Tyrosinase and its engineering bacteria, preparation method and application

Abstract

The invention belongs to the technical field of enzyme genetic engineering, and specifically relates to a tyrosinase mutant with improved enzyme activity and a preparation method thereof. The invention obtains a tyrosinase gene from Bacillus aryabhattai by molecular biological technology, mutates a mutant G43R tyrosinase gene by iterative saturation mutagenesis technology, obtains tyrosinase mutants G43R / M61H, G43R / M61H / A232C, G43R / M61H / A232C / Q214D, G43R / M61H / A232C / Q214D / V217A, G43R / M61H / A232C / Q214D / V217A / F197W, and encoding genes tyrm2, tyrm3, tyrm4, tyrm5, and tyrm6 thereof, reconstructs a recombinant plasmid, realizes efficient expression of the recombinant plasmid in Bacillus amyloliquefaciens, and obtains high-activity tyrosinase by fermentation, extraction, and other technologies.

Description

Technical field:

[0001] The invention belongs to the technical field of enzyme genetic engineering, and specifically relates to a tyrosinase mutant with improved enzyme activity obtained by iterative saturation mutation technology and its preparation. Background technology:

[0002] Tyrosinase (TYR, EC 1.14.18.1), also known as polyphenol oxidase and catechol oxidase, is a type 3 binuclear copper-containing metalloenzyme involved in the synthesis of melanin. In the presence of molecular oxygen, tyrosinase can perform two consecutive reactions with monophenol as substrate. First, it catalyzes monophenol as substrate to generate catechol, and then further oxidizes catechol as substrate to generate quinones, which spontaneously polymerize to form melanin. Tyrosinase is widely present in the human body, animals, plants and microorganisms. In tyrosinases from different sources, its active site is highly conserved, and there are six histidine residues coordinating two copper ions to activate tyrosinase activity.

[0003] In mammals, melanin produced by tyrosinase can play a dyeing role in animal skin, hair and other tissue cells, helping the eyes and skin to resist ultraviolet radiation. The decline and loss of tyrosinase function in mammals can also cause albinism and vitiligo, and the common Parkinson's disease is also related to it; in plants, tyrosinase is more commonly known as polyphenol oxidase, and is involved in the synthesis of phenolic polymers of tannins and flavonoids, causing browning of plants such as fruits and vegetables; in insects, tyrosinase often exists in the form of zymogen, completing different physiological functions in different parts of the insect, and is the only enzyme used for insect cuticle hardening. In arthropods, it is also involved in physiological processes such as wound healing.

[0004] At present, tyrosinase has been widely used in many fields. In terms of biomedical applications, the main method for synthesizing L-DOPA is chemical synthesis, which has problems such as complex reaction process and the need for metal catalysts. L-DOPA is the first product of tyrosinase with tyrosine as substrate. Therefore, tyrosinase can be used to catalyze the synthesis of L-DOPA from tyrosine. In terms of protein cross-linking, in food processing, proteins will undergo protein cross-linking due to changes in the conditions in which they are located, causing changes in their structure, which has a great impact on the characteristics of food and directly leads to changes in the nutrition and properties of the product. Compared with traditional cross-linking agents, tyrosinase has a wider substrate specificity. At present, there are many applications of using tyrosinase as a cross-linking agent for meat, dairy products and plant protein foods. In terms of environmental protection, phenolic compounds such as phenol and cresol are toxic and difficult to degrade. Most phenolic compounds are considered to be environmental pollutants, which have negative effects on the urinary system, digestive system, respiratory system and nervous system. Phenolic compounds are removed by ion exchange, photocatalysis and enzyme biocatalytic degradation. Tyrosinase can use phenolic substances as substrates to catalyze the production of quinones, and can catalyze the detoxification of toxic organic substances such as phenol. In terms of biological detection, tyrosinase can specifically recognize phenolic substances and some biological detectors have been developed to detect the polyphenol content in samples such as red wine.

[0005] Tyrosinase exists in almost every field of nature and participates in various biological functions. Currently, tyrosinases from a variety of sources, including fungi, bacteria, and animals, have been isolated and purified, and the crystal structures of tyrosinases from multiple sources have been successfully obtained. Studies have found that the number of amino acid residues in tyrosinases from different sources varies greatly, but the molecular weight of most mature tyrosinase proteins is around 35-50kDa.

[0006] Enzyme molecule modification is an important means to improve protein function and activity, which mainly includes directed evolution, rational design and semi-rational design. When the protein structure and its catalytic site are not understood, directed evolution is often used to modify the enzyme molecules. As more protein structures are solved and computer prediction of protein structure develops, more and more rational design and semi-rational design are used to modify enzyme molecules. Through the analysis of protein structure and its catalytic mechanism, rational analysis and selection of amino acid sites that may affect protein activity and function, fewer mutants are obtained through site-directed mutagenesis, and a screening system is established to achieve the purpose of quickly and efficiently screening ideal mutants. Compared with directed evolution, it has higher efficiency and less workload.

[0007] Compared with the commonly used Escherichia coli expression system, the Bacillus expression system can secrete the expression product outside the cell, which is beneficial to the separation and purification of the product. Bacillus has the advantages of clear genetic background research, fast growth rate, and low requirements for culture medium. It is widely used in agriculture, food, medicine, industry and other fields.

[0008] In the present invention, the wild-type tyrosinase gene is subjected to iterative saturation mutation to obtain a highly active tyrosinase mutant gene, and its efficient expression in the Bacillus amyloliquefaciens expression system is achieved to obtain a mutant strain producing highly active tyrosinase. Summary of the invention:

[0009] Based on the problems existing in the prior art, in order to further promote the application of tyrosinase in the industrial field, its existing properties need to be further improved. The purpose of the present invention is to provide a mutant of high-activity tyrosinase.

[0010] The technical route for achieving the purpose of the present invention is summarized as follows:

[0011] The mutant G43R obtained by mutating tyrosinase from Bacillus aryabhattai TCCC 111983 was subjected to iterative saturation mutagenesis. Its encoding gene is tyrm1. TYR mutants G43R / M61H, G43R / M61H / A232C, G43R / M61H / A232C / Q214D, G43R / M61H / A232C / Q214D / V217A, G43R / M61H / A232C / Q214D / V217A, G43R / M61H / A232C / Q214D / V217A / F197W and their encoding genes tyrm2, tyrm3, tyrm4, tyrm5, and tyrm6 were obtained by screening using an Escherichia coli expression system. Efficient expression of each mutant was achieved in Bacillus amyloliquefaciens, and TYR mutants with improved enzyme activity were obtained through fermentation, extraction and other techniques.

[0012] One of the technical solutions provided by the present invention is a tyrosinase mutant, wherein the mutant is obtained by causing at least one of the mutations G43R, M61H, A232C, Q214D, V217A, F197W, etc. to occur on the basis of the wild-type tyrosinase described in SEQ ID NO.1;

[0013] Further, the tyrosinase mutant is a G43R / M61H mutant having an amino acid sequence shown in SEQ ID NO.5;

[0014] Furthermore, the encoding gene tyrm2 of the G43R / M61H mutant has the nucleotide sequence shown in SEQ ID NO.6;

[0015] Furthermore, the tyrosinase mutant is a G43R / M61H / A232C mutant having an amino acid sequence shown in SEQ ID NO.7;

[0016] Furthermore, the encoding gene tyrm3 of the G43R / M61H / A232C mutant has the nucleotide sequence shown in SEQ ID NO.8;

[0017] Furthermore, the tyrosinase mutant is a G43R / M61H / A232C / Q214D mutant having an amino acid sequence shown in SEQ ID NO.9;

[0018] Furthermore, the encoding gene tyrm4 of the G43R / M61H / A232C / Q214D mutant has a nucleotide sequence shown in SEQ ID NO.10;

[0019] Further, the tyrosinase mutant is a G43R / M61H / A232C / Q214D / V217A mutant having an amino acid sequence shown in SEQ ID NO.11;

[0020] Furthermore, the encoding gene tyrm5 of the G43R / M61H / A232C / Q214D / V217A mutant has a nucleotide sequence shown in SEQ ID NO.12;

[0021] Further, the tyrosinase mutant is a G43R / M61H / A232C / Q214D / V217A / F197W mutant having an amino acid sequence shown in SEQ ID NO.13;

[0022] Furthermore, the encoding gene tyrm6 of the G43R / M61H / A232C / Q214D / V217A / F197W mutant has a nucleotide sequence shown in SEQ ID NO.14.

[0023] The second technical solution provided by the present invention is a recombinant plasmid or recombinant strain containing the above mutant encoding gene;

[0024] Furthermore, the expression vector used by the recombinant plasmid is pET22b, or pBSA43 plasmid;

[0025] Furthermore, the host cell used by the recombinant strain is Escherichia coli BL21 or Bacillus amyloliquefaciens CGMCC No.11218;

[0026] Preferably, the recombinant strain is obtained by connecting the mutant encoding gene to the expression vector pBSA43 and expressing it in the host Bacillus amyloliquefaciens CGMCC No.11218.

[0027] The third technical solution provided by the present invention is the application of the above-mentioned recombinant plasmid or recombinant strain, especially in the production of tyrosinase.

[0028] The fourth technical solution provided by the present invention is the application of the tyrosinase mutant described in the first technical solution, specifically the application in monophenol oxidation, especially in the technical fields of biomedicine, food processing, environmental protection, biological detection, etc.

[0029] The experimental scheme of the present invention is as follows:

[0030] 1. Obtaining the gene encoding the TYR mutant comprises the following steps:

[0031] (1) Using the G43R mutant encoding gene tyrm1 shown in SEQ ID NO.4 as the starting gene, the expression vector pET22b-tyrm1 was constructed and iterative saturation mutagenesis was performed;

[0032] (2) The mutated TYR encoding gene was transferred into Escherichia coli BL21 by constructing a recombinant plasmid, and TYR mutants with improved enzyme activity were obtained by screening. The TYR mutant encoding genes tyrm2, tyrm3, tyrm4, tyrm5, and tyrm6 were obtained by sequencing, and the plasmids pET22b-tyrm2, pET22b-tyrm3, pET22b-tyrm4, pET22b-tyrm5, and pET22b-tyrm6 containing the TYR mutant encoding genes with improved enzyme activity were preserved.

[0033] 2. The enzymatic properties of the TYR mutants G43R, G43R / M61H, G43R / M61H / A232C, G43R / M61H / A232C / Q214D, G43R / M61H / A232C / Q214D / V217A, and G43R / M61H / A232C / Q214D / V217A / F197W are as follows:

[0034] (1) Specific activity: The specific activity of TYR mutant G43R was 218.7 U / mg, and the specific activities of TYR mutants G43R / M61H, G43R / M61H / A232C, G43R / M61H / A232C / Q214D, G43R / M61H / A232C / Q214D / V217A, and G43R / M61H / A232C / Q214D / V217A / F197W were 292.0 U / mg, 377.4 U / mg, 469.9 U / mg, 550.4 U / mg, and 677.8 U / mg, respectively.

[0035] (2) Optimum reaction temperature: 60℃.

[0036] (3) Optimum pH: 5.0.

[0037] 3. A recombinant strain of Bacillus amyloliquefaciens containing a tyrosinase encoding gene and a process for preparing tyrosinase with improved enzyme activity using the recombinant strain of Bacillus amyloliquefaciens containing a tyrosinase encoding gene, comprising the following steps:

[0038] (1) The TYR mutant encoding genes tyrm2, tyrm3, tyrm4, tyrm5, and tyrm6 were connected with the Bacillus amyloliquefaciens expression plasmid pBSA43 to obtain new recombinant plasmids pBSA43-tyrm2, pBSA43-tyrm3, pBSA43-tyrm4, pBSA43-tyrm5, and pBSA43-tyrm6;

[0039] (2) The recombinant plasmid was transferred into Bacillus amyloliquefaciens CGMCC No.11218, and the recombinant strain was obtained by kanamycin (Kan) resistance screening and enzyme digestion verification. The recombinant strain was then cultured and fermented to obtain tyrosinase.

[0040] The following definitions are used in the present invention:

[0041] 1. Nomenclature of amino acid and DNA sequences

[0042] The generally accepted IUPAC nomenclature for amino acid residues is used, either as a single letter or three letter code. The generally accepted IUPAC nomenclature for DNA nucleic acid sequences is used.

[0043] 2. Identification of tyrosinase mutants

[0044] The mutated amino acid in the TYR mutant is represented by "amino acid replaced by the original amino acid position", such as Gly43Arg, which means that the amino acid at position 43 is replaced by Arg from Gly of the wild-type TYR, and the position number corresponds to the amino acid sequence number of the wild-type TYR in SEQ ID No. 1.

[0045] In the present invention, lowercase italic tyr represents the coding gene of wild-type tyrosinase TYR, lowercase italic tyrm1 represents the coding gene of mutant G43R, lowercase italic tyrm2, tyrm3, tyrm4, tyrm5, and tyrm6 represent the coding genes of mutants G43R / M61H, G43R / M61H / A232C, G43R / M61H / A232C / Q214D, G43R / M61H / A232C / Q214D / V217A, and G43R / M61H / A232C / Q214D / V217A, respectively, and the information is as shown in the following table.

[0046]

[0047] Beneficial effects:

[0048] 1. The present invention uses iterative saturation mutagenesis technology to mutate the TYR mutant G43R to obtain mutants G43R / M61H, G43R / M61H / A232C, G43R / M61H / A232C / Q214D, G43R / M61H / A232C / Q214D / V217A, and G43R / M61H / A232C / Q214D / V217A, and G43R / M61H / A232C / Q214D / V217A / F197W with improved enzyme activity. The maximum values ​​of fermentation enzyme activity in the Bacillus amyloliquefaciens expression system are 3040.4 U / mL, 3591.7 U / mL, 4798.8 U / mL, 5228.6 U / mL, and 6901.9 U / mL, respectively.

[0049] 2. The present invention uses the Bacillus amyloliquefaciens expression system to achieve efficient expression of TYR mutants with improved enzyme activity. Description of the drawings:

[0050] Figure 1 It is an SDS-PAGE image of the purified samples of the wild-type TYR, mutant G43R and mutants G43R / M61H, G43R / M61H / A232C, G43R / M61H / A232C / Q214D, G43R / M61H / A232C / Q214D / V217A, and G43R / M61H / A232C / Q214D / V217A / F197W of the present invention;

[0051] Among them: M is Protein Marker, 1 is the wild-type TYR ultrafiltration and concentration sample, 2 is the mutant G43R ultrafiltration and concentration sample, 3 is the mutant G43R / M61H ultrafiltration and concentration sample, 4 is the mutant G43R / M61H / A232C ultrafiltration and concentration sample, 5 is the mutant G43R / M61H / A232C / Q214D ultrafiltration and concentration sample, 6 is the mutant G43R / M61H / A232C / Q214D / V217A ultrafiltration and concentration sample, and 7 is the mutant G43R / M61H / A232C / Q214D / V217A / F197W ultrafiltration and concentration sample. Specific implementation method:

[0052] The technical content of the present invention is further described below in conjunction with the embodiments, but the present invention is not limited to these embodiments, and the protection scope of the present invention cannot be limited by the following embodiments.

[0053] The culture medium used in the embodiments of the present invention is as follows:

[0054] LB medium (g / L): 5.0% yeast extract, 10.0% tryptone, 10.0% NaCl, and the rest water.

[0055] For solid medium, add 2% agar.

[0056] The wild-type tyrosinase TYR of the present invention is from Bacillus aryabhattai, and the amino acid sequence is shown in SEQ ID NO.1:

[0057] MSNKYKVRKNVLSLTDAEKRDFIRAVLILKKKGIYDRYIAWHGAAGKFHTPPSSDRNAAHMSSAFLPWHREYLLRFERDLQSIDSEVTLPYWEWETDAQLQDPSQSQIWSADFMGGNGNPKKDFIVDTGPFVAGRWTTIDEQGNPSGGL KRNFGATKEAPTLPTRDDVLNALKITKYDTPPWDMTSQNSFRNQLEGFINGPQLHNRVHRWVGGQMGVVPTAPNDPVFFLHHANVDRIWAVWQMVHRNQNYQPMKNGPFGQNFRDPMYPWNTTPEDVMNHRKLGYVYDIELRKSKRSS.

[0058] The present invention will be further explained below through specific examples.

[0059] Example 1: Acquisition of TYR mutants

[0060] 1. Selection of mutation sites: The protein structure and catalytic mechanism of TYR were analyzed, and the amino acid residues around the copper ion binding site (Trp41, Gly43, Ala59, Met61, Trp68, Arg70, Leu203, Asn205, Val207, Arg209, His230 and Ala232), the amino acid residues around the substrate binding site (Gln214, Met215, Gly216, Val217, Val218 and Pro219) and the amino acid residues around the conserved amino acid site that mediates the deprotonation of water molecules (Glu195, Gly196, Phe197, Ile198, Asn199, Gly200, Pro201, Gln202, Leu203, Asn205) were selected. According to the order of the amino acid residues around the copper ion binding site, the amino acid residues around the substrate binding site and the amino acid residues around the conserved amino acid site that mediates the deprotonation of water molecules, iterative saturation mutations were performed one by one on the sites selected in each region.

[0061] 2. Inverse PCR: The G43R mutant encoding gene tyrm1 shown in SEQ ID NO.4 was used as the starting gene and connected with the pET22b plasmid to construct the expression vector pET22b-tyrm1. The recombinant plasmid pET22b-tyrm1 was used as the starting template to perform iterative saturation mutation using inverse PCR. The primers and reaction system used for inverse PCR are as follows:

[0062]

[0063]

[0064] Element Volume (μL) Plasmid (50ng / μL) 0.5 10×buffer 1.25 Primer F 0.5 Primer R 0.5 dNTPs 1.25 KOD-Plus 0.25 <![CDATA[ddH2O]]> 8.25

[0065] Note: The reagents required for the above inverse PCR are from KOD-Plus Muragensis Kit of Toyobo (Shanghai) Biotechnology Co., Ltd.

[0066] After the reaction system is mixed according to the above ratio, the reverse PCR reaction is performed, and the program setting is as follows:

[0067]

[0068] After the PCR reaction, the PCR product was digested to remove the template and then self-circularized and connected, and transformed into Escherichia coli BL21, coated on LB solid culture medium containing ampicillin (100 μg / mL), and cultured in a 37°C incubator for 12 hours to obtain transformants.

[0069] 3. Screening method: This experiment uses fluorescence detection to determine the activity of tyrosinase monophenolase. The fluorescence spectrometer can measure the fluorescence of stable tyrosine at an excitation wavelength of 275nm, and the emission light is obtained by scanning at 280-420nm. The slit width of the excitation and emission monochromator is set at 3-5nm. Since the target protein exists in the fermentation supernatant, the fermentation supernatant can be directly used for screening. The lower the fluorescence intensity, the greater the substrate consumption, which proves that the enzyme activity is higher.

[0070] 4. Screening of mutant library: Add 200 μL of LB liquid culture medium containing ampicillin (100 μg / mL) to each well of 96-well plate 1, then pick each single clone of the transformant into the 96-well plate with a sterilized toothpick, and try to get just a small amount of bacteria each time during the picking process. Transfer the 96-well plate to a shaker for culture at 160 rpm and 37°C for 48 hours. Then use a low-temperature centrifuge (4°C) and centrifuge at 4000 rpm for 10 minutes, take 10 μL of the fermentation supernatant and add it to 200 μL of the reaction solution, react at 60°C for 5 minutes, and detect the fluorescence intensity of the reaction system.

[0071] Solutions: (1) 2 mmol / L L-tyrosine solution: accurately weigh 36 mg of L-tyrosine, dissolve it in Tris-HCl (pH 7.0) and make up to 100 mL, and store in a refrigerator at 4°C;

[0072] (2) 0.2 mol / L boric acid solution: Dissolve 12.37 g of boric acid in 700 mL of deionized water and make up to 900 mL.

[0073] (3) 0.2 mol / L borax solution: Dissolve 1.907 g of borax in 80 mL of deionized water and make up to 100 mL.

[0074] (4) Borate buffer: Mix 900 mL of boric acid solution with 100 mL of borax solution. The pH after mixing is 7.4.

[0075] (5) Reaction solution: Take 1.4 mL of solution (1) and 28.6 mL of solution (4) and mix them with 10 μL of hydroxylamine and mix thoroughly to prepare a reaction solution.

[0076] 5. Select mutants with improved enzyme activity. According to the fluorescence detection results, calculate the enzyme activity of each mutant, select mutants with improved enzyme activity compared with the previous generation mutants, inoculate the mutants into plates, and send bacterial samples for sequencing. After the sequencing is verified to be correct, perform saturation mutation of the next site on this basis.

[0077] After iterative saturation mutation in the above steps, mutants with improved enzyme activity are selected for sequencing, and then mutants with improved enzyme activity are obtained:

[0078] G43R / M61H( G G T → C G G / ATG → CAC );

[0079] G43R / M61H / A232C( G G T → C G G / ATG → CAC / GCA → TGC );

[0080] G43R / M61H / A232C / Q214D( G G T → C G G / ATG → CAC / GCA → TGC / C A G → G A C );

[0081] G43R / M61H / A232C / Q214D / V217A( G G T → C G G / ATG → CAC / GCA → TGC / C A G → G A C / G TT →G CG );

[0082] G43R / M61H / A232C / Q214D / V217A / F197W( G G T → C G G / ATG → CAC / GCA → TGC / CAG → G A C / G TT →G CG / TT T →T GG )。

[0083] Thus, the TYR mutants G43R / M61H (amino acid sequence SEQ ID NO.5), G43R / M61H / A232C (amino acid sequence SEQ ID NO.7), G43R / M61H / A232C / Q214D (amino acid sequence SEQ ID NO.9), G43R / M61H / A232C / Q214D / V217A (amino acid sequence SEQ ID NO.11), G43R / M61H / A232C / Q214D / V217A / F197W (amino acid sequence SEQ ID NO.13), and their encoding genes tyrm2 (SEQ ID NO.6), tyrm3 (SEQ ID NO.8), tyrm4 (SEQ ID NO.10), tyrm5 (SEQ ID NO.12), tyrm6 (SEQ ID NO.13) were obtained. NO.14), the specific activities were 292.0U / mg, 377.4U / mg, 469.9U / mg, 550.4U / mg and 677.8U / mg respectively. The specific activities of the wild type and mutant G43R in the control group were 131.0U / mg and 218.7U / mg respectively.

[0084] Example 2: Construction of recombinant Bacillus amyloliquefaciens tyrosinase

[0085] 1. Construction of expression plasmid

[0086] Plasmids pET22b-tyrm2, pET22b-tyrm3, pET22b-tyrm4, and pET22b-tyrm5 were extracted from the strains obtained by the above screening to obtain mutants G43R / M61H, G43R / M61H / A232C, G43R / M61H / A232C / Q214D, G43R / M61H / A232C / Q214D / V217A, and G43R / M61H / A232C / Q214D / V217A / F197W. tyrm4, pET22b-tyrm5, and pET22b-tyrm6 were used to obtain the target genes tyrm2, tyrm3, tyrm4, tyrm5, and tyrm6, which were then connected with the Bacillus amyloliquefaciens expression vector pBSA43 to construct the recombinant expression plasmids pBSA43-tyrm2, pBSA43-tyrm3, pBSA43-tyrm4, pBSA43-tyrm5, and pBSA43-tyrm6.

[0087] 2. Transformation of expression plasmid into Bacillus amyloliquefaciens CGMCC No.11218

[0088] 1 μL (50 ng / μL) of recombinant plasmids pBSA43-tyrm1, pBSA43-tyrm2, pBSA43-tyrm3, pBSA43-tyrm4, pBSA43-tyrm5, and pBSA43-tyrm6 were added to 50 μL of Bacillus amyloliquefaciens CGMCC No. 11218 competent cells and mixed, then transferred to a pre-cooled electroporation cup (1 mm), ice-bathed for 1-1.5 min, and then electroporated once (25 μF, 200 Ω, 4.5-5.0 ms). After the electroporation, 1 mL of recovery medium (LB + 0.5 mol / L sorbitol + 0.38 mol / L mannitol) was added immediately. After shaking and culturing at 37°C for 3 h, the revived material was spread on an LB plate containing kanamycin and cultured at 37°C for 12-24 h. Positive transformants were picked to obtain recombinant strains of Bacillus amyloliquefaciens expressing genes encoding different mutants: CGMCC No.11218 / pBSA43-tyrm1, CGMCC No.11218 / pBSA43-tyrm2, CGMCC No.11218 / pBSA43-tyrm3, CGMCC No.11218 / pBSA43-tyrm4, CGMCC No.11218 / pBSA43-tyrm5, and CGMCC No.11218 / pBSA43-tyrm6.

[0089] Example 3: Expression and preparation of tyrosinase with improved enzyme activity in recombinant Bacillus amyloliquefaciens

[0090] 1. The recombinant strains of Bacillus amyloliquefaciens CGMCC No.11218 / pBSA43-tyrm1, CGMCC No.11218 / pBSA43-tyrm2, CGMCC No.11218 / pBSA43-tyrm3, CGMCC No.11218 / pBSA43-tyrm4, CGMCC No.11218 / pBSA43-tyrm5 and CGMCC No.11218 / pBSA43-tyrm6 were inoculated into LB liquid medium containing kanamycin (50 μg / mL) and cultured at 37°C and 220 r / min overnight;

[0091] 2. Transfer the inoculum to 50 mL of LB medium at 1%, culture at 37°C, 220 r / min for 48 h, centrifuge at 4000 r / min for 15 min, collect the supernatant to obtain high-activity TYR crude enzyme solution, and determine the enzyme activity of the TYR mutant crude enzyme solution using L-tyrosine as substrate at 60°C, pH = 7.0.

[0092] 3. Then collect the fermentation supernatant, first remove the impurities by salting out with 25% saturation ammonium sulfate, and then increase the saturation to 65% to precipitate the target protein. After dissolution, dialyze to remove salt, and then dissolve the active components obtained after salting out and desalting with 0.02mol / LTris-HCl (pH 8.0) buffer, load it onto the ion exchange chromatography column, first elute the unadsorbed protein with the same buffer, and then gradient elute with 0.02mol / LTris-HCl (pH 8.0) buffer containing 0-1mol / LNaCl to collect the target protein. The active components obtained by ion exchange are first balanced with 0.02mol / L Tris-HCl (pH8.0) buffer containing 0.15mol / L NaCl, and then loaded onto the gel column and eluted with the same buffer at a rate of 0.5mL / min to obtain a purified enzyme solution. The purified enzyme solution is freeze-dried to obtain high-activity TYR pure enzyme powder. SDS-PAGE image of protein purification sample Figure 1 .

[0093] Example 4: Tyrosinase activity assay

[0094] 1. Principle of tyrosinase activity assay

[0095] The fluorescence spectrometer can measure the fluorescence of stable tyrosine at an excitation wavelength of 275nm, and the emission light is obtained by scanning at 280-420nm. The slit width of the excitation and emission monochromator is set at 3-5nm. Since the target protein exists in the fermentation supernatant, the fermentation supernatant can be directly used for screening. The lower the fluorescence intensity, the greater the substrate consumption, which proves that the enzyme activity is higher.

[0096] 2. Tyrosinase activity assay method and steps used in the present invention

[0097] In a reaction system with a final volume of 3 mL, 0.14 mL of 2 mmol / L tyrosine solution (final concentration of 70 μM) and 1 μL of hydroxylamine were added, and the volume was made up to 3 mL with borate solution, and the mixture was kept at 60°C for 1 min, and 20 μL of enzyme solution (TYR crude enzyme solution obtained in Example 3) was added and mixed by pipetting, and the reaction was continued for 5 min, and the initial fluorescence intensity of the reaction and the fluorescence intensity after the reaction were recorded. Each sample contained 3 sets of parallel experiments.

[0098] Blank control: Treat the enzyme solution at 100°C for 10 min to inactivate the enzyme, and use the heat-inactivated enzyme solution as a control. The reaction system and method are the same as above.

[0099] Note: Solution: (1) 2 mmol / L L-tyrosine solution: Accurately weigh 36 mg of L-tyrosine, dissolve it in Tris-HCl (pH 7.0) and make up to 100 mL, and store in a refrigerator at 4°C;

[0100] (2) 0.2 mol / L boric acid solution: Dissolve 12.37 g of boric acid in 700 mL of deionized water and make up to 900 mL.

[0101] (3) 0.2 mol / L borax solution: Dissolve 1.907 g of borax in 80 mL of deionized water and make up to 100 mL.

[0102] (4) Borate buffer: Mix 900 mL of boric acid solution with 100 mL of borax solution. The pH after mixing is 7.4.

[0103] 3. Definition and calculation of tyrosinase activity

[0104] Definition of enzyme activity: Under certain reaction conditions (unless otherwise specified, the conditions are: 60°C, pH 7.0), the amount of enzyme required to consume 1 μmol of L-tyrosine per minute is defined as one enzyme activity unit, recorded as U / mL.

[0105] Enzyme activity calculation:

[0106] Enzyme activity: U / mL = x×V1 / y×Δt×V2

[0107] Where: x represents the change in fluorescence intensity from the beginning to the end of the reaction;

[0108] V1 represents the total volume of the reaction system (μL);

[0109] Δt represents the time taken from the start to the end of the reaction (min);

[0110] V2 represents the volume of enzyme solution in the reaction system (μL);

[0111] y represents the slope of the standard curve for detecting tyrosine fluorescence intensity.

[0112] Enzyme specific activity (U / mg) = enzyme activity / protein concentration.

[0113] 4. The enzyme activity of the fermentation broth obtained from the Bacillus amyloliquefaciens expression system in Example 3 was measured. The enzyme activity of the G43R mutant was 2087.5 U / mL, and the enzyme activities of the G43R / M61H, G43R / M61H / A232C, G43R / M61H / A232C / Q214D, G43R / M61H / A232C / Q214D / V217A, and G43R / M61H / A232C / Q214D / V217A / F197W mutants were 3040.4 U / mL, 3591.7 U / mL, 4798.8 U / mL, 5228.6 U / mL, and 6901.9 U / mL, respectively.

[0114] Example 5: Synthesis of bisphenol using high-activity mutant G43R / M61H / A232C / Q214D / V217A / F197W with monophenol as substrate

[0115] Taking L-tyrosine as substrate and TYR as catalyst to prepare L-dopa as an example, in a reaction system with 50 mL L-tyrosine concentration of 0.5 mmol / L, copper ion concentration of 2 μmol / L, and ascorbic acid concentration of 2 mmol / L, the addition amount of TYR mutant G43R / M61H / A232C / Q214D / V217A / F197W enzyme was 250 U, the reaction conditions were 35°C, pH 7.0, and the yield of L-dopa was detected by sampling at 5 min, 15 min, 30 min, 60 min, and 120 min of reaction, and the yields of L-dopa were 27.39%, 48.63%, 65.27%, 81.64%, and 84.5%, respectively.

[0116] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A tyrosinase mutant, characterized in that: The mutant is obtained by causing one of the following mutations to occur on the basis of the wild-type tyrosinase shown in SEQ ID NO.1: G43R / M61H, G43R / M61H / A232C, G43R / M61H / A232C / Q214D, G43R / M61H / A232C / Q214D / V217A, or G43R / M61H / A232C / Q214D / V217A / F197W.

2. The gene encoding the tyrosinase mutant according to claim 1.

3. The gene encoding the tyrosinase mutant according to claim 2, characterized in that: As shown in the sequence listing SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12 or SEQ ID NO.

14.

4. A recombinant vector or recombinant strain comprising the gene according to claim 2.

5. The recombinant vector or recombinant strain according to claim 4, characterized in that: The expression vector is pET22b or pBSA43 plasmid, and the host cell is Escherichia coli BL21 or Bacillus amyloliquefaciens CGMCC No.11218.

6. Use of the recombinant vector or recombinant strain according to claim 4 in producing the tyrosinase mutant according to claim 1.

7. The use of the tyrosinase mutant according to claim 1, characterized in that: The tyrosinase mutants were applied to monophenol oxidation.

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