Tyrosinase mutant derived from Presteria and application of tyrosinase mutant

Abstract

The invention discloses a tyrosinase mutant derived from Presteria and application of the tyrosinase mutant, and belongs to the technical field of genetic engineering. The mutant is obtained by mutating the 46th amino acid of SEQ ID NO: 3 from glycine to glutamic acid, mutating the 65th amino acid of SEQ ID NO: 3 from phenylalanine to tyrosine, mutating the 218th amino acid of SEQ ID NO: 3 from valine to tyrosine and keeping other amino acid sequences unchanged. The tyrosinase provided by the invention has obviously higher catalytic capability on free tyrosine than that of wild type recombinant clostridium butyricum, and can be applied to industrialization of production and modification of levodopa.

Description

Technical Field

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

[0002] Tyrosinase (EC 1.14.18.1) is a monophenol monooxygenase. Among them, tyrosinase plays an important role and economic value in the field of tyrosine modification, such as modifying tyrosine to form levodopa, a drug for Parkinson's disease. It also modifies tyrosine on proteins to form dopa groups. It is widely used in organic synthesis, biosensors, synthetic biology and food fields.

[0003] Computer-aided semi-rational design refers to the use of bioinformatics and computer programs to compare known protein sequences, three-dimensional modeling, and point mutation Gibbs free energy change simulation methods. Protein mutation points are selected based on the calculation results. Recombinant proteins are verified through genetic engineering technology to obtain highly active or highly stable recombinant protein mutants according to needs. This technical means has been widely used in the field of synthetic biology. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a protein that converts the substrate L-tyrosine into an L-DOPA enzyme. The technical problem to be solved is not limited to the described technical subject matter, and those skilled in the art can clearly understand other technical subjects not mentioned 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, wherein 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. 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 the use of the aforementioned protein or a substance regulating the expression of a gene encoding the protein or a substance regulating the activity or content of the protein in the preparation of levodopa.

[0010] In the above application, 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 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 or enhances or improves the expression of the mutant protein encoding gene or upregulates or 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, and keeping the remaining nucleotide sequence of pET-32a(+) unchanged.

[0022] In the above-mentioned biological material, the expression cassette described in B2) refers to a single-stranded or double-stranded nucleic acid molecule that can express the above-mentioned protein in a host cell. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule of all regulatory sequences necessary for expressing the nucleic acid molecule of any of the above-mentioned proteins or the DNA of the RNA molecule. The regulatory sequence can guide the coding sequence to express any of the above-mentioned proteins or the DNA of the RNA molecule in a suitable host cell under its compatible conditions. The regulatory sequence includes, but is 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 should include a promoter and termination signals for transcription and translation. In order to introduce specific restriction enzyme sites into the vector so as to connect the regulatory sequence to the coding region of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule, a regulatory sequence with a joint can be provided. The regulatory sequence can be a suitable promoter sequence, that is, a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates the expression of the protein or the DNA of the 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 obtained 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 can be recognized by the host cell to terminate transcription. The termination sequence can be operably connected to the 3' end of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule. Any terminator that can function 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 mRNA non-translated region that is very important for the translation of the host cell. The leader sequence can be operably connected to the 5' end of the nucleic acid sequence encoding the protein or the DNA of the RNA molecule. Any leader sequence that can function 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 connected to the amino terminus of the protein, which can guide the DNA encoding the protein or the RNA molecule into the cell secretory pathway. Signal peptide coding regions that can guide the expressed protein or the DNA of the RNA molecule into the secretory pathway of the host cell used can be used in the present invention. It may also be desirable to add regulatory sequences that regulate the expression of the protein or the RNA molecule according to the growth 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 one of the aforementioned proteins in the preparation of levodopa, wherein the biomaterial is any one 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 preparing levodopa by fermenting the aforementioned recombinant bacteria or the aforementioned biological materials.

[0031] The above method includes the following reaction system: adding 10 ml of reaction mixture (containing 0.4 g / L L-Tyr, 0.01 M PBS buffer solution, 0.06 mM CuSO4, and solvent is water) into a 15 ml centrifuge tube, adding 150 mg / L polyphenol oxidase (mushroom), 150 mg / L Trx-Bmtyr cell lysis solution, and 1 mL of 150 mg / L Trx-Bmtyr (E46\Y65\Y218) cell lysis solution, respectively, adjusting the pH of the mixture to 7.0 with 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 L-dopa production and modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The SDS-PAGE gel electrophoresis diagram of Trx-Bmtyr and Trx-Bmtyr (E46\Y65\Y218) protein lysate. M: TSINGKE TSP021 Prestained Protein Ladder 180kda-17kda Lane 1: Trx-Bmtyr protein lysate; Lane 2: Trx-Bmtyr (E46\Y65\Y218) protein lysate.

[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 assay 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 is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0040] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

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

[0042] The following examples used GraphPad Prism 8 statistical software to process the data, and the experimental results were expressed as mean ± standard deviation. One-way ANOVA test was used, and P < 0.05 (*) indicated a significant difference.

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

[0044] Levodopa: Levodopa purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number D111048-100g, referred to as L-DOPA.

[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 McLean 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., catalog 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) of the tyrosinase mutant Bmtyr (E46\Y65\Y218) was synthesized by Beijing Qingke Biotechnology Co., Ltd. The DNA molecule shown in SEQ ID NO: 2 was constructed into pET-32a (+) to obtain a recombinant expression vector pET-32a (+)-Bmtyr (E46\Y65\Y218). The recombinant expression vector pET-32a (+)-Bmtyr (E46\Y65\Y218) is a recombinant expression vector pET-32a (+)-Bmtyr (E46\Y65\Y218) obtained by replacing the nucleotide fragment between the BamH I and Hind III restriction recognition sites of the vector pET-32a (+) with a DNA molecule having a nucleotide sequence of SEQ ID NO: 2, and keeping the rest of the nucleotide sequence of pET-32a (+) unchanged. The recombinant vector expresses a fusion protein, and the fusion protein 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 other amino acids unchanged, and its amino acid sequence is SEQ ID NO: 1.

[0053] The optimized coding sequence (SEQ ID NO: 4) of tyrosinase Bmtyr (NCBI accession number WP_053487522.1, i.e., SEQ ID NO: 3) from Priestia was synthesized by Beijing Qingke Biotechnology Co., Ltd. The DNA molecule shown in SEQ ID NO: 4 was constructed into pET-32a (+) to obtain a recombinant expression vector pET-32a (+)-Bmtyr. The recombinant expression vector pET-32a (+)-Bmtyr is a recombinant expression vector pET-32a (+)-Bmtyr obtained by replacing the nucleotide fragment between the BamHI and Hind III restriction recognition sites of the vector pET-32a (+) with a DNA molecule having a nucleotide sequence of SEQ ID NO: 4, and keeping the remaining nucleotide sequences of pET-32a (+) unchanged. The recombinant vector expresses a fusion protein, and the fusion protein is T rx protein-tyrosinase Bmtyr. The amino acid sequence of tyrosinase Bmtyr is 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 ) were inoculated into 5 mL of LB medium containing 100 mg / L sodium ampicillin, shaken at 220 r / min, and cultured at 37°C for 8 h. Sterilized glycerol was added when OD610nm: 0.8-1.0 until the bacterial solution contained a final concentration of 20%. Glycerol was stored at -80°C in sterile tubes. 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 culture medium composition: tryptone 10g / L (OXOID), yeast extract 5g / L (OXOID), sodium chloride 10g / L, and the rest is distilled water.

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

[0057] TB medium composition: tryptone 12 g / L (OXOID), yeast extract 24 g / L (OXOID), 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 shaking 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 shaking flask containing 50 mL of TB medium at 200 rpm and cultured for 2 h at 37°C until OD 600nm When the cell density reaches about 4-6, add 0.1mM isopropyl-β-D-thiogalactoside (IPTG) in the clean bench and change to 220rpm, 25℃ shaker, add 0.1mM IPTG and culture for 24h, collect the bacterial solution into a centrifuge tube, centrifuge at 5000rpm for 5min, and then centrifuge at 8000rpm in a centrifuge (Lu Xiangyi 50ml centrifuge) for 10min to obtain the bacterial precipitation of BL21(DE3) / pET-32a(+)-Bmtyr. Take 2g of the bacterial precipitation of BL21(DE3) / pET-32a(+)-Bmtyr, dilute with 0.01M PBS solution, and make up to 100mL to obtain the bacterial mixture of BL21(DE3) / pET-32a(+)-Bmtyr. The mixed solution was homogenized for 2 min at 800 bar using a high pressure homogenizer (ATS AH1500 homogenizer), and then the homogenate was centrifuged at 8000 rpm for 10 min 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 Biotech Co., Ltd.) to the above protein sample. Mix thoroughly and heat at 100°C for 20 minutes. Prepare 1.0 mm 10% SDS-PAGE gel using the protein gel electrophoresis kit of BIOSS Co., Ltd. for electrophoresis verification of protein samples. Prestained ProteinLadder was used as a protein marker (product number TSP021). The gel after electrophoresis was stained with Coomassie stain (microwave oven, medium-high heat for 90s), then the stain was discarded and destaining solution was added for destaining. Destaining was performed for 2 hours each time until the bands were clear. The destaining solution consisted of 450mL methanol, 100mL glacial acetic acid, and 450ml water. The gel stained with Coomassie stain was photographed by Shanghai Qinxiang Scientific Instrument Co., Ltd., GenoSens 2250 gel imaging system.

[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 to replace the recombinant strain BL21(DE3) / pET-32a(+)-Bmtyr, and 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 This is the gel electrophoresis pattern for the verification of the expression product of the recombinant strain, where 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 protein sizes 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 150mg / L polyphenol oxidase (mushroom): weigh 15mg polyphenol oxidase (mushroom) and dissolve it in 0.01M PBS solution and make up to 100mL.

[0065] Preparation method of 150 mg / L Trx-Bmtyr cell lysis solution: collect BL21(DE3) / pET-32a(+)-Bmtyr bacterial cell precipitate, determine the protein concentration of the cell lysis solution with BCA protein concentration determination kit, and dilute it to 150 mg / L with 0.01M PBS solution.

[0066] Preparation method of 150mg / L Trx-Bmtyr(E46\Y65\Y218) cell lysis solution: Collect BL21(DE3) / pET-32a-Bmty r(E46\Y65\Y218) bacterial precipitate, determine the protein concentration of the cell lysis solution with BCA protein concentration determination kit, and dilute it to 150mg / L with 0.01MP BS solution.

[0067] DOPA detection method (Arnow method) principle: Substances containing 3,4-dihydroxyphenylalanine (DOPA) structure are 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 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] Nitrite 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 0mL, 0.1mL, 0.5mL, 1.0mL, 1.5mL, and 2.0mL of DOPA standard solution, dilute to 10mL 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 min after the addition of the nitrous acid reagent), and the test tube was shaken to obtain each series of standard solutions to be tested. Tube No. 0 was used as a blank, and the absorbance of the above series of standard solutions to be tested was measured at a wavelength of 500 nm using a 1 cm cuvette, and a standard curve was drawn with OD500 nm as the ordinate and L-DOPA content (mg / L) as the abscissa (standard curve, regression expectation should be 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 for catalyzing L-Tyr to form L-DOPA

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

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

[0081] Preparation method of 150mg / L Trx-Bmtyr (E46\Y65\Y218) cell lysis solution: Use BCA protein concentration determination kit to determine the protein concentration of cell lysis solution, and dilute it to 150mg / L with 0.01M PBS solution. Repeat the test three times, and each repetition is as follows:

[0082] Bmtyr (E46\Y65\Y218) group: add 10 ml of reaction mixture (containing 0.4 g / L L-Ty r, 0.01 M PBS buffer solution, 0.06 mM CuSO4, and the solvent is water) to a 15 ml centrifuge tube, 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℃100rpm for 1h, 1ml of the reaction solution was sampled after 0.5h and the content of L-DOPA in it was determined by Arnow method. The specific operation is as follows: 1mL of the reaction solution was placed in a 10mL test tube, and then 0.5mL of acidic reagent, 1.5mL of nitrous acid reagent and 2mL of alkaline reagent were added (the alkaline reagent should be added within 5min after the addition of nitrous acid reagent), and the test solution was obtained. Tube No. 0 was used as a blank, and the OD500nm value of the above-mentioned test solution was measured at a wavelength of 500nm using a 1cm colorimetric dish. Three replicates were set, and the average value of the results was taken. Then, according to the measured absorbance of the test solution, the DOPA content of the sample was calculated in combination with the above-mentioned standard curve. Note: If the sample interferes with the colorimetric phenomenon, centrifuge at 3000r / min for 10min, and take the supernatant for colorimetric.

[0083] Bmtyr group: The difference between this group and the Bmtyr (E46\Y65\Y218) group is that 1 mL of 150 mg / L Trx-Bmtyr (E46\Y65\Y218) cell lysis solution was used instead of 1 mL of 150 mg / L Trx-Bmtyr (E46\Y65\Y218) cell lysis solution, and the rest of the operations were the same as those of the Bmtyr (E46\Y65\Y218) group.

[0084] Polyphenol oxidase group: The difference between this group and the Bmtyr (E46\Y65\Y218) group is that 1 mL of 150 mg / L Trx-Bmtyr cell lysis solution was replaced by 1 mL of 150 mg / L polyphenol oxidase solution, and the rest of the operations 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 one-way ANOVA test results 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 of Bmtyr (E46\Y65\Y218) compared with Bmtyr and polyphenol oxidase (mushroom) groups were extremely significant (P < 0.0001). It can be seen that compared with Trx-Bm tyr fusion protein, mutant Trx-Bmtyr (E46\Y65\Y218) has the ability to convert substrate L-tyrosine into L-DOPA, and the enzyme activity of the enzyme is higher than that of polyphenol oxidase (mushroom) under the conditions of pH 7.0 reaction temperature 30°C, 0.06mM CuSO4, and 100rpm in 0.01MPBS buffer system.

[0086] Table 1. Enzyme activity assay results

[0087]

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

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

[0090] In a 15 ml centrifuge tube, add 10 ml of reaction mixture (containing 0.4 g / L L-Tyr, 0.01 M PBS buffer solution, 0.06 mM CuSO4, and the solvent is water), 1 mL of 150 mg / L Trx-Bmtyr (E46\Y65\Y218) cell lysate, and 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. Oscillate the reaction on a shaker at 30°C for 0.5 h to determine the L-DOPA content by the Arnow method.

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

[0092] Different pH 0D Repeat OD replicate 2 OD replicate 3 L-DOPA(mg / L) pH5.0 0.273 0.321 0.365 21.400±2.9513 pH5.5 0.338 0.294 0.299 20.833±1.5373 pH 6.0 0.332 0.344 0.285 21.467±2.0033 pH 6.5 0.366 0.335 0.404 24.533±2.2030 pH7.0 0.408 0.437 0.430 28.167±0.9452 pH7.5 0.371 0.338 0.268 21.800±3.3719 pH8.0 0.134 0.151 0.143 10.067±0.5508 PH8.5 0.149 0.165 0.157 11.000±0.5000

[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 with pH 7.0 as the control group. The results were as follows: Figure 4As shown, the comparison results of Trx-Bmtyr (E46\Y65\Y218) in pH 7.0 group to pH 5.0, pH 5.5, pH 6.0 and pH 7.5 were significant (P<0.01), and extremely significant to pH 8.0 and pH 8.5 (P<0.0001). This indicates that at pH 7.0, the catalytic ability of Trx-Bmtyr (E46\Y65\Y218) has significant differences in the above pH conditions, while the comparison results for pH 6.5 are not significant (P>0.05), and the concentration of L-DOPA generated by the pH 6.5 and pH 7.0 control groups is higher than that of other pH condition groups. Therefore, the optimal enzyme activity range of Trx-Bmtyr (E46\Y65\Y218) is pH 6.5-7.0.

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

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

[0096] In a 15 ml centrifuge tube, add 10 ml of reaction mixture (containing 0.4 g / L L-Tyr, 0.01 M PBS buffer solution, 0.06 mM CuS04, and the solvent is water), 1 ml of 150 mg / L Trx-Bmtyr (E46\Y65\Y218) cell lysate, and adjust the pH of the mixture to 7.0 with 1 M phosphoric acid solution or 2 M NaOH solution. Shake on a shaker at 26, 28, 30, 32, 34, 37, 39, 41, 43 ° C for 0.5 h to measure the L-DOPA content by the Arnow method.

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

[0098] Reaction temperature 0D Repeat 1 0D Repeat 2 0D Repeat 3 L-DOPA(mg / L) 26℃ 0.213 0.201 0.201 14.067±0.4619 28℃ 0.285 0.287 0.294 19.433±0.3215 30℃ 0.416 0.424 0.425 27.967±0.3215 32℃ 0.439 0.429 0.435 28.767±0.3512 34℃ 0.454 0.471 0.474 30.800±0.7000 37℃ 0.488 0.497 0.491 32.467±0.3055 39℃ 0.138 0.143 0.145 10.033±0.2082 41℃ 0.134 0.131 0.135 9.467±0.1528 43℃ 0.127 0.124 0.123 8.933±0.1528

[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) was significantly higher at 37°C than at 34°C (P<0.001), and significantly higher at other experimental temperature conditions (P<0.0001). Moreover, the L-DOPA production of Trx-Bmtyr (E46\Y65\Y218) at 37°C was higher than that of other temperature groups, indicating that the optimal temperature of the enzyme was 37°C. One enzyme activity unit (U) was defined as the production of 1 micromole of substrate DOPA per minute, and the enzyme activity of each mg of Trx-Bmtyr (E46\Y65\Y218) reached 0.366U based on the production of 3.47mg / L L-DOPA at 37°C.

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

[0101]

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

[0103]

[0104]

[0105] SEQ ID NO: 3 (amino acid sequence of Bmtyr)

[0106] MSNKYRVRKNVLRLTDTEKRDFVRTVLILKEKGIYDRYIAWHGAAGKFHTPPGSDRNAAHMSSAFLPWHREYLLRFERDLQSINPEVTLPYWEWETDAQLQDPSQSQIWSADFMGGNGNPKKDFIVDTGPFAAGRWTTIDEQGNPSGGL KRNFGATKEAPTLPTRDDVLDALKITQYDTPPWDMTSQNSFRNQLEGFINGPQLHNRVHRWVGGQMGVVPTAPNDPVFFLHHANVDRIWAVWQIVHRNQNYQPMKNGPFGQNFRDPMYPWNTTPEDVMNHRKLGYVYDIELRKSKRSS.

[0107] SEQ ID NO:4 (nucleotide sequence of Bmtyr)

[0108] 5'-ATGGGCAACAAATACCGTGTTCGTAAGAACGTGCTGCACCTGACTGACACCGAGAAACGTGACTTCGTTCGTA CCGTTCTGATCTTGAAAGAGAAAGGTATCTACGACCGTTACATCGCATGGCACGGTGCTGCGGGTAAATTCCACACTCCGCCGGGTTCTGACCGTAACGCAGCGCACATGAGTTCTGCGTTCTTGCCGTGGCACCGTGAATACCTGCTGCGTTTCGAACGTGACCTGCAGTCTATCAACCCGGAAGTTACTCTGCCGTACTGGGAATGGGAAACCGATGCGCAGATGCAAGATCCGTCTCAGTCTCAGATTTGGTCCGCAGACTTCATGGGTGGTAACGGTAATCCAATCAAAGACTTCATCGTTGACACTGGTCCGTTCGCTGCGGGTCGTTGGACCACCATCGATGAACAGGGTAACCCGTCTGGTGGTTTGAAACGTAACTTCGGTGCTACCAAAGAAGCTCCGACCTTGCCGACTCGTGACGACGTACTGAACGCGCTGAAGATCACTCAGTACGACACTCCACCGTGGGATATGACCAGCCAGAACTCTTTCCGTAACCAGCTGGAAGGTTTCATCAACGGTCCACAGCTGCACAATCGCGTACACCGTTGGGTTGGTGGTCAGATGGGTGTGGTTCCGACTGCACCGAACGATCCGGTGTTCTTTCTGCACCACGCGAACGTTGACCGTATCTGGGCAGTTTGGCAGATCATCCACCGTAACCAGAACTACCAGCCGATGAAGAACGGTCCATTCGGTCAGAACTTCCGTGATCCGATGTATCCGTGGAACACCACTCCGGAAGATGTGATGAACCACCGTAAACTGGGTTACGTTTACGACATCGAACTGCGTAAATCCAAACGTAGCTCTTAA-3'。

[0109] 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 the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with 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, the 218th amino acid from valine to tyrosine, and keeping other amino acid sequences unchanged.

2. Use of the protein as claimed in claim 1 or a substance regulating the expression of a gene encoding the protein or a substance regulating the activity or content of the protein in the preparation of levodopa.

3. The use according to claim 2, characterized in that: The regulation is to upregulate, enhance or increase the expression of the gene encoding the protein in claim 1 or the content or activity of the protein.

4. The use according to claim 2, characterized in that: The substance is any 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).

5. The use according to claim 4, characterized in that: B1) The coding sequence of the nucleic acid molecule is SEQ ID NO:

2.

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

7. The recombinant bacterium according to claim 6, characterized in that The recombinant bacteria contains the gene encoding the protein according to claim 1.

8. The recombinant bacterium according to claim 6 or 7, characterized in that The recombinant bacteria include bacteria.

9. Use of a biological material related to the protein of claim 1 in the preparation of levodopa, characterized in that: The biological material is any 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).

10. A method for preparing levodopa, comprising fermenting levodopa using the recombinant bacteria according to any one of claims 6 to 8 or the biological material according to claim 9.

Images