L-tyrosine oxidase double mutant, coding gene, self-assembled immobilized composite material and application thereof

Abstract

The present application relates to a kind of L-tyrosine oxidase double mutant, coding gene, self-assembled immobilized composite material and its application, belong to enzyme engineering and biological catalysis field.The L-tyrosine oxidase double mutant is derived from wild-type L-tyrosine oxidase Ralstonia of Raoultella Rs TYR is based on, by site-directed mutagenesis, the 301st leucine is mutated into phenylalanine, the 333rd phenylalanine is mutated into leucine, and double mutant Rs TYR is obtained.Compared with wild-type L-tyrosine oxidase Rs TYR, the enzyme activity of L-tyrosine oxidase Rs TYR double mutant is increased by 3.59 times.The present application constructs immobilized L-tyrosine oxidase composite material by the self-assembly of metal ion and enzyme molecule, and can efficiently catalyze L-tyrosine to convert into L-DOPA.

Description

Technical Field

[0001] This invention relates to the fields of enzyme engineering and biocatalysis, specifically to an L-tyrosine oxidase double mutant, its encoding gene, a self-assembled immobilized composite material, and its application in the biosynthesis of L-DOPA. Background Technology

[0002] L-3,4-Dihydroxyphenylalanine (L-DOPA) is an important precursor to the neurotransmitter dopamine. Due to its ability to cross the blood-brain barrier, it is widely used in the clinical treatment of Parkinson's disease (PD). Simultaneously, studies have shown that L-DOPA also has certain therapeutic effects on amblyopia, non-drug-induced Parkinson's syndrome, anti-aging, and acute hepatic encephalopathy, and can be used as an adjunct therapy for schizophrenia and drug dependence. Therefore, L-DOPA has high application value and broad development prospects in the pharmaceutical field, and its preparation methods have always been a research hotspot. As early as 1911, Casimir Funk first synthesized L-DOPA and D-DOPA using 3,4-carbonyldioxybenzaldehyde via chemical methods; in 1913, Marcus Guggenheim first isolated L-DOPA from pea pod membranes. With continuous advancements in related technologies, the preparation processes of L-DOPA have gradually become more diverse and applied to industrial production. Currently, its main production routes include plant extraction, chemical synthesis, and biosynthesis.

[0003] In recent years, with the rapid development of biotechnology, compared with plant extraction and chemical synthesis methods, biological methods have received widespread attention due to their advantages such as mild reaction conditions, low environmental pollution, and green safety, and are considered a more promising method for L-DOPA preparation. Since the 1960s, foreign scholars have begun to explore the biosynthesis of L-DOPA. Larway et al. first discovered tyrosinophilic microspirilla (… Microspira tyrasinatica Tyrosine oxidase in *Cephalosporium* can catalyze the production of L-DOPA; subsequently, Krishnaveni et al. found that in *Cephalosporium* (… Acremonium rutilum Similar enzyme activity was also found in Bacillus cereus; subsequently, John et al. also found it in Bacillus cereus ( Bacillus cereusTyrosine oxidases with the same catalytic function were identified in [the study]. Japanese scholars Yamada et al. also conducted systematic research on the biosynthesis of L-DOPA. Research on the biological preparation of L-DOPA in my country started relatively late, gradually beginning around the 1980s. With the development of new synthetic routes and the continuous optimization of traditional processes, the biological preparation of L-DOPA currently mainly includes five pathways: aminoacylase (EC 3.5.1.14), tyrosinase (EC 1.14.18.1), tyrosine phenol lyase (TPL, EC 4.1.99.2), transaminase (EC 2.6.1.5), and 4-hydroxyphenylacetic acid 3-hydroxylase (PHAH, EC 1.14.14.9).

[0004] Tyrosine oxidase is a type of redox enzyme containing a binuclear copper active site. It catalyzes the ortho-hydroxylation of L-tyrosine, introducing a hydroxyl group at the 3-position of the benzene ring, thereby generating L-DOPA. This enzyme is widely distributed in various organisms and is currently used in [various fields]. Microspira tyrasinatica , Acremonium rutilum , Bacillus cereus Tyrosine oxidases capable of catalyzing L-DOPA synthesis have been found in microorganisms such as [list of microorganisms]; in addition, Surwase et al. also reported on *Shortwave Monotrophus* (*list of microorganisms*). Brevundimonas sp. The enzymes originating from this source are related to tyrosine oxidase. To further improve enzyme catalytic efficiency, researchers often use immobilization techniques to modify tyrosine oxidase. For example, Valdes et al. used sodium alginate as an immobilization carrier; other studies have used calcium aluminosilicate and polyacrylamide materials for enzyme immobilization; Rahman et al. used glassy carbon electrodes to immobilize tyrosine oxidase. These methods have all achieved certain results in improving catalytic performance.

[0005] Nevertheless, tyrosine oxidases possess both monophenolase and bisphenolase activities, exhibiting a broad substrate adaptability. After catalyzing the conversion of L-tyrosine to L-DOPA, they further oxidize L-DOPA to dopaquinone, leading to further consumption of the target product and significantly reducing L-DOPA accumulation. This issue has become the most critical limiting factor in the efficient one-step preparation of L-DOPA. Therefore, it is crucial to discover novel tyrosine oxidase genes or utilize protein engineering and molecular modification strategies to obtain enzyme mutants with high efficiency and low bisphenol oxidation activity.

[0006] However, traditional immobilization methods still suffer from problems such as low immobilization efficiency, severe enzyme activity loss, limited mass transfer, and unstable enzyme microenvironment. This is especially true for tyrosine oxidases with bisphenol oxidation activity, where conventional immobilization methods struggle to balance catalytic activity with L-DOPA accumulation efficiency. Therefore, developing a self-assembled immobilized tyrosine oxidase system that combines high catalytic activity, high stability, and good recyclability is of great significance for achieving the green and efficient preparation of L-DOPA. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides an L-tyrosine oxidase mutant, its encoding gene, and its applications. This invention also provides a self-assembled immobilized L-tyrosine oxidase composite material, which is formed by the self-assembly of an L-tyrosine oxidase double mutant and metal ions to create a nanoflower-like composite structure.

[0008] This invention is achieved through the following technical solution: This invention provides a double mutant of L-tyrosine oxidase, the amino acid sequence of which differs from the L-tyrosine oxidase sequence shown in SEQ ID NO.3 by only the following mutations: leucine at position 301 is replaced by phenylalanine, and phenylalanine at position 333 is replaced by leucine; the amino acid sequence of the L-tyrosine oxidase double mutant is shown in SEQ ID NO.4.

[0009] The single mutant has leucine at position 301 replaced with phenylalanine, and the amino acid sequence of the L-tyrosine oxidase mutant is shown in SEQ ID NO.1 (L301F). Alternatively, phenylalanine at position 333 can be replaced with leucine, and the amino acid sequence of the L-tyrosine oxidase mutant is shown in SEQ ID NO.2 (F333L).

[0010] The present invention provides the encoding gene of the L-tyrosine oxidase mutant, the nucleotide sequence of which is shown in SEQ ID NO. 8.

[0011] A double mutant recombinant expression vector is developed by inserting the coding gene of the above-mentioned L-tyrosine oxidase double mutant into a plasmid vector.

[0012] According to a preferred embodiment of the present invention, the plasmid vector is pET-28a(+).

[0013] A recombinant strain is obtained by transforming the above-mentioned recombinant vector into a host cell.

[0014] According to a preferred embodiment of the present invention, the host cell is Escherichia coli.

[0015] The present invention also provides a method for preparing the above-mentioned L-tyrosine oxidase mutant, comprising the following steps: (1) Using the plasmid of one mutant as the amplification template (L301F) and the primer of the other mutant as the amplification primer (F333L), a site-directed mutagenesis PCR amplification reaction was performed. The reaction product was then circularized and ligated to construct a mutant expression vector. (2) The mutant expression vector constructed in step (1) is introduced into the host cell to obtain a double mutant recombinant strain; positive single clones after verification are selected for induced expression culture to obtain the fermentation broth of the recombinant strain; (3) Collect the bacterial cells in the fermentation broth of the recombinant strain described in step (2), resuspend them in buffer solution, break the bacterial cells, centrifuge to remove cell debris, and purify the supernatant by nickel column affinity chromatography to obtain the L-tyrosine oxidase double mutant.

[0016] The application of the encoding genes of the above-mentioned L-tyrosine oxidase double mutant and / or L-tyrosine oxidase mutant in the preparation of L-DOPA.

[0017] According to a preferred embodiment of the present invention, the application uses L-tyrosine as a reaction substrate to prepare L-DOPA, and the specific method is as follows: A reaction system was constructed, comprising L-tyrosine at a final concentration of 1–10 mmol / L and L-tyrosine oxidase at a final concentration of 1–3 mg / mL. Rs The TYR double mutant and 80-120 mmol / L Tris-HCl buffer were reacted and the reaction system was incubated at 45-55℃ for 10-15 h. After inactivation to terminate the reaction, the mixture was centrifuged and filtered to obtain L-DOPA.

[0018] The present invention also provides a self-assembled immobilized L-tyrosine oxidase Rs The method for producing TYR double mutant MT4 composite materials includes the following steps: (1) Dissolve the L-tyrosine oxidase double mutant in PBS buffer to obtain an enzyme protein solution; (2) Add CuSO4 solution to the enzyme protein solution and incubate at 25°C to allow the enzyme molecules and copper ions to self-assemble into an organic-inorganic hybrid nanoflower structure. (3) The precipitate was collected by centrifugation, washed with deionized water, and freeze-dried to obtain immobilized L-tyrosine oxidase. Rs TYR double mutant MT4 composite material.

[0019] The beneficial effects of this invention are as follows: (1) This invention uses bacteria derived from the genus *Raulella* (… Ralstonia Wild-type L-tyrosine oxidase RsBased on TYR, site-directed mutagenesis was performed, replacing leucine at position 301 with phenylalanine and phenylalanine at position 333 with leucine, resulting in L-tyrosine oxidase. Rs TYR double mutant. Similar to wild-type L-tyrosine oxidase. Rs Compared to TYR, L-tyrosine oxidase Rs The enzyme activity of the TYR mutant was increased by 3.59 times; (2) The L-tyrosine oxidase provided by the present invention Rs The TYR double mutant is easy to express and purify heterologously, and can efficiently and specifically catalyze the production of L-DOPA using only L-tyrosine as a substrate, with significantly improved catalytic efficiency. This effectively reduces enzyme usage and costs during the synthesis of L-DOPA. Furthermore, the operating conditions are simple and mild, making it a promising candidate for large-scale industrial production of L-DOPA. (3) The present invention simultaneously utilizes L-tyrosine oxidase Rs The TYR double mutant was self-assembled and immobilized. Using the self-assembly immobilization method, it was immobilized on a self-assembly support, and a self-assembled nanoflower structure was successfully prepared. Experimental catalysis verified that immobilization successfully improved the stability and recycling number of the enzyme. Attached Figure Description

[0020] Figure 1 L-tyrosine oxidase Rs Polyacrylamide gel electrophoresis image of TYR double mutant; Figure 2 L-tyrosine oxidase Rs Enzyme activity of TYR double mutant; Figure 3 L-tyrosine oxidase Rs Graph of products generated from L-tyrosine by TYR double mutant catalyzing L-DOPA; Figure 4 L-tyrosine oxidase Rs SEM results of TYR double mutant MT4 composite material; Figure 5 (a) is L-tyrosine oxidase Rs (a) Relative activity of TYR double mutant MT4 composite material in different buffer solutions at different pH values; (b) L-tyrosine oxidase Rs Thermal stability of TYR double mutant MT4 composite material at different temperatures; Figure 6 L-tyrosine oxidase Rs The TYR double mutant MT4 composite material was used to catalyze a certain amount of immobilized product, and the relative activity of the immobilized product was obtained after 12 cycles. Detailed Implementation

[0021] The following embodiments and accompanying drawings are only used to further illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Any equivalent substitutions, improvements or adjustments made to the present invention without departing from the core ideas and basic principles of the present invention shall be deemed to fall within the scope of protection of the present invention.

[0022] Unless otherwise specified, the experimental operations and methods involved in this embodiment were all carried out using conventional experimental techniques in the field; the raw materials, reagents and related materials used could be purchased through legitimate commercial channels unless otherwise specified.

[0023] In this embodiment, high-performance liquid chromatography (HPLC) was used to determine enzyme activity. Enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the production of 1 μmol L-DOPA per minute under standard reaction conditions.

[0024] This example uses a self-assembly immobilization method to study L-tyrosine oxidase. Rs Immobilized TYR double mutants were successfully prepared into immobilized nanoflowers, and the enzyme activity was determined by high performance liquid chromatography (HPLC) to assess the enzyme stability and number of cycles after immobilization.

[0025] Example 1 L-Tyrosine Oxidase Rs Design of TYR double mutants The inventors screened for strains from the genus *Raulella* through gene mining. Ralstonia L-tyrosine oxidase Rs TYR, this enzyme has low sequence similarity to reported prokaryotic tyrosine oxidases and can specifically catalyze the production of L-DOPA from L-tyrosine. Its amino acid sequence is shown in SEQ ID NO.3 and its nucleotide sequence is shown in SEQ ID NO.5.

[0026] The inventors of this application employed a rational design strategy, combining it with L-tyrosine oxidase... RsTYR Analysis of crystal structure and structure-activity relationship revealed that wild-type L-tyrosine oxidase RsTYR The amino acid sequences at positions 301 and 333 are likely key sites affecting catalytic efficiency. Subsequently, site-directed mutagenesis was used to target L-tyrosine oxidase. Rs The TYR protein undergoes a single-point gene mutation.

[0027] Therefore, two L-tyrosine oxidases were designed simultaneously. RsThe amino acid sequences of the TYR mutants L301F and F333L are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, and their nucleotide sequences are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively.

[0028] Example 2 L-Tyrosine Oxidase Rs Preparation of TYR double mutants An L-tyrosine oxidase Rs The method for preparing TYR double mutants includes the following steps: (1) The amino acid sequences of the single mutants L301F and F333L obtained from the previous experiments are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, and their nucleotide sequences are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively; (2) Using the single mutant L301F obtained in step (1) as a template, the site-directed mutagenesis primer F333L-F / R was designed for site-directed mutagenesis PCR amplification. After the reaction, 2 μL of the PCR product was taken for agarose gel electrophoresis band verification. 0.4 μL of Dpn I was added to the verified PCR product and the enzyme digestion reaction was carried out at 37℃ for 1 h. After the enzyme digestion reaction, the gene fragment of the digested product was circularized and ligated into the pET-28a(+) vector. The circularized ligation product was reacted at 37℃ for 1 h. After the reaction was completed, the reaction product was transformed into pET-28a(+) vector. E.coli JM109 competent cells were used, and positive clones were selected for plasmid extraction and DNA sequencing. Individuals with correct sequencing were identified as successfully constructed double mutant expression vectors pET-28a(+)-L301F / F333L. The mutant PCR amplification reaction system is shown in Table 1 below; Table 1 The PCR reaction program was as follows: 94℃, 2 min (pre-denaturation); 98℃, 10 s (denaturation); 68℃, 6 min (extension); 6 cycles; 4℃ (storage). The cyclization linkage reaction system is shown in Table 2 below; Table 2 (3) Transform the mutant expression vector pQE-80L-L301F / F333L constructed in step (2) into Escherichia coli BL21(DE3) cells to obtain recombinant strains; pick positive transformants and culture them overnight in LB liquid medium at 37°C and 200 rpm to obtain seed culture; inoculate the seed culture into LB liquid medium at a volume ratio of 2% and culture at 37°C until OD. 600The value was 0.6~0.8, then the temperature was lowered to 16℃, and IPTG with a final concentration of 1.0 mM was added to induce fermentation for 16~18 h to obtain the fermentation broth of the recombinant strain; (4) The fermentation broth was centrifuged at 4℃ and 4000 rpm for 15 min to collect the cells. After resuspending the cells in solution A (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 20 mM imidazole, 2 mM M DTT), lysozyme (final concentration 200 µg / mL) and IPTG (final concentration 1 mM) were added and the cells were placed on ice for 30 min. Then, the cells were sonicated on ice (2 s, 5 s interval, 400 W power) and finally centrifuged at low temperature and high speed (4℃, 13000 r / min) to remove cell debris and obtain the supernatant.

[0029] The supernatant was used for Ni affinity chromatography: 1 mL of Ni-NTA resin (QIAGEN) was added to each open column; the resin was equilibrated with 20 mL of solution A, and then the supernatant and 1 mL of resin were incubated at 4°C for 50 min; the mixture was passed through the open column, and the protein-bound resin was retained; the resin was then washed with 20 mL of solution A; finally, the protein was eluted with 15 mL of solution B (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 400 mM imidazole, 2 mM DTT). The eluted protein was then transferred to HEPES buffer (pH 8.0) using an ultrafiltration tube to remove imidazole and other metal ions from the eluent, yielding L-tyrosine oxidase. Rs The TYR double mutant was stored at 4°C for later use.

[0030] The purified L-tyrosine oxidase was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE). Rs The expression of TYR double mutants was identified, and the results are as follows: Figure 1 As shown in the figure: lanes 1-4 are the dissolution solution of the purified L-tyrosine oxidase RsTYR double mutant, lane 10 is the ultrasonically disrupted solution obtained in step (4) of Example 2, lane 9 is the supernatant obtained in step (4) of Example 2, lane M is Maker, lane 8 is the precipitate obtained in step (4) of Example 2, lane 7 is the liquid obtained after Ni affinity chromatography in step (4) of Example 2, lane 6 is the liquid obtained after washing with solution A in step (4) of Example 2, and lane 5 is the liquid obtained after washing with solution B in step (4) of Example 2. Depend on Figure 1 It can be seen that the present invention successfully expressed and purified L-tyrosine oxidase using this method. Rs The TYR double mutant has a single band and a molecular weight consistent with the theoretical value of 54 kDa.

[0031] Example 3 L-Tyrosine Oxidase Rs TYR double mutant activity assay Determination of L-tyrosine oxidase Rs TYR double mutant and wild-type L-tyrosine oxidase Rs Kinetic parameters of TYR on the substrate L-tyrosine. Among them... K m This indicates the affinity between the protease and its substrate; a smaller value indicates a higher affinity. K cat / K m The ratio indicates the catalytic efficiency of an enzyme; the higher the value, the higher the catalytic activity.

[0032] The specific assay method is as follows: L-tyrosine is diluted with buffer to prepare a gradient concentration of L-tyrosine substrate ranging from 200 to 1000 μmol / L. L-tyrosine oxidase is then added to the prepared gradient concentration L-tyrosine substrate to a final concentration of 1 mg / mL. Rs The TYR double mutant was thoroughly mixed and catalyzed at 50°C for 15 min. The reaction was then immediately terminated by heating in a boiling water bath. The catalytic reaction solution was centrifuged, filtered, and subjected to chromatographic analysis. Enzyme kinetic parameters were calculated using nonlinear regression analysis of the experimental data based on the Michaelis-Menten equation. K m , k cat V max All the above experiments were conducted in triplicate, with the experimental group consisting of L-tyrosine oxidase. Rs TYR double mutant (MT4). Simultaneously using wild-type L-tyrosine oxidase Rs TYR (WT) and the single mutant MT1 (L301F) were used as control groups, and the results were as follows: Figure 2 As shown.

[0033] Depend on Figure 2 It can be seen that wild-type L-tyrosine oxidase Rs TYR for substrate L-tyrosine K m The value was 174.5 μmol / L, L-tyrosine oxidase Rs TYR double mutants exhibit resistance to the substrate L-tyrosine. K m The value was 124.7 μmol / L. This indicates that compared to wild-type L-tyrosine oxidase... Rs TYR, L-tyrosine oxidase RsThe TYR double mutant exhibits a higher affinity for the substrate L-tyrosine. This is likely because the mutated amino acid makes it easier for the substrate to enter the active site, thus increasing the affinity between the two. Furthermore, wild-type L-tyrosine oxidase... Rs TYR and L-tyrosine oxidase Rs TYR double mutant k cat / K m The values ​​are 3.5 × 10 -1 μM -1 s -1 12.57×10 -1 μM -1 s -1 This further illustrates the difference between wild-type L-tyrosine oxidase and... Rs TYR, L-tyrosine oxidase Rs The TYR double mutant showed a 3.59-fold increase in enzyme activity, L-tyrosine oxidase. Rs The TYR double mutant exhibits higher catalytic activity toward the substrate L-tyrosine.

[0034] Example 4: Utilization of L-tyrosine oxidase Rs L-DOPA preparation using TYR double mutants A 5 mL reaction system was constructed using L-tyrosine as the reaction substrate. The reaction system comprised L-tyrosine at a final concentration of 7.5 mmol / L and L-tyrosine oxidase at a concentration of 2 mg / mL. Rs TYR and 100 mmol / L Tris-HCl buffer were used. The reaction system was then incubated at 50 °C for 12 h, with samples taken every two hours. After terminating the reaction by boiling in a water bath for 5 min, high-performance liquid chromatography (HPLC) analysis was performed. The reaction solution after catalytic reaction was centrifuged, filtered through a 0.22 μm filter membrane, and the supernatant was retained for HPLC analysis. The results are shown below. Figure 3 As shown in the figure: the purple elution line represents the experimental blank group, containing only buffer and substrate, with a peak position of 9.876 min, which is basically consistent with the pink elution line (tyrosine substrate standard) peak position of 9.877 min. The blue line represents the first peak of the experimental group at 8.049 min, which is the product L-DOPA. High-performance liquid chromatography (HPLC) analysis was performed using a Shimadzu SIL-20A HPLC system, and the chromatographic conditions are shown in Table 3 below. Table 3 Example 5 Self-assembled immobilized L-tyrosine oxidase Rs Preparation of TYR double mutant MT4 composite material Purified L-tyrosine oxidase RsThe TYR double mutant was dissolved in 10 mM PBS buffer (pH 7.4) to achieve a final protein concentration of 1.0 mg / mL.

[0035] Subsequently, CuSO4 solution was slowly added to the protein solution to bring the final concentration of CuSO4 to 150 mM. After slight mixing, the mixture was placed in a constant temperature incubator at 25°C and allowed to react for 48 h, allowing the enzyme protein to undergo a self-assembly reaction with copper ions to form a blue organic-inorganic hybrid nanoflower precipitate.

[0036] After the reaction was completed, the mixture was centrifuged at 12000 g for 10 min, the supernatant was discarded, and the precipitate was washed three times with deionized water to remove unbound proteins and free ions.

[0037] The resulting precipitate was then freeze-dried in a vacuum freeze dryer for 12 h to obtain immobilized L-tyrosine oxidase. Rs TYR double mutant MT4 composite material, Figure 4 The SEM images clearly show that the MT4 composite material forms a nanoflower morphology. Figure 5 It can be seen that L-tyrosine oxidase Rs The relative activity of the TYR double mutant MT4 composite material at different pH values ​​in different buffer solutions and its thermal stability at different temperatures.

[0038] The resulting composite material was stored at 4°C for later use.

[0039] Example 6 Self-assembled immobilized L-tyrosine oxidase Rs Activity determination of TYR double mutant MT4 composite material A 5 mL reaction system was constructed using L-tyrosine as the reaction substrate. The reaction system comprised L-tyrosine at a final concentration of 7.5 mmol / L and L-tyrosine oxidase at a concentration of 2 mg / mL. Rs TYR (self-assembled immobilized L-tyrosine oxidase) Rs The TYR double mutant MT4 composite material and 100 mmol / L Tris-HCl buffer were reacted at 50 °C for 12 h.

[0040] After the reaction was completed, the immobilized enzyme material was recovered by centrifugation, washed with PBS buffer, and then added to a new reaction system for the next round of catalysis.

[0041] The reaction was repeated 12 times, and the relative enzyme activity was measured after each cycle.

[0042] Figure 6 The results showed that immobilized L-tyrosine oxidase RsThe TYR double mutant MT4 composite material retained more than 70% of the relative enzyme activity after 12 cycles, indicating that the immobilized enzyme has good reusability and stability.

Claims

1. An L-tyrosine oxidase Rs TYR double mutant, characterized by, The amino acid sequence is shown in SEQ ID NO.

4.

2. The L-tyrosinase oxidase as described in claim 1 Rs TYR mutant, characterized by, Its amino acid sequence differs from that of the L-tyrosine oxidase shown in SEQ ID NO.3 only in the following mutations: leucine at position 301 is replaced by phenylalanine, and phenylalanine at position 333 is replaced by leucine.

3. An L-tyrosinase oxidase according to claim 1 Rs The coding gene of the TYR mutant is characterized by, The nucleotide sequence of the L-tyrosine oxidase double mutant is shown in SEQ ID NO.

8.

4. A double-mutant recombinant expression vector, characterized in that, Insert the L-tyrosine oxidase of claim 3 into the plasmid vector Rs The encoding gene of the TYR double mutant.

5. A recombinant bacterial strain, characterized in that, The double mutant recombinant expression vector of claim 4 is obtained by transforming it into host cells.

6. A self-assembled immobilized L-tyrosine oxidase Rs TYR double mutant MT4 composite material, characterized in that, include: The L-tyrosine oxidase double mutant and metal ions as described in claim 1; The metal ion is Cu. 2+ ; The composite material has an organic-inorganic hybrid nanoflower structure.

7. A self-assembled immobilized L-tyrosine oxidase as described in claim 6 Rs A method for preparing TYR double mutant MT4 composite material, characterized in that, include: (1) Add the L-tyrosine oxidase double mutant to a buffer solution containing metal ions; (2) Self-assembly reaction at 25℃; (3) Centrifugation to collect immobilized composite materials.

8. An L-tyrosinase oxidase according to claim 1 Rs The encoding gene of the TYR double mutant, the L-tyrosine oxidase RsTYR double mutant of claim 3, or the self-assembled immobilized L-tyrosine oxidase of claim 6. Rs Application of TYR double mutant MT4 composite material in the preparation of L-3,4-dihydroxyphenylalanine.

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