ ID NO.2 was predicted using the Swissmodel online tool, and the crystal structure of the protein shown in SEQ ID NO.2 was observed using PyMOL. The mutation sites related to thermal stability were screened out as: P155K, S242Y, T378D, and P438K.
[0018] The sixth technical solution provided by the present invention is a genetically engineered bacterium, wherein the genetically engineered bacterium uses Escherichia coli BL21(DE3) as a host and pET28a plasmid as an expression vector to express the mutant described in the first technical solution.
[0019] The seventh technical solution provided by the present invention is a method for preparing an L-tryptophan oxidase mutant, wherein the method involves culturing the genetically engineered bacteria described in the sixth technical solution and inducing the expression of the mutant described in the first technical solution.
[0020] The eighth technical solution provided by this invention is the application of the mutant described in the first technical solution, the gene described in the second technical solution, the recombinant plasmid described in the third technical solution, the recombinant cell described in the fourth technical solution, the method described in the fifth technical solution, or the genetically engineered bacteria described in the sixth technical solution in the production of L-tryptophan oxidase products.
[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The L-tryptophan oxidase mutants with improved thermal stability provided by the present invention include single-point mutants and combined mutants. Compared with wild-type L-tryptophan oxidase, both single-point mutants and combined mutants have longer half-lives at 50°C. In particular, the combined mutants exhibit the superimposed effect of the thermal stability of the single-point mutants, and their half-life is about 8 times that of the wild type.
[0022] 2. The method for constructing a thermostable L-tryptophan oxidase mutant provided by this invention differs from rational design based on the precise structure-function relationship of proteins. This invention is guided by the Consensus Concept theory, analyzes information that can improve the thermostability of enzymes from an evolutionary perspective, integrates and analyzes the sequences of thermostable reverse transcriptase family, and combines bioinformatics and crystallography methods to obtain a novel L-tryptophan oxidase mutant with high stability.
[0023] 3. The L-tryptophan oxidase mutant with improved thermal stability provided by this invention has excellent catalytic activity and good application prospects. Attached Figure Description
[0024] Figure 1 This is a schematic diagram of the simulated crystal structure of L-tryptophan oxidase protein provided in Embodiment 2 of the present invention.
[0025] Figure 2 This is the gene map of L-tryptophan oxidase provided in Example 2 of the present invention.
[0026] Figure 3 The equation for the L-tryptophan oxidase-catalyzed hydrolysis of L-tryptophan enzyme-linked reaction provided in Example 3 of the present invention is shown.
[0027] Figure 4 This is the substrate spectrum of 20 natural amino acids catalyzed by L-tryptophan oxidase provided in Example 4 of the present invention.
[0028] Figure 5The half-life of L-tryptophan oxidase catalyzed at 50°C is provided in Example 4 of this invention. Detailed Implementation
[0029] Reference Appendix Figures 1-5 The preferred embodiments of the present invention will be described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.
[0030] Raw materials used in the examples: The L-tryptophan oxidase gene was provided by Suzhou Genewiz Biotechnology Co., Ltd.
[0031] The PCR amplification enzyme was a KOD high-fidelity polymerase provided by Toyobo.
[0032] The DpnI enzyme was supplied by Fermentas.
[0033] pET28a plasmid and Escherichia coli BL21(DE3) are both commercially available plasmids and strains.
[0034] LB medium formulation: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride (ThermoScientific™ Oxoid).
[0035] The primer information involved in the following examples is shown in Table 1.
[0036] Table 1. Primer sequences for amplification of single-point mutations
[0037] Example 1 1. Cloning of the wild-type L-tryptophan oxidase gene Originating from anthrax bacteria Bacillus anthracis The wild-type L-tryptophan oxidase gene was codon optimized using Escherichia coli as the host cell to obtain the optimized L-tryptophan oxidase gene, whose nucleic acid sequence is SEQ ID NO.1 and the expressed amino acid sequence is SEQ ID NO.2; using SEQ ID NO.1 as the target gene, the target gene was amplified using upstream and downstream amplification primers; The upstream amplification primer nucleic acid sequence is as follows: 5'-ACTGCT CATATG ATGATGCAGCCGCTGACCATGG-3' (where the underlined part is the NdeI restriction enzyme recognition site); The nucleic acid sequences of the amplification primers are as follows: 5'-TCAGCT CTCGAGTTTTTCGTTCACTTCATGCGCCACG-3' (where the underlined part is the restriction endonuclease XhoI recognition site).
[0038] The amplification conditions were as follows: amplification at 98℃ for 30 seconds, then amplification at 98℃ for 30 seconds, amplification at 60℃ for 10 seconds, amplification at 72℃ for 70 seconds, for a total of 35 cycles, and finally amplification at 72℃ for 5 minutes.
[0039] After the reaction was complete, the PCR amplification product was detected by 1.5% agarose gel electrophoresis, yielding a 1.5kb band, the length of which met the expected result. Following the kit's standard operating procedure, the target fragment was recovered and purified. The target fragment and the pET28a plasmid were double-digested using restriction endonucleases XhoI and NdeI, and then ligated using T4 DNA ligase. The ligation product was transformed into *E. coli* BL21(DE3) competent cells. The transformed cells were plated on LB agar plates containing 50 μg / ml kanamycin, and positive clone plasmids were extracted and sequenced. The results showed that the cloned L-tryptophan oxidase gene sequence was correct and correctly inserted into the pET28a plasmid, yielding the recombinant plasmid pET28a-RebO. Figure 2 .
[0040] 2. Expression and purification of L-tryptophan oxidase protein The engineered bacteria from the glycerol tube were inoculated at a volume ratio of 1% into a 4 mL LB medium tube containing 100 μg / mL Kan, and cultured at 37℃ and 220 rpm for 12 h. 4 mL of the bacterial suspension was then transferred to a 1 L LB medium shake flask containing 50 μg / mL Kan, and cultured at 37℃ and 220 rpm for 2.5 h until the OD600 reached approximately 0.9. Then, 0.1 mM IPTG inducer was added, and the culture was induced at 25℃ and 200 rpm for 14 h. The harvested *E. coli* bacterial suspension was ultrasonically disrupted, followed by a one-step Ni-NTA affinity chromatography to obtain L-tryptophan oxidase protein with a purity >95%, the amino acid sequence of which is SEQ ID NO.2.
[0041] 3. Multiple sequence alignment and consensus sequence analysis of L-tryptophan oxidase homologous proteins 3.1 Go to the Pfam database homepage (http: / / pfam.xfam.org / ), enter the amino acid sequence of L-tryptophan oxidase in the SEQUENCE SEARCH tool, and the server will directly return the alignment results of the amino acid sequences of the entire protein family, displaying the abundance of various amino acids at each mutation site in the form of a bar chart. The website can also automatically generate the consensus sequence of the protein family.
[0042] 3.2 Input the amino acid sequence shown in SEQ ID NO.2 into the NCBI protein database and Pfam database. Use the Blast tool to find all L-tryptophan oxidase proteins whose amino acid sequence (SEQ ID NO.2) has a similarity of more than 30%. Delete any duplicate sequences and organize the remaining amino acid sequences into fasta. format. Input the sequences into ClustalX 1.83 software for multiple sequence alignment. The alignment results are output in alan., dnd., and fasta. formats. The dnd. file is the phylogenetic tree file, while the alan. and fasta. files are sequence files in different formats.
[0043] Upload the aforementioned fasta.file to the Consensus Maker v2.0.0 server (http: / / www.hiv.lanl.gov / content / sequence / CONSENSUS / consensus.html). After modifying the settings as needed, the online software will generate a consensus sequence that can be edited later.
[0044] 3.3 The amino acid sequence (SEQ ID NO.2) of L-tryptophan oxidase protein was compared with the consensus sequence of the family and the amino acid abundance map of each site.
[0045] 4. Simulation of the three-dimensional structure of L-tryptophan oxidase protein and selection of mutation hotspots 4.1 The three-dimensional structure of L-tryptophan oxidase protein (amino acid sequence SEQ ID NO.2) was predicted using the Swissmodel online tool.
[0046] 4.2 The crystal structure of L-tryptophan oxidase (amino acid sequence SEQ ID NO.2) was observed using PyMOL. Based on the structural information, the above-mentioned candidate mutation sites and mutation modes were reviewed, and the mutant sites most likely to improve the thermostability of L-tryptophan oxidase were screened out. The screening conditions are as follows: (1) The criteria for determining a site as a candidate site are: ①Most proteins in this family have a generally high amino acid abundance at this site; ②The amino acid at this site is conserved; ③ The amino acids that appear more frequently at this site have significant differences in physicochemical properties compared to the amino acids at this site in L-tryptophan oxidase, such as differences in charge, polarity, and steric hindrance.
[0047] (2) Remove amino acid residues near the active site, i.e., within 10 Å of the catalytic residues, and remove amino acid residues that are in an embedded or semi-embedded state.
[0048] After the above two screening steps, a total of 10 differential sites remain, most of which are located on the surface of the L-tryptophan oxidase protein molecule, such as... Figure 1 As shown, the arrow points to the mutation site.
[0049] (3) Based on the crystal structure of L-tryptophan oxidase protein, analyze each of the above 10 mutation forms in detail and screen out mutants that may improve the thermal stability of L-tryptophan oxidase protein.
[0050] The main criteria for judgment are: ① Mutations should eliminate existing forces that are detrimental to thermal stability, such as electrostatic repulsion and charge accumulation; ② Mutations should not destroy existing forces that are beneficial to thermal stability and stable protein structures; ③ Mutations should introduce new forces that are beneficial to thermal stability, such as hydrogen bonds, salt bridges, and hydrophobic interactions.
[0051] Four single-point mutants were designed, with mutation sites of P155K, S242Y, T378D, and P438K.
[0052] The activity of the four L-tryptophan oxidase mutants was determined, and four L-tryptophan oxidases with improved thermostability were screened out. The mutation sites were P155K, S242Y, T378D, and P438K. The amino acid sequences of the corresponding single-point mutants were SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.
[0053] Example 2: Construction, Expression, and Purification of Mutants 1. Construction of L-tryptophan oxidase single-point mutant Using the recombinant plasmid pET28a-RebO from step 1 as a template, and a pair of complementary oligonucleotides with mutation sites as amplification primers, KOD high-fidelity enzyme was used to perform whole plasmid PCR amplification to obtain a recombinant plasmid with specific mutation sites. The amplification primers used are shown in Table 1.
[0054] The amplification conditions were as follows: amplification at 98℃ for 30 seconds, followed by amplification at 98℃ for 30 seconds, 60℃ for 10 seconds, and 72℃ for 70 seconds, for a total of 35 cycles, and finally amplification at 72℃ for 5 minutes. The PCR amplification products were recovered from the gel and digested with DpnI enzyme at 37℃ for 2 hours to degrade the initial template. The digested products were transformed into E. coli BL21(DE3) competent cells, plated on LB agar plates containing 50 μg / mL kanamycin, and cultured overnight at 37℃. Positive clones were screened and sequenced to verify the results, yielding recombinant bacteria containing a single-point mutant of L-tryptophan oxidase.
[0055] 2. Construction of L-tryptophan oxidase protein combinatorial mutants Using a construction method similar to that of single-point mutants, single-point mutants with improved stability are cumulatively combined. Multiple mutation sites are selected and combined in the amino acid sequence shown in SEQ ID NO.2. For example, 2 to 4 mutation sites are selected from the above 4 mutation sites for combination to obtain different L-tryptophan oxidase combination mutants.
[0056] (1) By selecting two mutation sites for combination, six thermostability-enhanced L-tryptophan oxidase mutants can be constructed. The combined mutation sites are: P155K / S242Y, P155K / T378D, P155K / P438K, S242Y / T378D, S242Y / P438K, and T378D / P438K. The amino acid sequences of these six thermostability-enhanced L-tryptophan oxidase combined mutants are SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively.
[0057] (2) By selecting three mutation sites for combination, four thermostability-enhanced L-tryptophan oxidase combination mutants can be constructed. The combination mutation sites are: P155K / S242Y / T378D, P155K / S242Y / P438K, P155K / T378D / P438K, and S242Y / T378D / P438K. The amino acid sequences of these four thermostability-enhanced L-tryptophan oxidase combination mutants are SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16, respectively.
[0058] (3) Four mutation sites were selected and combined to construct a thermo-stable L-tryptophan oxidase combination mutant. The combination mutation sites were: P155K / S242Y / T378D / P438K. The amino acid sequence of the thermo-stable L-tryptophan oxidase combination mutant was SEQ ID NO.17.
[0059] Example 3: Enzymatic characterization of L-tryptophan oxidase mutant The thermostability of wild-type L-tryptophan oxidase and various L-tryptophan oxidase mutants provided in Example 1 was tested. First, the activity of L-tryptophan oxidase was determined using standard methods, specifically: The catalytic activity of L-tryptophan oxidase for L-tryptophan was determined by colorimetric analysis of the rate at which pretreated L-tryptophan oxidase catalyzes the oxidation of L-tryptophan to hydrogen peroxide at different temperatures. One enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the production of 1 micromole of H₂O₂ per minute under specific assay conditions (pH 7.2, temperature 37 °C, L-tryptophan 10 mM). Figure 3 As shown. Wild-type and mutant L-tryptophan oxidase (0.5 U / mL) were heated in a 50°C water bath for different times, then removed and placed on ice. L-tryptophan oxidase and L-tryptophan were added to a reaction solution containing 4-aminoantipyrine (4-AA, 450 μM), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (TOOS, 500 μM), and HRP (0.9 U / mL), along with phosphate buffer (pH 7.0), to a final volume of 1 mL. Absorbance was measured at 555 nm using a spectrophotometer. The entire reaction was conducted at 37°C.
[0060] Secondly, the enzyme solution was incubated at 50 °C, and samples were taken at different treatment times to determine the percentage of residual activity of L-tryptophan oxidase or L-tryptophan oxidase mutant. The ln value of the residual activity percentage was plotted against time t (min), and the slope of the straight line was the inactivation constant kinact. The half-life of the wild-type L-tryptophan oxidase or L-tryptophan oxidase mutant at this temperature was obtained by t1 / 2=ln2 / kinact.
[0061] The experimental results show that among the various L-tryptophan oxidase mutants, the thermal stability of 4 single-point mutants and 11 combined mutants was significantly improved, as shown in Table 2.
[0062] Table 2 Enzymatic properties of wild-type L-tryptophan oxidase, single-point mutants, and combined mutants
[0063] As shown in Table 1, the L-tryptophan oxidase mutants provided by this invention include single-point mutants and combined mutants. Compared with wild-type L-tryptophan oxidase, both single-point mutants and combined mutants have longer half-lives at 50°C. In particular, the combined mutants exhibit the superimposed effect of the thermal stability of the single-point mutants, and their half-life is about 8 times that of the wild type.
[0064] Example 4: Performance Evaluation of L-Tryptophan Oxidase and its Mutants The performance of wild-type L-tryptophan oxidase and the L-tryptophan oxidase mutant (S242Y / T378D / P438K) with the highest thermostability provided in Example 3 were tested, and the L-tryptophan oxidase activity was determined according to the method in Example 3.
[0065] Experimental results show that the substrate specificity is as follows: Figure 4 The L-tryptophan oxidase mutant (S242Y / T378D / P438K) exhibits the highest thermal stability and shows significantly improved performance compared to the wild type, as shown in Table 3. The half-life of L-tryptophan oxidase (S242Y / T378D / P438K) at 50°C is as follows: Figure 5 As shown.
[0066] Table 3 Performance evaluation of L-tryptophan oxidase and its mutants
[0067] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. An L-tryptophan oxidase mutant, characterized in that, Compared to the L-tryptophan oxidase parent with the amino acid sequence shown in SEQ ID NO.2, the L-tryptophan oxidase mutant has lysine at position 155, tyrosine at position 242, aspartic acid at position 378, and / or lysine at position 438.
2. The gene encoding the L-tryptophan oxidase mutant of claim 1.
3. A recombinant plasmid containing the gene described in claim 2.
4. The recombinant plasmid according to claim 3, characterized in that, The recombinant plasmid was expressed using pET28a plasmid as the expression vector.
5. A recombinant cell expressing the L-tryptophan oxidase mutant of claim 1, or carrying the gene of claim 2, or transformed with the recombinant plasmid of claim 3 or 4.
6. The recombinant cell according to claim 5, characterized in that, The recombinant cells used Escherichia coli as the host.
7. A method for improving the thermal stability of L-tryptophan oxidase, characterized in that, The method involves performing at least one of the following mutations on the L-tryptophan oxidase parent with the amino acid sequence shown in SEQ ID NO.2: (1) The phenylalanine at position 155 is mutated to lysine; (2) Serine at position 242 is mutated to tyrosine; (3) Threonine at position 378 is mutated to aspartic acid; (4) Phenylalanine at position 438 is mutated to lysine.
8. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria use Escherichia coli BL21(DE3) as the host and pET28a plasmid as the expression vector to express the mutant described in claim 1.
9. A method for preparing an L-tryptophan oxidase mutant, characterized in that, The method is used to culture the genetically engineered bacteria of claim 8 and induce the expression of the mutant of claim 1.
10. The use of the mutant of claim 1, the gene of claim 2, the recombinant plasmid of claim 3 or 4, the recombinant cell of claim 5 or 6, the method of claim 7, or the genetically engineered bacteria of claim 8 in the production of L-tryptophan oxidase products.