A tyrosinase mutant derived from Priesteria and its application

Abstract

The present invention discloses a tyrosinase mutant derived from the genus Priesteria and its application, belonging to the field of genetic engineering technology. The mutant is obtained by mutating the amino acid at position 46 of SEQ ID NO:3 from glycine to glutamate, the amino acid at position 65 from phenylalanine to tyrosine, and the amino acid at position 218 from valine to tyrosine, while keeping the other amino acid sequences unchanged. The tyrosinase provided by the present invention has a significantly higher catalytic activity for free tyrosine than wild-type recombinant tyrosinase and can be applied to the industrialization of levodopa production and modification.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a tyrosinase mutant derived from Priesteria and application thereof. Background Art

[0002] Tyrosinase (EC 1.14.18.1) is a monophenol monooxygenase. Tyrosinase plays an important and economically valuable role in tyrosine modification, such as forming the Parkinson's drug L-dopa. It also modifies tyrosine on proteins to form dopa groups. It is widely used in organic synthesis, biosensors, synthetic biology, and the food industry.

[0003] Computer-assisted semi-rational design uses bioinformatics and computer programs to compare known protein sequences, perform three-dimensional modeling, and simulate Gibbs free energy changes for point mutations. Protein mutation points are then selected based on the calculated results. The recombinant protein is then validated through genetic engineering techniques to obtain mutants with high activity or stability, as desired. This technique has been widely used in synthetic biology. Summary of the Invention

[0004] The present invention aims to provide a protein that converts the substrate L-tyrosine into an L-DOPA enzyme. The present invention is not limited to the technical subject matter described herein, and those skilled in the art will readily appreciate other technical subject matter not described herein through the following description.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a mutant protein obtained by mutating amino acid position 46 of SEQ ID NO: 3 from glycine to glutamic acid, amino acid position 65 from phenylalanine to tyrosine, and amino acid position 218 from valine to tyrosine, while keeping the other amino acid sequences unchanged. The protein is derived from the genus Priesteria.

[0007] In the above protein, the nucleotide sequence of the protein is SEQ ID NO: 2.

[0008] In the above protein, the amino acid sequence of the protein is SEQ ID NO: 1.

[0009] The present invention also provides use of the aforementioned protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in the preparation of levodopa.

[0010] In the above applications, the regulation is to upregulate, enhance or increase the expression of the gene encoding the aforementioned protein or the content or activity of the protein.

[0011] In the above application, the substance is any one of the following:

[0012] B1), a nucleic acid molecule encoding the aforementioned protein;

[0013] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0014] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0015] B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

[0016] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium contains any one of the aforementioned proteins.

[0017] In the above, the recombinant bacteria contains a gene encoding any one of the aforementioned proteins.

[0018] In the above, the recombinant bacteria include bacteria.

[0019] In the above, the recombinant bacteria can be prepared by the following method: introducing a substance that upregulates, enhances or improves the expression of the mutant protein encoding gene or upregulates, enhances or improves the activity and / or content of the mutant protein into the starting strain to obtain the recombinant bacteria.

[0020] In the above, the substance that upregulates, enhances or improves the expression of the gene encoding the aforementioned mutant protein or upregulates, enhances or improves the activity and / or content of the protein may be an overexpression vector of the aforementioned mutant protein.

[0021] In the above, the overexpression vector can specifically be the recombinant expression vector pET-32a (+) -Bmtyr (E46\Y65\Y218), which is a recombinant expression vector pET-32a (+) -Bmtyr (E46\Y65\Y218) obtained by replacing the nucleotide fragment between the BamH I and Hind III enzyme recognition sites of the vector pET-32a (+) with a DNA molecule having a nucleotide sequence of SEQ ID NO: 2, while keeping the remaining nucleotide sequence of pET-32a (+) unchanged.

[0022] In the above-mentioned biological materials, the expression cassette described in B2) refers to a single-stranded or double-stranded nucleic acid molecule capable of expressing the aforementioned proteins in host cells. The expression cassette may also include all regulatory sequences necessary for expressing the nucleic acid molecule for any of the aforementioned proteins or the DNA for the aforementioned RNA molecule. These regulatory sequences are capable of directing the expression of the coding sequence for any of the aforementioned proteins or the DNA for the aforementioned RNA molecule in a suitable host cell under compatible conditions. These regulatory sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the regulatory sequence must include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for ligation of the regulatory sequence to the coding region of the protein-encoding nucleic acid sequence or the DNA for the aforementioned RNA molecule, a linker may be provided. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence recognized by the host cell in which the nucleic acid sequence is to be expressed. A promoter sequence contains transcriptional regulatory sequences that mediate expression of the protein or the DNA for the aforementioned RNA molecule. The promoter can be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutant, truncated, and hybrid promoters, and can be derived from genes encoding extracellular or intracellular proteins that are homologous or heterologous to the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that is recognized by the host cell to terminate transcription. The termination sequence can be operably linked to the 3' end of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule. Any terminator that is functional in the selected host cell can be used in the present invention. The regulatory sequence can also be a suitable leader sequence, i.e., an untranslated region of an mRNA that is important for translation in the host cell. The leader sequence can be operably linked to the 5' end of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule. Any leader sequence that is functional in the selected host cell can be used in the present invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence attached to the amino terminus of the protein that directs the DNA encoding the protein or the RNA molecule into the cellular secretory pathway. Any signal peptide coding region that directs the expressed protein or the DNA of the RNA molecule into the secretory pathway of the selected host cell can be used in the present invention. It may also be desirable to add regulatory sequences that regulate the expression of the protein or RNA molecule according to the growth conditions of the host cell. Examples of regulatory sequences are systems that can turn gene expression on or off in response to chemical or physical stimuli, including in the presence of regulatory compounds. Other examples of regulatory sequences are those that allow gene amplification.

[0023] The present invention also provides use of a biomaterial related to any of the aforementioned proteins in the preparation of levodopa, wherein the biomaterial is any of the following:

[0024] B1), a nucleic acid molecule encoding the aforementioned protein;

[0025] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0026] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0027] B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

[0028] The microorganisms include bacteria.

[0029] The bacteria may specifically be BL21 (DE3) Escherichia coli.

[0030] The present invention also provides a method for preparing levodopa, comprising using the aforementioned recombinant bacteria for fermentation or the aforementioned biological materials to prepare levodopa.

[0031] The above method includes the following reaction system: adding 10 ml of a reaction mixture (containing 0.4 g / L L-Tyr, 0.01 M PBS buffer solution, 0.06 mM CuSO4, and a solvent of water) to a 15 ml centrifuge tube, adding 150 mg / L polyphenol oxidase (mushroom), 150 mg / L Trx-Bmtyr lysate, and 1 mL of 150 mg / L Trx-Bmtyr (E46\Y65\Y218) lysate, respectively, adjusting the pH of the mixture to 7.0 with a 2 M NaOH solution, and reacting at 30° C. and 100 rpm.

[0032] The present invention proves through experiments that the tyrosinase provided by the present invention has significantly higher catalytic ability for free tyrosine than wild-type recombinant tyrosinase, and can be applied to the industrialization of levodopa production and modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 SDS-PAGE gel electrophoresis of lysed cell suspensions of Trx-Bmtyr and Trx-Bmtyr(E46\Y65\Y218) proteins. M: TSINGKE TSP021 Trelief® Prestained Protein Ladder 180 kda-17 kda. Lane 1: lysed cell suspension of Trx-Bmtyr protein; Lane 2: lysed cell suspension of Trx-Bmtyr(E46\Y65\Y218) protein.

[0034] Figure 2 Spectrophotometer standard curve established for the Arnow method.

[0035] Figure 3 These are the enzyme activity assay results of Trx-Bmtyr and Trx-Bmtyr(E46\Y65\Y218) proteins.

[0036] Figure 4 These are the results of enzyme activity determination of Trx-Bmtyr (E46\Y65\Y218) protein under different pH conditions.

[0037] Figure 5 These are the results of enzyme activity determination of Trx-Bmtyr (E46\Y65\Y218) protein under different temperature conditions.

[0038] Figure 6 These are the sequencing results of the coding genes of Bmtyr and Bmtyr(E46\Y65\Y218). DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0040] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0041] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0042] The data in the following examples were processed using GraphPad Prism 8 statistical software. The experimental results were expressed as mean ± standard deviation and tested using One-way ANOVA. P < 0.05 (*) indicated a significant difference.

[0043] L-Tyrosine: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number T103976-25g, referred to as L-Tyr in the examples.

[0044] Levodopa: Levodopa (abbreviated as L-DOPA) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. under the product number D111048-100g.

[0045] Polyphenol oxidase (mushroom): Polyphenol oxidase (mushroom) purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number S10171-100KU was used.

[0046] Ampicillin sodium: purchased from Shanghai MacLean Biochemical Technology Co., Ltd., product number: A800429-25g.

[0047] IPTG: purchased from Shanghai MacLean Biochemical Technology Co., Ltd., product number: I811719-5g.

[0048] BCA protein concentration determination kit was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., product number: B917925.

[0049] Example 1. Construction of strains expressing tyrosinase Bmtyr and its mutants

[0050] pET-32a (+) plasmid: purchased from the Qingkekebangbang Mall of Beijing Qingke Biotechnology Co., Ltd., with the product number P3100-10ug.

[0051] BL21 (DE3) Escherichia coli strain: purchased from the Qingkekebangbang Mall of Beijing Qingke Biotechnology Co., Ltd., with the product number TSC-E01.

[0052] The coding sequence (SEQ ID NO: 2) for the tyrosinase mutant Bmtyr(E46\Y65\Y218) was synthesized by Beijing Qingke Biotechnology Co., Ltd. The DNA molecule represented by SEQ ID NO: 2 was inserted into pET-32a(+) to generate the recombinant expression vector pET-32a(+)-Bmtyr(E46\Y65\Y218). The recombinant expression vector pET-32a(+)-Bmtyr(E46\Y65\Y218) was constructed by replacing the nucleotide fragment between the BamH I and Hind III restriction sites of the pET-32a(+) vector with the DNA molecule represented by SEQ ID NO: 2, while maintaining the remaining nucleotide sequence of the pET-32a(+) vector unchanged. The recombinant vector expresses a fusion protein, which is a Trx protein-tyrosinase mutant Bmtyr (E46\Y65\Y218). The tyrosinase mutant Bmtyr (E46\Y65\Y218) is a mutant protein obtained by mutating the 46th amino acid of SEQ ID NO: 3 from glycine to glutamic acid, the 65th amino acid from phenylalanine to tyrosine, and the 218th amino acid from valine to tyrosine, while keeping the other amino acids unchanged. The amino acid sequence of the mutant protein is SEQ ID NO: 1.

[0053] The optimized coding sequence (SEQ ID NO:4) of the Priestia-derived tyrosinase Bmtyr (NCBI accession number WP_053487522.1, also known as SEQ ID NO:3) was synthesized by Beijing Qingke Biotechnology Co., Ltd. The DNA molecule represented by SEQ ID NO:4 was inserted into pET-32a(+) to generate the recombinant expression vector pET-32a(+)-Bmtyr. The recombinant expression vector pET-32a(+)-Bmtyr was constructed by replacing the nucleotide fragment between the BamH I and Hind III restriction sites of the pET-32a(+) vector with the DNA molecule represented by SEQ ID NO:4, while retaining the remaining nucleotide sequence of the pET-32a(+) vector unchanged. This recombinant vector expresses a fusion protein consisting of the Trx protein and the tyrosinase Bmtyr. The amino acid sequence of the tyrosinase Bmtyr is represented by SEQ ID NO:3.

[0054] The recombinant expression vector pET-32a (+) -Bmtyr and the recombinant expression vector pET-32a (+) -Bmtyr (E46\Y65\Y218) were transformed into the host cell E. coli BL21 (DE3), and single clones were collected and sequenced. Figure 6 Recombinant single-clone strains BL21(DE3) / pET-32a(+)-Bmtyr and BL21(DE3) / pET-32a-Bmtyr(E46\Y65\Y218) were inoculated into 5 mL of LB medium containing 100 mg / L ampicillin sodium and incubated at 37°C with shaking at 220 rpm for 8 hours. Sterile glycerol was added to a final concentration of 20% glycerol when the OD610nm reached 0.8-1.0. The glycerol was stored in sterile tubes at -80°C. In the following examples, the recombinant strain BL21(DE3) / pET-32a-Bmtyr(E46\Y65\Y218) is also referred to as the E46\Y65\Y218 mutant strain.

[0055] LB liquid medium composition: tryptone 10 g / L (OXOID), yeast extract 5 g / L (OXOID), sodium chloride 10 g / L, and the rest is distilled water.

[0056] Example 2: Preparation of crude enzyme solution of genetically engineered bacteria and SDS-PAGE electrophoresis analysis of total protein

[0057] TB medium composition: tryptone 12 g / L (OXOID), yeast extract 24 g / L (OXOI D), glycerol 5 g / L, potassium hydrogen phosphate trihydrate 16.43 g / L, potassium dihydrogen phosphate 2.31 g / L, and the rest is distilled water.

[0058] Bmtyr group: The stored recombinant strain BL2 (DE3) 1 / pET-32a (+) -Bmtyr was inoculated into a shake tube containing 5 mL of LB medium containing 100 mg / L ampicillin sodium, and cultured on a shaker at 37°C for 24 h at 220 rpm to obtain a culture solution. The culture solution was then inoculated into a 100 mL shake flask containing 50 mL of TB medium at a 2% inoculum volume and cultured at 200 rpm at 37°C for 2 h. 600nm When the cell count reaches approximately 4-6, add 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) in a shaker at 220 rpm and 25°C in a clean hood. Cultivate for 24 hours with 0.1 mM IPTG. Collect the bacterial solution into a centrifuge tube and centrifuge at 5000 rpm for 5 minutes. Then centrifuge at 8000 rpm for 10 minutes in a Lu Xiangyi 50ml centrifuge to obtain a BL21(DE3) / pET-32a(+)-Bmtyr bacterial pellet. Dilute 2 g of the BL21(DE3) / pET-32a(+)-Bmtyr bacterial pellet with 0.01 M PBS and dilute to 100 mL to obtain a BL21(DE3) / pET-32a(+)-Bmtyr bacterial mixture. The mixture was homogenized for 2 minutes using a high-pressure homogenizer (ATS AH1500 homogenizer) at 800 bar pressure. The homogenate was then centrifuged at 8000 rpm for 10 minutes using a Lu Xiangyi 50 ml centrifuge. The protein supernatant was collected and stored at 4°C to obtain a supernatant containing tyrosinase Bmtyr.

[0059] Add 20 μL of 5* protein loading buffer (Sangon Biotechnology) to the protein sample. Mix thoroughly and heat at 100°C for 20 minutes. Prepare a 1.0 mm 10% SDS-PAGE gel using a Bioss protein gel electrophoresis kit. Use the TSINGKE TSP021 Trelief® Prestained Protein Ladder (Cat. No. TSP021) from Beijing Qingke Company as a protein marker. After electrophoresis, stain the gel with Coomassie stain (microwave oven, medium-high for 90 seconds). Discard the stain and destain with destaining buffer. Destain for 2 hours each time until the bands are clear. The destaining buffer consists of: 450 mL of methanol, 100 mL of glacial acetic acid, and 450 mL of water. The Coomassie-stained gel was imaged using a GenoSens 2250 gel imaging system from Shanghai Qinxiang Scientific Instrument Co., Ltd.

[0060] Bmtyr (E46\Y65\Y218) group: This group differs from the Bmtyr group in that the recombinant strain BL21 (DE3) / pET-32a-Bmtyr (E46\Y65\Y218) was used instead of the recombinant strain BL21 (DE3) / pET-32a (+) -Bmtyr. The rest of the operations were the same as those of the Bmtyr group.

[0061] The results are as follows Figure 1 shown. Figure 1 The figure shows the gel electrophoresis pattern for verification of the expression product of the recombinant strain. M is a marker. The sizes of the bands from bottom to top are 180, 130, 95, 70, 55, 43, 33, 20, 17, and 10 kDa. Lane 1 is the homogenate of the recombinant strain BL21(DE3) / pET-32a(+)-Bmtyr, and lane 2 is the homogenate of the recombinant strain BL21(DE3) / pET-32a-Bmtyr(E46\Y65\Y218). The target proteins of Trx-Bmtyr and Trx-Bmtyr(E46\Y65\Y218) are both 43 kDa.

[0062] The tyrosinase expressed by the recombinant strain BL21 (DE3) / pET-32a (+)-Bmtyr was named Trx-Bmtyr; the tyrosinase expressed by the recombinant strain BL21 (DE3) / pET-32a-Bmtyr (E46\Y65\Y218) was named Trx-Bmtyr (E46\Y65\Y218).

[0063] Example 3, enzyme activity determination

[0064] Preparation method of 150 mg / L polyphenol oxidase (mushroom): Weigh 15 mg of polyphenol oxidase (mushroom), dissolve it in 0.01M PBS solution and dilute to 100 mL.

[0065] Preparation of 150 mg / L Trx-Bmtyr lysis solution: Collect BL21 (DE3) / pET-32a (+) -Bmtyr bacterial pellet, determine the protein concentration of the lysis solution using a BCA protein concentration assay kit, and dilute to 150 mg / L with 0.01 M PBS solution.

[0066] Preparation of 150 mg / L Trx-Bmtyr(E46\Y65\Y218) cell lysis solution: Collect BL21(DE3) / pET-32a-Bmtyr(E46\Y65\Y218) bacterial pellet, determine the protein concentration of the lysis solution using a BCA protein concentration assay kit, and dilute to 150 mg / L with 0.01 M PBS solution.

[0067] DOPA detection method (Arnow method) principle: Substances containing 3,4-dihydroxyphenylalanine (DOPA) structure appear yellow with sodium nitrite under acidic conditions, and turn into deep orange when excess alkali is added.

[0068] 1. Prepare standard curve

[0069] Solution preparation:

[0070] Hydrochloric acid solution (0.012 mol / L): Take 0.2 mL of hydrochloric acid and dilute it to 200 mL with pure water.

[0071] DOPA standard solution (200 μg / mL): Weigh 20 mg of DOPA standard and dilute to 100 mL with hydrochloric acid solution. Prepare it before use.

[0072] Acidic reagent (0.516 mol / L): Take 4.3 mL of hydrochloric acid and dilute it to 100 mL with pure water.

[0073] Alkaline reagent (1 mol / L): Weigh 4.0 g of sodium hydroxide (NaOH) and dilute to 100 mL with pure water.

[0074] Nitrous acid reagent: Weigh 10 g of sodium molybdate dihydrate and 10 g of sodium nitrite, dissolve them in pure water and make up to 100 mL.

[0075] Preparation of DOPA standard solution series: Take 0 mL, 0.1 mL, 0.5 mL, 1.0 mL, 1.5 mL, and 2.0 mL of DOPA standard solution, dilute to 10 mL with hydrochloric acid solution, shake well, and set aside.

[0076] The experiment was repeated three times, and each repetition was as follows: 1 mL of the above series of standard solutions was taken, 0.5 mL of acidic reagent was added to each test tube, and then 1.5 mL of nitrous acid reagent and 2 mL of alkaline reagent were added to each test tube in sequence (the alkaline reagent should be added within 5 minutes after the addition of nitrous acid reagent). The mixture was shaken to obtain each series of standard solutions to be tested. Tube 0 was used as a blank, and the absorbance of each series of standard solutions to be tested was measured at a wavelength of 500 nm using a 1 cm cuvette. A standard curve was drawn with the OD500 nm value as the ordinate and the L-DOPA content (mg / L) as the abscissa (standard curve, regression expected response R 2 >0.997).

[0077] The results are as follows Figure 2 As shown, the standard curve y = 0.0156x-0.0145, where x is the L-DOPA content, R 2 =0.997.

[0078] 2. Construction of a reaction system that catalyzes the formation of L-DOPA from L-Tyr

[0079] Preparation method of 150 mg / L polyphenol oxidase (mushroom): Weigh 15 mg of polyphenol oxidase (mushroom), dissolve it in 0.01M PBS solution and dilute to 100 mL.

[0080] Preparation of 150mg / L Trx-Bmtyr lysis solution: Determine the protein concentration of the lysis solution using the BCA protein concentration assay kit and dilute it to 150mg / L with 0.01M PBS solution.

[0081] Preparation of 150mg / L Trx-Bmtyr (E46\Y65\Y218) lysis solution: Determine the protein concentration of the lysis solution using the BCA protein concentration assay kit and dilute it to 150mg / L with 0.01M PBS. Repeat the test three times, with the following results for each replicate:

[0082] Bmtyr(E46\Y65\Y218) group: To a 15-ml centrifuge tube, add 10 ml of the reaction mixture (containing 0.4 g / L L-Tyr, 0.01 M PBS buffer, 0.06 mM CuSO4, and water as the solvent), add 1 ml of 150 mg / L Trx-Bmtyr(E46\Y65\Y218) cell lysis solution, and adjust the pH of the mixture to 7.0 with 2 M NaOH solution. Reaction conditions: 30°C, 100 rpm, for 1 hour. After 0.5 hours, sample 1 mL of the reaction solution and determine the L-DOPA content using the Arnow method. The procedure is as follows: Place 1 mL of the reaction solution in a 10 mL test tube. Add 0.5 mL of an acidic reagent, 1.5 mL of a nitrous acid reagent, and 2 mL of an alkaline reagent (the alkaline reagent should be added within 5 minutes of the nitrous acid reagent). Shake well to obtain the test solution. Using tube 0 as a blank, measure the OD500nm value of the test solution using a 1 cm cuvette at a wavelength of 500 nm. Perform three replicates and average the results. Calculate the DOPA content of the sample based on the absorbance of the test solution and the standard curve. Note: If the sample interferes with the colorimetric analysis, centrifuge at 3000 rpm for 10 minutes and collect the supernatant for colorimetric analysis.

[0083] Bmtyr group: This group differs from the Bmtyr (E46\Y65\Y218) group in that 1 mL of 150 mg / L Trx-Bmtyr (E46\Y65\Y218) lysis buffer was used instead of 1 mL of 150 mg / L Trx-Bmtyr lysis buffer. The rest of the procedures were the same as those in the Bmtyr (E46\Y65\Y218) group.

[0084] Polyphenol oxidase group: This group differs from the Bmtyr (E46\Y65\Y218) group in that 1 mL of 150 mg / L Trx-Bmtyr cell lysis solution was replaced with 1 mL of 150 mg / L polyphenol oxidase solution. The rest of the procedures were the same as those of the Bmtyr (E46\Y65\Y218) group.

[0085] The results are as follows Figure 3 As shown in Figure 2, the L-DOPA content in the reaction solution of the Bmtyr (E46\Y65\Y218) group was significantly higher than that of the Bmtyr group and the polyphenol oxidase (mushroom) group. The results of the one-way ANOVA test of the experiment (P < 0.0001) were compared by Dunnett-t test with Bmtyr (E46\Y65\Y218) as the control. The results are shown in Figure 2. Figure 3As shown, the results for Bmtyr(E46\Y65\Y218) compared to the Bmtyr and polyphenol oxidase (mushroom) groups were highly significant (P < 0.0001). This indicates that compared to the Trx-Bmtyr fusion protein, the mutant Trx-Bmtyr(E46\Y65\Y218) has the ability to convert the substrate L-tyrosine to L-DOPA. Furthermore, the enzyme activity is higher than that of polyphenol oxidase (mushroom) in a 0.01 M PBS buffer system at pH 7.0, a reaction temperature of 30°C, 0.06 mM CuSO₄, and 100 rpm.

[0086] Table 1. Enzyme activity assay results

[0087]

[0088] 3. Effect of different reaction pH on L-DOPA content

[0089] For the preparation of 150 mg / L Trx-Bmtyr (E46\Y65\Y218) cell lysis solution, see "2. Construction of a reaction system for catalyzing the formation of L-DOPA from L-Tyr."

[0090] To a 15-ml centrifuge tube, add 10 ml of the reaction mixture (containing 0.4 g / L L-Tyr, 0.01 M PBS buffer solution, 0.06 mM CuSO4, and water as the solvent) and 1 ml of 150 mg / L Trx-Bmtyr (E46\Y65\Y218) cell lysate. Adjust the pH of the mixture to 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5 with 1 M phosphoric acid solution or 2 M NaOH solution. Incubate the reaction on a shaker at 30°C for 0.5 h. Determine the L-DOPA content using the Arnow method.

[0091] Table 2. Enzyme activity determination results at different reaction pH

[0092]

[0093] The results showed that the one-way ANOVA test results (P < 0.0001) showed that the catalytic ability of Trx-Bmtyr (E46\Y65\Y218) at different pH values ​​was significantly different. The Dunnett-t test was used as the control group to compare the results. Figure 4As shown, the Trx-Bmtyr(E46\Y65\Y218) group showed significant differences in catalytic activity at pH 7.0 compared to those at pH 5.0, pH 5.5, pH 6.0, and pH 7.5 (P < 0.01), and extremely significant differences at pH 8.0 and pH 8.5 (P < 0.0001). This indicates that at pH 7.0, the catalytic activity of Trx-Bmtyr(E46\Y65\Y218) significantly differed across these pH conditions, while the difference at pH 6.5 was not significant (P > 0.05). Furthermore, the concentrations of L-DOPA generated in the pH 6.5 and pH 7.0 control groups were higher than those in the other pH groups. Therefore, the optimal range of Trx-Bmtyr(E46\Y65\Y218) activity is between pH 6.5 and 7.0.

[0094] 4. Effect of different reaction temperatures on L-DOPA content

[0095] For the preparation of 150 mg / L Trx-Bmtyr (E46\Y65\Y218) cell lysis solution, see "2. Construction of a reaction system for catalyzing the formation of L-DOPA from L-Tyr."

[0096] To a 15-ml centrifuge tube, add 10 ml of the reaction mixture (containing 0.4 g / L L-Tyr, 0.01 M PBS buffer, 0.06 mM CuSO₄, and water) and 1 ml of 150 mg / L Trx-Bmtyr (E46, Y65, or Y218) cell lysate. Adjust the pH of the mixture to 7.0 with 1 M phosphoric acid or 2 M NaOH. Incubate on a shaker at 26, 28, 30, 32, 34, 37, 39, 41, or 43°C for 0.5 h. Determine the L-DOPA content using the Arnow method.

[0097] Table 3. Enzyme activity assay results at different reaction temperatures

[0098]

[0099] The results showed that the one-way ANOVA test results (P < 0.0001) showed that the catalytic ability of Trx-Bmtyr (E46\Y65\Y218) under different temperature conditions was significantly different. Using 37℃ as the control group, the Dunnett-t test was used to compare the results. Figure 5As shown, Trx-Bmtyr(E46\Y65\Y218) showed a significant difference at 37°C compared to 34°C (P < 0.001) and at all other experimental temperatures (P < 0.0001). Furthermore, the amount of L-DOPA produced by Trx-Bmtyr(E46\Y65\Y218) at 37°C was higher than that at other temperatures, indicating that the optimal temperature for the enzyme is 37°C. One unit (U) of enzyme activity is defined as the production of 1 micromole of substrate DOPA per minute. Based on the production of 3.47 mg / L of L-DOPA at 37°C, the enzyme activity per mg of Trx-Bmtyr(E46\Y65\Y218) reached 0.366 U.

[0100] SEQ ID NO: 1 (amino acid sequence of Bmtyr (E46\Y65\Y218))

[0101] MSNKYRVRKNVLRLTDTEKRDFVRTVLILKEKGIYDRYIAWHGAAEKFHTPPGSDRNAAHMSSAYLPWHREYLLRFERDLQSINPEVTLPYWEWETDAQLQDPSQSQIWSADFMGGNGNPKKDFIVDTGPFAAGRWTTIDEQGNPSGGL KRNFGATKEAPTLPTRDDVLDALKITQYDTPPWDMTSQNSFRNQLEGFINGPQLHNRVHRWVGGQMGVYPTAPNDPVFFLHHANVDRIWAVWQIVHRNQNYQPMKNGPFGQNFRDPMYPWNTTPEDVMNHRKLGYVYDIELRKSKRSS.

[0102] SEQ ID NO: 2 (nucleotide sequence of Bmtyr (E46\Y65\Y218))

[0103] 5'-ATGTCTAACAAATACCGTGTTCGTAAGAACGTTCTGCGTTTGACCGACACCGAGAAACGTGACTTCGTTCGTACCGTTCTGATTCTGAAAGAGAAAGGTATCTACGACCGTTACATCGCATGGCACGGTGCTGCGGAGAAATTCCACACTCCGCCGGGTTCTGATCGTAACGCGGCTCACATGAGTTCTGCGTACTTGCCGTGGCACCGTGAATACCTGCTGCGTTTCGAACGTGACCTTCAGTCTATCAATCCGGAAGTTACTCTGCCGTACTGGGAATGGGAAACCGATGCACAGCTGCAAGATCCGTCTCAGTCTCAAATCTGGTCTGCGGACTTCATGGGTGGTAACGGTAATCCGAAAAAAGACTTCATCGTTGACACTGGTCCGTTTGCTGCTGGTCGTTGGACCACCATCGACGAACAGGGTAATCCGTCTGGTGGTCTGAAACGTAACTTCGGTGCGACCAAAGAAGCGCCGACCTTGCCGACCCGTGATGACGTTCTGGACGCGCTGAAGATCACTCAGTACGACACTCCACCGTGGGACATGACCAGCCAGAACTCTTTCCGTAACCAGCTGGAAGGTTTCATCAACGGTCCGCAGCTGCATAACCGTGTTCACCGCTGGGTTGGTGGTCAAATGGGTGTGTATCCGACCGCACCAAACGATCCGGTGTTCTTTCTGCATCACGCTAACGTTGACCGTATCTGGGCTGTTTGGCAGATCGTGCACCGTAACCAGAACTACCAGCCGATGAAGAACGGTCCGTTCGGTCAGAACTTCCGTGATCCGATGTATCCGTGGAACACTACTCCGGAAGACGTTATGAACCACCGTAAACTGGGTTACGTTTACGACATCGAACTGCGTAAATCTAAACGTAGCTCTTAA-3'.<00002�1>

[0104] SEQ ID NO:3 (Amino acid sequence of Bmtyr)

[0105] It should be noted that there seems to be an unclear "�" in the original text at position . It might be a typo. If it's a specific symbol with a known meaning in the original context, it needs to be corrected for a more accurate translation. Here, it's translated as "3" tentatively.MSNKYRVRKNVLRLTDTEKRDFVRTVLILKEKGIYDRYIAWHGAAGKFHTPPGSDRNAAHMSSAFLPWHREYLLRFERDLQSINPEVTLPYWEWETDAQLQDPSQSQIWSADFMGGNGNPKKDFIVDTGPFAAGRWTTIDEQGNPSGGLKRNFGATKEAPTLPTRDDVLDALKITQYDTPPWDMTSQNSFRNQLEGFINGPQLHNRVHRWVGGQMGVVPTAPNDPVFFLHHANVDRIWAVWQIVHRNQNYQPMKNGPFGQNFRDPMYPWNTTPEDVMNHRKLGYVYDIELRKSKRSS。

[0106] SEQ ID NO:4 (Nucleotide sequence of Bmtyr)

[0107] 5'-ATGTCTAACAAATACCGTGTTCGTAAGAACGTGCTGCGACTGACTGACACCGAGAAACGTGACTTCGTTCGTACCGTTCTGATCTTGAAAGAGAAAGGTATCTACGACCGTTACATCGCATGGCACGGTGCTGCGGGTAAATTCCACACTCCGCCGGGTTCTGACCGTAACGCAGCGCACATGAGTTCTGCGTTCTTGCCGTGGCACCGTGAATACCTGCTGCGTTTCGAACGTGACCTGCAGTCTATCAACCCGGAAGTTACTCTGCCGTACTGGGAATGGGAAACCGATGCGCAGTTGCAAGATCCGTCTCAGTCTCAGATTTGGTCCGCAGACTTCATGGGTGGTAACGGTAATCCAAAGAAAGACTTCATCGTTGACACTGGTCCGTTCGCTGCGGGTCGTTGGACCACCATCGATGAACAGGGTAACCCGTCTGGTGGTTTGAAACGTAACTTCGGTGCTACCAAAGAAGCTCCGACCTTGCCGACTCGTGACGACGTACTGGACGCGCTGAAGATCACTCAGTACGACACTCCACCGTGGGATATGACCAGCCAGAACTCTTTCCGTAACCAGCTGGAAGGTTTCATCAACGGTCCACAGCTGCACAATCGCGTACACCGTTGGGTTGGTGGTCAGATGGGTGTGGTTCCGACTGCACCGAACGATCCGGTGTTCTTTCTGCACCACGCGAACGTTGACCGTATCTGGGCAGTTTGGCAGATCGTTCACCGTAACCAGAACTACCAGCCGATGAAGAACGGTCCATTCGGTCAGAACTTCCGTGATCCGATGTATCCGTGGAACACCACTCCGGAAGATGTGATGAACCACCGTAAACTGGGTTACGTTTACGACATCGAACTGCGTAAATCCAAACGTAGCTCTTAA-3'。

[0108] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A protein, characterized in that The protein is obtained by mutating the 46th amino acid of SEQ ID NO: 3 from glycine to glutamic acid, the 65th amino acid from phenylalanine to tyrosine, and the 218th amino acid from valine to tyrosine, while keeping other amino acid sequences unchanged.

2. Use of the protein according to claim 1 or a biomaterial related to the protein in the preparation of levodopa, wherein the biomaterial is any one of the following: B1), a nucleic acid molecule encoding the protein according to claim 1; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

3. The use according to claim 2, characterized in that B1) The coding sequence of the nucleic acid molecule is SEQ ID NO:

2.

4. A recombinant bacterium, characterized in that The recombinant bacteria contains the protein according to claim 1.

5. The recombinant bacterium according to claim 4, characterized in that The recombinant bacteria contains the gene encoding the protein according to claim 1.

6. The recombinant bacterium according to claim 4 or 5, characterized in that The recombinant bacteria include bacteria.

7. Use of a biomaterial related to the protein of claim 1 in the preparation of levodopa, characterized in that: The biological material is any one of the following: B1), a nucleic acid molecule encoding the protein according to claim 1; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

8. A method for preparing levodopa, comprising fermenting levodopa using the recombinant bacteria according to any one of claims 4 to 6 or the biological material according to claim 2.

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