CN113373123A - Tyrosinase mutant and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of enzyme engineering, and relates to a tyrosinase mutant and application thereof. The tyrosinase mutant mutates a plurality of amino acid sites in wild type tyrosinase of an amino acid sequence shown in SEQ ID NO. 1. By utilizing the tyrosinase mutant and the method for preparing theaflavin from the tyrosinase mutant, the mutant has higher specific activity than wild tyrosinase and higher yield in the process of preparing theaflavin through catalysis.
Description
Technical Field
The invention belongs to the field of enzyme engineering, and relates to a tyrosinase mutant and application thereof in synthesis of theaflavin.
Background
In China, tea has a long history of culture as a national drink. The tea can be divided into six kinds of tea, namely black tea, green tea, oolong tea, yellow tea, black tea and white tea according to different making and properties of the tea, while the black tea is the second kind of tea in China, the production region is wide, including more than ten provinces such as Fujian, Guangdong, Yunnan and Taiwan, and the Yunnan and the Fujian are main planting regions of the tea. The most obvious characteristic of the black tea is that the black tea has the characteristics of black tea, red soup, red leaves, sweet and mellow taste and the like, and is deeply loved by the consumers. In recent years, researches show that the black tea has a plurality of physiological functions beneficial to health, such as effects of inhibiting obesity, regulating blood fat, reducing blood pressure, reducing blood sugar, resisting bacteria, viruses and cancers, and the like, wherein the key function of the black tea is an effective functional component, namely theaflavin, in the black tea. Theaflavin was extracted from black tea by Roberts in 1957, is a kind of compound which is easily dissolved in ethyl acetate, has orange yellow color and contains a tropolone structure and is formed in the fermentation process of black tea (figure 1), and the formation mechanism is that in the preparation process of black tea, intracellular tea polyphenols are formed by self-polymerization after oxidation reaction under the action of polyphenol oxidase and peroxidase. Currently, there are twenty more than one theaflavin species that have been discovered and analyzed and identified, the most predominant of which are four (fig. 1), respectively: theaflavin (Theaflavin, TF1), Theaflavin-3-gallate (Theaflavin 3-O-gallate, TF2a), Theaflavin-3 ' -gallate (Theaflavin 3 ' -O-gallate, TF2b) and Theaflavin digallate (Theaflavin 3 ' 3-di-O-gallate, TF 3). The different theaflavins are formed by oxidative autopolymerization of the different substrates catechol under the action of oxidase. With the structural elucidation of different theaflavins, the functional research of the theaflavins is increasingly deep, and particularly in the aspects of health care and pharmacological functions, the research shows that the theaflavins have the functions of reducing blood fat, resisting aging, resisting oxidation, resisting cancer, preventing cardiovascular and cerebrovascular diseases and the like. Based on this, many theaflavin health care products are on the market, for example, the American life extension company develops a series of theaflavin extract products, the theaflavin extract products are applied to the fields of health care products and the like, and along with the continuous market expansion and the increasing attention of people to health, the development of theaflavin related products has great market prospect in the future.
The current industrial production method for preparing theaflavin mainly comprises plant extraction method and enzymatic oxidation. The method for separating and extracting theaflavin from plants is adopted to prepare theaflavin, and because the content of theaflavin in tea is low (0.2% -2%), the method has the defects of high cost, complex purification process, low product purity, low yield and the like, obviously, the method is not economic enough, and the product cost and quality are difficult to reach the expected level of people, so that the requirement of large-scale industrial production cannot be met.
A polyphenol oxidation enzymatic oxidation method is adopted, leaves or other tissue cells of plant sources (tea, pear and the like) are crushed and then put into a reaction kettle containing tea polyphenol, reaction parameters are set, oxygen is introduced for reaction, the tea polyphenol is catalyzed by using polyphenol oxidase existing in the plant to synthesize theaflavin, but the content of the tea polyphenol oxidase of the plant sources is limited, and other components existing in the plant tissues can interfere with the catalytic reaction to cause large difficulty and high cost in subsequent separation and purification, so that the quality and purity of the theaflavin product can be influenced.
The enzymes currently generally recognized as involved in the theaflavin enzymatic synthesis pathway are the polyphenol oxidases (EC1.10.3.1, polyphenolic oxidases, PPO,). Polyphenol oxidases, also known as Catechol oxidases, can be divided into three major classes according to their substrate preference, namely Catechol enzyme (EC1.10.3.1, cathechol Oxidase), Laccase (EC 1.10.3.2, lacccase), Tyrosinase enzyme (EC 1.14.18.1, Tyrosinase). In the early work, the research on tyrosinase mainly focuses on the fields of medical health care and fine chemical engineering such as oxidation resistance and melanin formation inhibition: for example, the inhibition effect of the effective components in the tea on tyrosinase is researched, and whitening and skin-care products are developed. At present, the commercialized tyrosinase comes from fungal microorganisms such as mushrooms, and related researches on preparation of theaflavin by oxidation of tyrosinase are reported. The method selects the tyrosinase gene from Bacillus megaterium for the first time, obtains the mutant enzyme with high catalytic activity and good substrate tolerance by directed evolution and high-throughput screening by utilizing a genetic engineering technical means, and has very important industrial value and significance when being applied to efficient directed synthesis of theaflavin or monomers thereof.
Disclosure of Invention
The invention mainly aims to provide a tyrosinase mutant capable of efficiently synthesizing theaflavin and monomers thereof, wherein the mutant has higher specific activity and higher product conversion rate than wild tyrosinase.
To achieve this object, in a basic embodiment, the present invention provides a tyrosinase mutant, which comprises a mutation in a wild-type tyrosinase having the amino acid sequence shown in SEQ ID No.1 at a plurality of amino acid positions including one or more of N205D, D166E, D167G and V285I.
In a preferred embodiment, the mutation pattern of the mutant of the present invention includes any one of the following 10 types:
N205D; D166E; D167G; V285I; N205D and D166E; N205D and D167G; N205D and V285I; N205D and D166E and D167G; N205D and D166E and V285I; N205D and D166E and D167G and V285I.
Further: the mutation mode comprises any one of the following 7 types:
N205D; N205D and D166E; N205D and D167G; N205D and V285I; N205D and D166E and D167G; N205D and D166E and V285I; N205D and D166E and D167G and V285I.
Further wherein said mutant has the amino acid sequence N205D; D166E; D167G; V285I; N205D and D166E; N205D and D167G; N205D and V285I; N205D and D166E and D167G; N205D and D166E and V285I; N205D and D166E and D167G and V285I; sequentially shown as SEQ ID NO. 2-11.
The second purpose of the invention is to provide a polynucleotide for coding the tyrosinase mutant, so that the coded tyrosinase mutant has higher specific activity than wild tyrosinase, and has higher product yield and higher substrate concentration tolerance when the theaflavin is prepared through catalysis.
To achieve this object, in a basic embodiment, the present invention provides polynucleotides encoding the foregoing tyrosinase mutants.
The third purpose of the invention is to provide the application of the tyrosinase mutant, so as to better prepare theaflavin.
To achieve the purpose, in a basic embodiment, the tyrosinase mutant provided by the invention is used for catalyzing catechin reaction by the tyrosinase mutant in a reaction system to prepare theaflavin.
In a preferred embodiment, the invention provides application of the tyrosinase mutant, wherein catechins are used as substrates, and the theaflavin products are catalytically synthesized under the aerobic condition.
The main substrates of catechins include: epicatechin (EC), Epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), reaction substrates and synthesis products of either:
epicatechin + epigallocatechin producing Theaflavin (Theaflavin, TF 1);
epicatechin + epigallocatechin gallate to form Theaflavin-3-gallate (Theaflavin 3-O-gallate, TF2 a);
the epicatechin gallate and epigallocatechin produce Theaflavin-3 '-gallate (Theaflavin 3' -O-gallate, TF2 b);
the epicatechin gallate and epigallocatechin gallate form Theaflavin digallate (Theaflavin 3' 3-di-O-gallate, TF 3).
In a preferred embodiment, in the reaction system of the present invention, the tyrosinase mutant activity is 200-500U/L, EC, EGC and EGCG are all 10-100mM, and ECG is 2-10 mM.
In a preferred embodiment, the reaction temperature of the invention is 25-30 ℃, the reaction pH is 4.0-6.0, the reaction stirring speed is 150-.
In a preferred embodiment, the tyrosinase mutant according to the invention is an immobilized tyrosinase mutant.
The invention has the advantages that the tyrosinase mutant has higher specific activity than wild tyrosinase and higher product yield in the process of preparing theaflavin through catalysis.
The invention selects the tyrosinase Bmtyrc from Bacillus megaterium as a starting point, and has the following advantages compared with polyphenol oxidases from other sources: high heterologous expression efficiency, good stability and high catalytic efficiency for catechin as a substrate. On the basis, the Bmtyrc is mutated by means of gene engineering and enzyme engineering technology, and compared with the Bmtyrc, the obtained tyrosinase mutant has the advantages of higher enzyme specific activity, higher catalytic efficiency and conversion yield, higher substrate tolerance and the like, so that the method is more suitable for synthesizing theaflavin.
Drawings
FIG. 1 is a schematic diagram of the synthesis of theaflavins catalyzed by catechins as different substrates.
Detailed Description
The following description will further describe embodiments of the present invention with reference to the accompanying drawings.
The method for measuring the enzyme activity of the tyrosinase and the mutant thereof comprises the following steps:
preparing a 10mM substrate polyphenol solution (EC, EGC, ECG or EGCG): accurately weighing tea polyphenol with corresponding weight, adding 90mL of 0.1mol/L citric acid-phosphoric acid buffer solution with pH of 5.0 for dissolution, preheating to 30 ℃, and adding the buffer solution for constant volume to 100 mL;
0.01mol/L copper sulfate solution: accurately weigh 0.249g CuSO4·5H2O, dissolved in 100mL of water.
4mL of 0.01mol/L polyphenol solution preheated to 30 ℃ and 0.1mL of 0.01mol/L copper sulfate solution are sequentially added into a quartz cuvette with the specification of 1cm, after uniform mixing, zero calibration is carried out at the position with the wavelength of 420nm, 100 mu L of enzyme solution (or 0.1g of immobilized enzyme is directly added) is added, the measurement is started after rapid mixing, and the absorbance A420 is recorded every 10 seconds.
Unit of enzyme activity: the amount of enzyme required to achieve a change in OD420nm of 0.001 per minute at 30 ℃ and pH5.0 was defined as one unit (1U).
HPLC analysis was performed using an Agilent liquid chromatograph, column: 5C18-AR-II (250 mm. times.4.6 mm, 5 μm); column temperature: 40 ℃; sample introduction amount: 20 mu L of the solution; detection wavelength: 280 nm; mobile phase: a: 50mmol/L phosphoric acid; b: acetonitrile-ethyl acetate (7: 1, V/V); flow rate: 1.0 mL/min; elution procedure: 0-25min, 82% -68% of phase A.
Example 1: construction of tyrosinase Bmtyrc prokaryotic expression strain derived from Bacillus megaterium
The amino acid sequence of tyrosinase derived from Bacillus megaterium in GenBank (SEQ ID NO.1, corresponding to GenBank accession No.: ACC86108.1) was downloaded and submitted to Beijing Ongzhike Biotechnology Co., Ltd for total gene sequence synthesis (using codon preferred by E.coli). The C-end of the synthetic gene is provided with a His label, and is constructed into a prokaryotic expression vector pET30a (+), and the restriction enzyme site of the prokaryotic expression vector is as follows: nde I at the 5 'end, Xho I at the 3' end. Passing the constructed plasmid pET30a (+) -Bmtyrc through CaCl2The heat shock transformation method is used for transforming the strain into an escherichia coli expression strain BL21(DE3), the strain is coated on an LB solid medium plate containing 50 mu g/ml Kanamycin, the plate is cultured overnight at 37 ℃, and a colony growing on the plate is the tyrosinase zymogen nucleus expression recombinant strain E.coli BL21(DE3)/pET30a (+) -Bmtyrc.
Carefully picking out a single colony of the zymogen tyrosine nucleus expression recombinant strain in the LB solid culture medium plate by using a sterilized gun head, inoculating the single colony into a triangular flask containing 20mL of LB liquid culture medium, culturing at 37 ℃ at 200r/min, and shaking overnight. Inoculating the shake flask bacterial liquid into a triangular flask containing 100ml of LTB liquid culture medium the next day according to the inoculation amount of 1%, carrying out shaking culture at 37 ℃ at 220r/min, measuring the OD value of the culture solution every 1h, supplementing lactose with the final concentration of 1% (m/v) when the OD value of the culture solution is 1.5, continuously culturing at 25 ℃ at 220rpm for 4-6 h, and stopping culturing.
Example 2: purification and immobilization of tyrosinase (Bmtyrc)
The activated IDA Resin (purchased from Annu (Beijing) Biotechnology Inc., with a specific model of His. bind Resin, Ni-charged) is adopted by using His label carried in Bmtyrc recombinant protein, and the adopted specific method and steps are as follows: centrifuging the fermentation liquid for 10min at 4 ℃ and 10000r/min, discarding the supernatant, collecting the thallus, repeatedly washing the thallus twice with phosphate buffer solution (pH 8.0 and 0.1mol/L), centrifuging, and concentrating the thallus by 5 times and suspending in 20mL of phosphate buffer solution (pH 8.0 and 0.1 mol/L). And (3) placing the treated bacterial liquid in ice water for ultrasonic crushing until the bacterial liquid is clarified, wherein the ultrasonic crushing conditions are as follows: work 2s, interval 5s, ultrasonic power 500W. And (3) placing the crushed lysate into a low-temperature high-speed centrifuge for centrifugation (12000rpm, 4 ℃ and 20min), and collecting supernatant to obtain crude protein. And loading the crude protein onto activated resin, performing gradient elution by using imidazole solution (200mM-500mM), performing real-time monitoring by using a protein chromatography system (Bio-Rad), and collecting the stable protein peak, namely Bmtyrc recombinant protein purified protein, for preparing immobilized enzyme.
The method for preparing the immobilized enzyme by using the purified Bmtyrc recombinant protein comprises the following steps:
(1) activating an immobilized carrier: accurately measuring 30mL of 60% (m/v) glutaraldehyde and dipotassium hydrogen phosphate (K)2HPO4·3H2O)4.76g is added into 600mL deionized water, dissolved and then the volume is adjusted to 1000mL by deionized water, and the pH value is adjusted to 8.0 by phosphoric acid solution. An epoxy-based carrier ECEP (Resindions S.r.l. Italy) of 250g was put into the above solution and activated at 25 ℃ with low stirring for 2 hours, and the carrier was collected by filtration, washed 2 to 3 times with sterile deionized water and vacuum-filtered to dryness for use.
(2) Immobilization of Bmtyrc recombinant protein: diluting a certain amount of purified Bmtyrc recombinant protein with a phosphate buffer solution (pH 8.0 and 0.1mol/L), adding 50g of activated carrier, immobilizing for 48h at 25 ℃ and 120rpm, washing the obtained immobilized enzyme with deionized water for 3-5 times, and carrying out vacuum filtration to obtain the final immobilized enzyme product.
Example 3: construction of Bmtyrc prokaryotic expression strain E.coli BL21(DE3)/pET30a (+) -Bmtyrc error-prone mutation library
pET30a (+) -Bmtyrc recombinant plasmid is used as PCR template, conventional T7F/R is used as universal primer (primer sequence: T7F: 5'-TAATACGACTCACTATAGGG-3', T7R: GCTAGTTATTGCTCAGCGG see SEQ ID NO.12 and 13) to carry out error-prone PCR amplification on Bmtyrc gene, and Mg in PCR amplification reaction system is adjusted2+、Mn2+dCTP and dTTP oligonucleotide concentration, making the base mismatching rate of the mutant library only two thousandth, namely ensuring that only 1 to 2 amino acids of one mutant are mutated.
Error-prone PCR reaction system:
error-prone PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; then denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 1min, and extension at 72 ℃ for 1.5min for 25 cycles; finally, extension is carried out for 10min at 72 ℃.
And sampling 2 mu L of the error-prone PCR product, detecting the error-prone PCR product through agarose gel electrophoresis, and purifying the error-prone PCR product by using a PCR product purification kit after the error-prone PCR product is detected. The PCR purified product and the prokaryotic expression vector pET30a (+) were subjected to double digestion with Nde I and Xho I restriction enzymes at 37 ℃ respectively, and the digestion product was subjected to gel cutting recovery (wherein the size of the recovered PCR purified product fragment was about 1200bp, and the size of the recovered vector pET30a (+) fragment was about 5400bp), followed by error-prone PCR: the prokaryotic expression vector pET30a (+) is 3: 1, and T4 DNA ligase was added to the mixture and ligated overnight at 16 ℃. The next day, the ligation product is transferred into escherichia coli BL21(DE3) by an electric shock transformation method to construct engineering bacteria, and a random mutant library with large library capacity can be obtained.
Example 4: screening of error-prone mutation library of Bmtyrc prokaryotic expression strain E.coli BL21(DE3)/pET30a (+) -Bmtyrc
Because the polyphenol substrate can be oxidized by tyrosinase under the participation of oxygen to form quinone substances, on one hand, the quinone substances have obvious color change, on the other hand, theaflavin substances formed by oxidation autopolymerization of the quinone substances have specific absorption peaks (the experiment adopts 420nm detection) at the positions of 400-500nm, on the basis, a corresponding high-throughput screening method can be established, the enzyme property in a reaction system is better, the higher the catalytic efficiency is, the more oxidation products are generated, the deeper the color is, and the positive target clone can be screened and obtained by the judgment of naked eyes and the measurement of an enzyme labeling instrument. The specific method adopted for high throughput screening is as follows:
using the sterilized toothpicks, single colonies of the mutant library (1 single colony per toothpick) were carefully picked and inoculated into different wells of a 96-well cell culture plate (LB liquid medium containing 50. mu.g/ml kanamycin had been added to each well). The 96-well cell culture plate was incubated at 37 ℃ for 6 hours at 700rpm in a constant temperature shaker, 50. mu.L of the resulting culture plate was collected by an 8-channel pipette and stored as a seed solution in a new 96-well plate, and lactose was added to each well to a final concentration of 1% (m/v), and the culture was induced at 25 ℃ for 8 hours at 250 rpm. After induction culture is finished, the 96-well cell culture plate is put into an ultralow temperature refrigerator with the temperature of 86 ℃ below zero for freezing for 2 hours, taken out and placed at room temperature for half an hour, then is centrifuged at 4000r/min at the temperature of 4 ℃ for 20 minutes, and 50 mu L of supernatant is taken out of each well. mu.L of the reaction solution (substrate tea polyphenol concentration: 3-5mg/mL, citric acid-phosphate buffer solution of pH 5.0) was added to 50. mu.L of the supernatant of each well, and incubated at 30 ℃ for 20-60 min. The color change was observed and analyzed with a microplate reader (detection wavelength 420nm), and wells with high absorbance were selected for further assay verification.
Through repeated mass screening verification (about 200000 clones), sequencing analysis and enzyme activity determination, 3 expression strains of mutants with significantly higher activities on EGCG and ECG substrates than wild Bmtyrc, namely Bmtyrc-1, Bmtyrc-2 and Bmtyrc-3, are obtained, and the following table 1 is summarized.
Table 1: bmtyrc mutant expression strain obtained by screening error-prone mutation library
As is obvious from Table 1, compared with the wild-type enzyme, the Bmtyrc-1 mutant has obviously improved catalytic activity on four polyphenol substrates EC, EGC, ECGC and ECG in the screened positive mutant enzyme, wherein the activity on the polyphenol substrate EC is improved by 2.16 times, the activity on the EGC is improved by 1.31 times, the activity on the EGCG is improved by 1.59 times, and the activity on the ECG is improved by 3.05 times. In addition, compared with the wild enzyme, the activity of the mutant Bmtyrc-2 and Bmtyrc-3 on the substrate is also improved, and the activity of the mutant Bmtyrc-4 is not obviously improved as compared with that of Bmtyrc-1, -2, -3. Therefore, the Bmtyrc-1 mutant gene is taken as a starting point, and the superposition mutation of other favorable amino acids is carried out on the basis, so that the catalytic activity on the substrate is further improved.
Example 5: construction and screening of different stacked mutant strains
The Bmtyrc-1 expression strains in the table 1 are subjected to amplification culture, plasmids are extracted by a plasmid kit (OMEGA), plasmids pET30a (+) -Bmtyrc-1 are taken as a template, overlapping mutation is carried out on obtained mutation sites, 166 th, 167 th and 285 th positions of Bmtyrc amino acid sequences are selected, fixed point mutation primers are respectively designed, full plasmid PCR reaction is carried out, full plasmid PCR products are digested by DpnI and then transformed into an expression strain BL21(DE3), sequencing verification is carried out, expression strains of all overlapping mutants based on Bmtyrc-1 mutants are obtained, and the specific conditions of the expressed and screened Bmtyrc mutants are shown in the table 2.
TABLE 2 mutation sites and Activity of Bmtyrc and its mutants
As can be seen from Table 2, the activity of each stacked mutant is obviously improved compared with that of Bmtyrc-1. The Bmtyrc-3A mutant shows the highest activity to four catechol substrates compared with other superposed mutants, and the Bmtyrc-4A mutant has lower activity than Bmtyrc-3A after being superposed with V285I on the basis of Bmtyrc-3A. In order to further verify the catalytic performance of each enzyme and the theaflavin synthesis capacity, expression strains of Bmtyrc (SEQ ID NO.1), Bmtyrc-1(SEQ ID NO.2), Bmtyrc-2A (SEQ ID NO.6), Bmtytc-2B (SEQ ID NO.7), Bmtyrc-2C (SEQ ID NO.8), Bmtyrc-3A (SEQ ID NO.9), Bmtyrc-3B (SEQ ID NO.10) and Bmtyrc-4A (SEQ ID NO.11) are selected for fermentation culture, and target proteins are respectively separated, purified and immobilized for catalyzing the theaflavin synthesis (TF3) reaction.
Example 6: fermentation and purification of different Bmtyrc mutants
Bmtyrc-1 and Bmtyrc-2A, Bmtyrc-2B, Bmtyrc-2C, Bmtyrc-3A, Bmtyrc-3B, Bmtyrc-4A are fermented and purified by a method basically the same as that of Bmtyrc, and the obtained protein purified samples and the Bmtyrc purified samples obtained in the example 2 are subjected to SDS-PAGE electrophoretic purity detection (electrophoretic conditions: 10% of separating gel concentration, 100V of voltage, 50mA of current and 120min of electrophoretic time), so that the purity is over 95%.
Example 7: theaflavin (TF3) synthesized by catalysis of immobilized enzymes
After Bmtyrc, Bmtyrc-1 and Bmtyrc-2A, Bmtyrc-2B, Bmtyrc-2C, Bmtyrc-3A, Bmtyrc-3B, Bmtyrc-4A are respectively prepared into immobilized enzymes (the method is the same as the example 2), the immobilized enzymes are catalyzed and synthesized to react, and a large amount of research work of researchers in the previous period shows that substrate polyphenols and theaflavin are very unstable under the condition of high pH value, high-temperature theaflavin is easy to degrade, and the reaction time is too long, so that the amount of theaflavin is reduced, therefore, in the theaflavin synthesis experiment, the reaction time is determined to be 20min, and the dosage of the immobilized enzymes is determined to be 4g/L (equivalent to 200-; the other parameter values are set as: pH values of 4.0, 5.0 and 6.0, reaction temperature of 25 ℃, 30 ℃ and 35 ℃, stirring speed of 150r/min, 175r/min and 200r/min, and reaction conditions and reaction results are shown in tables 3-4.
TABLE 3 reaction conditions for the immobilized enzymes to catalyze the theaflavin (TF3) synthesis reaction
TABLE 4 results of reaction of theaflavins (TF3) catalyzed by each immobilized enzyme
Note:
the amount of TF3 produced in the reaction, and b the amount of ECG added to the reaction.
As can be seen from the catalytic reaction results in Table 4, compared with wild Bmtyrc, each Bmtyrc mutant and each superposed mutant are obviously improved in product concentration and product yield compared with Bmtyrc-1, the best mutant is Bmtyrc-3A, and when the mutant is used for catalytically synthesizing theaflavin, the yield of the mutant can reach 13.45 percent and is 3.28 times of that of the wild type. From the above data, it can also be concluded that the mutant has better actual effect than the wild-type enzyme when applied to other theaflavin synthesis (TF1, TF2a, TF2b, etc.).
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is intended to include such modifications and variations. The foregoing examples or embodiments are merely illustrative of the present invention, which may be embodied in other specific forms or in other specific forms without departing from the spirit or essential characteristics thereof. The described embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. The scope of the invention should be indicated by the appended claims, and any changes that are equivalent to the intent and scope of the claims should be construed to be included therein.
Sequence listing
<110> Hunan Fulaige Biotechnology Ltd
Hunan Agricultural University
<120> tyrosinase mutant and application thereof
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 299
<212> PRT
<213> Bacillus megaterium (Bacillus megaterium)
<400> 1
Met Ser Asn Lys Tyr Arg Val Arg Lys Asn Val Leu His Leu Thr Asp
1 5 10 15
Thr Glu Lys Arg Asp Phe Val Arg Thr Val Leu Ile Leu Lys Glu Lys
20 25 30
Gly Ile Tyr Asp Arg Tyr Ile Ala Trp His Gly Ala Ala Gly Lys Phe
35 40 45
His Thr Pro Pro Gly Ser Asp Arg Asn Ala Ala His Met Ser Ser Ala
50 55 60
Phe Leu Pro Trp His Arg Glu Tyr Leu Leu Arg Phe Glu Arg Asp Leu
65 70 75 80
Gln Ser Ile Asn Pro Glu Val Thr Leu Pro Tyr Trp Glu Trp Glu Thr
85 90 95
Asp Ala Gln Met Gln Asp Pro Ser Gln Ser Gln Ile Trp Ser Ala Asp
100 105 110
Phe Met Gly Gly Asn Gly Asn Pro Ile Lys Asp Phe Ile Val Asp Thr
115 120 125
Gly Pro Phe Ala Ala Gly Arg Trp Thr Thr Ile Asp Glu Gln Gly Asn
130 135 140
Pro Ser Gly Gly Leu Lys Arg Asn Phe Gly Ala Thr Lys Glu Ala Pro
145 150 155 160
Thr Leu Pro Thr Arg Asp Asp Val Leu Asn Ala Leu Lys Ile Thr Gln
165 170 175
Tyr Asp Thr Pro Pro Trp Asp Met Thr Ser Gln Asn Ser Phe Arg Asn
180 185 190
Gln Leu Glu Gly Phe Ile Asn Gly Pro Gln Leu His Asn Arg Val His
195 200 205
Arg Trp Val Gly Gly Gln Met Gly Val Val Pro Thr Ala Pro Asn Asp
210 215 220
Pro Val Phe Phe Leu His His Ala Asn Val Asp Arg Ile Trp Ala Val
225 230 235 240
Trp Gln Ile Ile His Arg Asn Gln Asn Tyr Gln Pro Met Lys Asn Gly
245 250 255
Pro Phe Gly Gln Asn Phe Arg Asp Pro Met Tyr Pro Trp Asn Thr Thr
260 265 270
Pro Glu Asp Val Met Asn His Arg Lys Leu Gly Tyr Val Tyr Asp Ile
275 280 285
Glu Leu Arg Lys Ser Lys Arg Ser Ser Leu Glu
290 295
<210> 2
<211> 299
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Ser Asn Lys Tyr Arg Val Arg Lys Asn Val Leu His Leu Thr Asp
1 5 10 15
Thr Glu Lys Arg Asp Phe Val Arg Thr Val Leu Ile Leu Lys Glu Lys
20 25 30
Gly Ile Tyr Asp Arg Tyr Ile Ala Trp His Gly Ala Ala Gly Lys Phe
35 40 45
His Thr Pro Pro Gly Ser Asp Arg Asn Ala Ala His Met Ser Ser Ala
50 55 60
Phe Leu Pro Trp His Arg Glu Tyr Leu Leu Arg Phe Glu Arg Asp Leu
65 70 75 80
Gln Ser Ile Asn Pro Glu Val Thr Leu Pro Tyr Trp Glu Trp Glu Thr
85 90 95
Asp Ala Gln Met Gln Asp Pro Ser Gln Ser Gln Ile Trp Ser Ala Asp
100 105 110
Phe Met Gly Gly Asn Gly Asn Pro Ile Lys Asp Phe Ile Val Asp Thr
115 120 125
Gly Pro Phe Ala Ala Gly Arg Trp Thr Thr Ile Asp Glu Gln Gly Asn
130 135 140
Pro Ser Gly Gly Leu Lys Arg Asn Phe Gly Ala Thr Lys Glu Ala Pro
145 150 155 160
Thr Leu Pro Thr Arg Asp Asp Val Leu Asn Ala Leu Lys Ile Thr Gln
165 170 175
Tyr Asp Thr Pro Pro Trp Asp Met Thr Ser Gln Asn Ser Phe Arg Asn
180 185 190
Gln Leu Glu Gly Phe Ile Asn Gly Pro Gln Leu His Asp Arg Val His
195 200 205
Arg Trp Val Gly Gly Gln Met Gly Val Val Pro Thr Ala Pro Asn Asp
210 215 220
Pro Val Phe Phe Leu His His Ala Asn Val Asp Arg Ile Trp Ala Val
225 230 235 240
Trp Gln Ile Ile His Arg Asn Gln Asn Tyr Gln Pro Met Lys Asn Gly
245 250 255
Pro Phe Gly Gln Asn Phe Arg Asp Pro Met Tyr Pro Trp Asn Thr Thr
260 265 270
Pro Glu Asp Val Met Asn His Arg Lys Leu Gly Tyr Val Tyr Asp Ile
275 280 285
Glu Leu Arg Lys Ser Lys Arg Ser Ser Leu Glu
290 295
<210> 3
<211> 299
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Met Ser Asn Lys Tyr Arg Val Arg Lys Asn Val Leu His Leu Thr Asp
1 5 10 15
Thr Glu Lys Arg Asp Phe Val Arg Thr Val Leu Ile Leu Lys Glu Lys
20 25 30
Gly Ile Tyr Asp Arg Tyr Ile Ala Trp His Gly Ala Ala Gly Lys Phe
35 40 45
His Thr Pro Pro Gly Ser Asp Arg Asn Ala Ala His Met Ser Ser Ala
50 55 60
Phe Leu Pro Trp His Arg Glu Tyr Leu Leu Arg Phe Glu Arg Asp Leu
65 70 75 80
Gln Ser Ile Asn Pro Glu Val Thr Leu Pro Tyr Trp Glu Trp Glu Thr
85 90 95
Asp Ala Gln Met Gln Asp Pro Ser Gln Ser Gln Ile Trp Ser Ala Asp
100 105 110
Phe Met Gly Gly Asn Gly Asn Pro Ile Lys Asp Phe Ile Val Asp Thr
115 120 125
Gly Pro Phe Ala Ala Gly Arg Trp Thr Thr Ile Asp Glu Gln Gly Asn
130 135 140
Pro Ser Gly Gly Leu Lys Arg Asn Phe Gly Ala Thr Lys Glu Ala Pro
145 150 155 160
Thr Leu Pro Thr Arg Glu Asp Val Leu Asn Ala Leu Lys Ile Thr Gln
165 170 175
Tyr Asp Thr Pro Pro Trp Asp Met Thr Ser Gln Asn Ser Phe Arg Asn
180 185 190
Gln Leu Glu Gly Phe Ile Asn Gly Pro Gln Leu His Asn Arg Val His
195 200 205
Arg Trp Val Gly Gly Gln Met Gly Val Val Pro Thr Ala Pro Asn Asp
210 215 220
Pro Val Phe Phe Leu His His Ala Asn Val Asp Arg Ile Trp Ala Val
225 230 235 240
Trp Gln Ile Ile His Arg Asn Gln Asn Tyr Gln Pro Met Lys Asn Gly
245 250 255
Pro Phe Gly Gln Asn Phe Arg Asp Pro Met Tyr Pro Trp Asn Thr Thr
260 265 270
Pro Glu Asp Val Met Asn His Arg Lys Leu Gly Tyr Val Tyr Asp Ile
275 280 285
Glu Leu Arg Lys Ser Lys Arg Ser Ser Leu Glu
290 295
<210> 4
<211> 299
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Met Ser Asn Lys Tyr Arg Val Arg Lys Asn Val Leu His Leu Thr Asp
1 5 10 15
Thr Glu Lys Arg Asp Phe Val Arg Thr Val Leu Ile Leu Lys Glu Lys
20 25 30
Gly Ile Tyr Asp Arg Tyr Ile Ala Trp His Gly Ala Ala Gly Lys Phe
35 40 45
His Thr Pro Pro Gly Ser Asp Arg Asn Ala Ala His Met Ser Ser Ala
50 55 60
Phe Leu Pro Trp His Arg Glu Tyr Leu Leu Arg Phe Glu Arg Asp Leu
65 70 75 80
Gln Ser Ile Asn Pro Glu Val Thr Leu Pro Tyr Trp Glu Trp Glu Thr
85 90 95
Asp Ala Gln Met Gln Asp Pro Ser Gln Ser Gln Ile Trp Ser Ala Asp
100 105 110
Phe Met Gly Gly Asn Gly Asn Pro Ile Lys Asp Phe Ile Val Asp Thr
115 120 125
Gly Pro Phe Ala Ala Gly Arg Trp Thr Thr Ile Asp Glu Gln Gly Asn
130 135 140
Pro Ser Gly Gly Leu Lys Arg Asn Phe Gly Ala Thr Lys Glu Ala Pro
145 150 155 160
Thr Leu Pro Thr Arg Asp Gly Val Leu Asn Ala Leu Lys Ile Thr Gln
165 170 175
Tyr Asp Thr Pro Pro Trp Asp Met Thr Ser Gln Asn Ser Phe Arg Asn
180 185 190
Gln Leu Glu Gly Phe Ile Asn Gly Pro Gln Leu His Asn Arg Val His
195 200 205
Arg Trp Val Gly Gly Gln Met Gly Val Val Pro Thr Ala Pro Asn Asp
210 215 220
Pro Val Phe Phe Leu His His Ala Asn Val Asp Arg Ile Trp Ala Val
225 230 235 240
Trp Gln Ile Ile His Arg Asn Gln Asn Tyr Gln Pro Met Lys Asn Gly
245 250 255
Pro Phe Gly Gln Asn Phe Arg Asp Pro Met Tyr Pro Trp Asn Thr Thr
260 265 270
Pro Glu Asp Val Met Asn His Arg Lys Leu Gly Tyr Val Tyr Asp Ile
275 280 285
Glu Leu Arg Lys Ser Lys Arg Ser Ser Leu Glu
290 295
<210> 5
<211> 299
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Met Ser Asn Lys Tyr Arg Val Arg Lys Asn Val Leu His Leu Thr Asp
1 5 10 15
Thr Glu Lys Arg Asp Phe Val Arg Thr Val Leu Ile Leu Lys Glu Lys
20 25 30
Gly Ile Tyr Asp Arg Tyr Ile Ala Trp His Gly Ala Ala Gly Lys Phe
35 40 45
His Thr Pro Pro Gly Ser Asp Arg Asn Ala Ala His Met Ser Ser Ala
50 55 60
Phe Leu Pro Trp His Arg Glu Tyr Leu Leu Arg Phe Glu Arg Asp Leu
65 70 75 80
Gln Ser Ile Asn Pro Glu Val Thr Leu Pro Tyr Trp Glu Trp Glu Thr
85 90 95
Asp Ala Gln Met Gln Asp Pro Ser Gln Ser Gln Ile Trp Ser Ala Asp
100 105 110
Phe Met Gly Gly Asn Gly Asn Pro Ile Lys Asp Phe Ile Val Asp Thr
115 120 125
Gly Pro Phe Ala Ala Gly Arg Trp Thr Thr Ile Asp Glu Gln Gly Asn
130 135 140
Pro Ser Gly Gly Leu Lys Arg Asn Phe Gly Ala Thr Lys Glu Ala Pro
145 150 155 160
Thr Leu Pro Thr Arg Asp Asp Val Leu Asn Ala Leu Lys Ile Thr Gln
165 170 175
Tyr Asp Thr Pro Pro Trp Asp Met Thr Ser Gln Asn Ser Phe Arg Asn
180 185 190
Gln Leu Glu Gly Phe Ile Asn Gly Pro Gln Leu His Asn Arg Val His
195 200 205
Arg Trp Val Gly Gly Gln Met Gly Val Val Pro Thr Ala Pro Asn Asp
210 215 220
Pro Val Phe Phe Leu His His Ala Asn Val Asp Arg Ile Trp Ala Val
225 230 235 240
Trp Gln Ile Ile His Arg Asn Gln Asn Tyr Gln Pro Met Lys Asn Gly
245 250 255
Pro Phe Gly Gln Asn Phe Arg Asp Pro Met Tyr Pro Trp Asn Thr Thr
260 265 270
Pro Glu Asp Val Met Asn His Arg Lys Leu Gly Tyr Ile Tyr Asp Ile
275 280 285
Glu Leu Arg Lys Ser Lys Arg Ser Ser Leu Glu
290 295
<210> 6
<211> 299
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Met Ser Asn Lys Tyr Arg Val Arg Lys Asn Val Leu His Leu Thr Asp
1 5 10 15
Thr Glu Lys Arg Asp Phe Val Arg Thr Val Leu Ile Leu Lys Glu Lys
20 25 30
Gly Ile Tyr Asp Arg Tyr Ile Ala Trp His Gly Ala Ala Gly Lys Phe
35 40 45
His Thr Pro Pro Gly Ser Asp Arg Asn Ala Ala His Met Ser Ser Ala
50 55 60
Phe Leu Pro Trp His Arg Glu Tyr Leu Leu Arg Phe Glu Arg Asp Leu
65 70 75 80
Gln Ser Ile Asn Pro Glu Val Thr Leu Pro Tyr Trp Glu Trp Glu Thr
85 90 95
Asp Ala Gln Met Gln Asp Pro Ser Gln Ser Gln Ile Trp Ser Ala Asp
100 105 110
Phe Met Gly Gly Asn Gly Asn Pro Ile Lys Asp Phe Ile Val Asp Thr
115 120 125
Gly Pro Phe Ala Ala Gly Arg Trp Thr Thr Ile Asp Glu Gln Gly Asn
130 135 140
Pro Ser Gly Gly Leu Lys Arg Asn Phe Gly Ala Thr Lys Glu Ala Pro
145 150 155 160
Thr Leu Pro Thr Arg Glu Asp Val Leu Asn Ala Leu Lys Ile Thr Gln
165 170 175
Tyr Asp Thr Pro Pro Trp Asp Met Thr Ser Gln Asn Ser Phe Arg Asn
180 185 190
Gln Leu Glu Gly Phe Ile Asn Gly Pro Gln Leu His Asp Arg Val His
195 200 205
Arg Trp Val Gly Gly Gln Met Gly Val Val Pro Thr Ala Pro Asn Asp
210 215 220
Pro Val Phe Phe Leu His His Ala Asn Val Asp Arg Ile Trp Ala Val
225 230 235 240
Trp Gln Ile Ile His Arg Asn Gln Asn Tyr Gln Pro Met Lys Asn Gly
245 250 255
Pro Phe Gly Gln Asn Phe Arg Asp Pro Met Tyr Pro Trp Asn Thr Thr
260 265 270
Pro Glu Asp Val Met Asn His Arg Lys Leu Gly Tyr Val Tyr Asp Ile
275 280 285
Glu Leu Arg Lys Ser Lys Arg Ser Ser Leu Glu
290 295
<210> 7
<211> 299
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Met Ser Asn Lys Tyr Arg Val Arg Lys Asn Val Leu His Leu Thr Asp
1 5 10 15
Thr Glu Lys Arg Asp Phe Val Arg Thr Val Leu Ile Leu Lys Glu Lys
20 25 30
Gly Ile Tyr Asp Arg Tyr Ile Ala Trp His Gly Ala Ala Gly Lys Phe
35 40 45
His Thr Pro Pro Gly Ser Asp Arg Asn Ala Ala His Met Ser Ser Ala
50 55 60
Phe Leu Pro Trp His Arg Glu Tyr Leu Leu Arg Phe Glu Arg Asp Leu
65 70 75 80
Gln Ser Ile Asn Pro Glu Val Thr Leu Pro Tyr Trp Glu Trp Glu Thr
85 90 95
Asp Ala Gln Met Gln Asp Pro Ser Gln Ser Gln Ile Trp Ser Ala Asp
100 105 110
Phe Met Gly Gly Asn Gly Asn Pro Ile Lys Asp Phe Ile Val Asp Thr
115 120 125
Gly Pro Phe Ala Ala Gly Arg Trp Thr Thr Ile Asp Glu Gln Gly Asn
130 135 140
Pro Ser Gly Gly Leu Lys Arg Asn Phe Gly Ala Thr Lys Glu Ala Pro
145 150 155 160
Thr Leu Pro Thr Arg Asp Gly Val Leu Asn Ala Leu Lys Ile Thr Gln
165 170 175
Tyr Asp Thr Pro Pro Trp Asp Met Thr Ser Gln Asn Ser Phe Arg Asn
180 185 190
Gln Leu Glu Gly Phe Ile Asn Gly Pro Gln Leu His Asp Arg Val His
195 200 205
Arg Trp Val Gly Gly Gln Met Gly Val Val Pro Thr Ala Pro Asn Asp
210 215 220
Pro Val Phe Phe Leu His His Ala Asn Val Asp Arg Ile Trp Ala Val
225 230 235 240
Trp Gln Ile Ile His Arg Asn Gln Asn Tyr Gln Pro Met Lys Asn Gly
245 250 255
Pro Phe Gly Gln Asn Phe Arg Asp Pro Met Tyr Pro Trp Asn Thr Thr
260 265 270
Pro Glu Asp Val Met Asn His Arg Lys Leu Gly Tyr Val Tyr Asp Ile
275 280 285
Glu Leu Arg Lys Ser Lys Arg Ser Ser Leu Glu
290 295
<210> 8
<211> 299
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Met Ser Asn Lys Tyr Arg Val Arg Lys Asn Val Leu His Leu Thr Asp
1 5 10 15
Thr Glu Lys Arg Asp Phe Val Arg Thr Val Leu Ile Leu Lys Glu Lys
20 25 30
Gly Ile Tyr Asp Arg Tyr Ile Ala Trp His Gly Ala Ala Gly Lys Phe
35 40 45
His Thr Pro Pro Gly Ser Asp Arg Asn Ala Ala His Met Ser Ser Ala
50 55 60
Phe Leu Pro Trp His Arg Glu Tyr Leu Leu Arg Phe Glu Arg Asp Leu
65 70 75 80
Gln Ser Ile Asn Pro Glu Val Thr Leu Pro Tyr Trp Glu Trp Glu Thr
85 90 95
Asp Ala Gln Met Gln Asp Pro Ser Gln Ser Gln Ile Trp Ser Ala Asp
100 105 110
Phe Met Gly Gly Asn Gly Asn Pro Ile Lys Asp Phe Ile Val Asp Thr
115 120 125
Gly Pro Phe Ala Ala Gly Arg Trp Thr Thr Ile Asp Glu Gln Gly Asn
130 135 140
Pro Ser Gly Gly Leu Lys Arg Asn Phe Gly Ala Thr Lys Glu Ala Pro
145 150 155 160
Thr Leu Pro Thr Arg Asp Asp Val Leu Asn Ala Leu Lys Ile Thr Gln
165 170 175
Tyr Asp Thr Pro Pro Trp Asp Met Thr Ser Gln Asn Ser Phe Arg Asn
180 185 190
Gln Leu Glu Gly Phe Ile Asn Gly Pro Gln Leu His Asp Arg Val His
195 200 205
Arg Trp Val Gly Gly Gln Met Gly Val Val Pro Thr Ala Pro Asn Asp
210 215 220
Pro Val Phe Phe Leu His His Ala Asn Val Asp Arg Ile Trp Ala Val
225 230 235 240
Trp Gln Ile Ile His Arg Asn Gln Asn Tyr Gln Pro Met Lys Asn Gly
245 250 255
Pro Phe Gly Gln Asn Phe Arg Asp Pro Met Tyr Pro Trp Asn Thr Thr
260 265 270
Pro Glu Asp Val Met Asn His Arg Lys Leu Gly Tyr Val Tyr Asp Ile
275 280 285
Glu Leu Arg Lys Ser Lys Arg Ser Ser Leu Glu
290 295
<210> 9
<211> 299
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Met Ser Asn Lys Tyr Arg Val Arg Lys Asn Val Leu His Leu Thr Asp
1 5 10 15
Thr Glu Lys Arg Asp Phe Val Arg Thr Val Leu Ile Leu Lys Glu Lys
20 25 30
Gly Ile Tyr Asp Arg Tyr Ile Ala Trp His Gly Ala Ala Gly Lys Phe
35 40 45
His Thr Pro Pro Gly Ser Asp Arg Asn Ala Ala His Met Ser Ser Ala
50 55 60
Phe Leu Pro Trp His Arg Glu Tyr Leu Leu Arg Phe Glu Arg Asp Leu
65 70 75 80
Gln Ser Ile Asn Pro Glu Val Thr Leu Pro Tyr Trp Glu Trp Glu Thr
85 90 95
Asp Ala Gln Met Gln Asp Pro Ser Gln Ser Gln Ile Trp Ser Ala Asp
100 105 110
Phe Met Gly Gly Asn Gly Asn Pro Ile Lys Asp Phe Ile Val Asp Thr
115 120 125
Gly Pro Phe Ala Ala Gly Arg Trp Thr Thr Ile Asp Glu Gln Gly Asn
130 135 140
Pro Ser Gly Gly Leu Lys Arg Asn Phe Gly Ala Thr Lys Glu Ala Pro
145 150 155 160
Thr Leu Pro Thr Arg Glu Gly Val Leu Asn Ala Leu Lys Ile Thr Gln
165 170 175
Tyr Asp Thr Pro Pro Trp Asp Met Thr Ser Gln Asn Ser Phe Arg Asn
180 185 190
Gln Leu Glu Gly Phe Ile Asn Gly Pro Gln Leu His Asp Arg Val His
195 200 205
Arg Trp Val Gly Gly Gln Met Gly Val Val Pro Thr Ala Pro Asn Asp
210 215 220
Pro Val Phe Phe Leu His His Ala Asn Val Asp Arg Ile Trp Ala Val
225 230 235 240
Trp Gln Ile Ile His Arg Asn Gln Asn Tyr Gln Pro Met Lys Asn Gly
245 250 255
Pro Phe Gly Gln Asn Phe Arg Asp Pro Met Tyr Pro Trp Asn Thr Thr
260 265 270
Pro Glu Asp Val Met Asn His Arg Lys Leu Gly Tyr Val Tyr Asp Ile
275 280 285
Glu Leu Arg Lys Ser Lys Arg Ser Ser Leu Glu
290 295
<210> 10
<211> 299
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Met Ser Asn Lys Tyr Arg Val Arg Lys Asn Val Leu His Leu Thr Asp
1 5 10 15
Thr Glu Lys Arg Asp Phe Val Arg Thr Val Leu Ile Leu Lys Glu Lys
20 25 30
Gly Ile Tyr Asp Arg Tyr Ile Ala Trp His Gly Ala Ala Gly Lys Phe
35 40 45
His Thr Pro Pro Gly Ser Asp Arg Asn Ala Ala His Met Ser Ser Ala
50 55 60
Phe Leu Pro Trp His Arg Glu Tyr Leu Leu Arg Phe Glu Arg Asp Leu
65 70 75 80
Gln Ser Ile Asn Pro Glu Val Thr Leu Pro Tyr Trp Glu Trp Glu Thr
85 90 95
Asp Ala Gln Met Gln Asp Pro Ser Gln Ser Gln Ile Trp Ser Ala Asp
100 105 110
Phe Met Gly Gly Asn Gly Asn Pro Ile Lys Asp Phe Ile Val Asp Thr
115 120 125
Gly Pro Phe Ala Ala Gly Arg Trp Thr Thr Ile Asp Glu Gln Gly Asn
130 135 140
Pro Ser Gly Gly Leu Lys Arg Asn Phe Gly Ala Thr Lys Glu Ala Pro
145 150 155 160
Thr Leu Pro Thr Arg Glu Asp Val Leu Asn Ala Leu Lys Ile Thr Gln
165 170 175
Tyr Asp Thr Pro Pro Trp Asp Met Thr Ser Gln Asn Ser Phe Arg Asn
180 185 190
Gln Leu Glu Gly Phe Ile Asn Gly Pro Gln Leu His Asp Arg Val His
195 200 205
Arg Trp Val Gly Gly Gln Met Gly Val Val Pro Thr Ala Pro Asn Asp
210 215 220
Pro Val Phe Phe Leu His His Ala Asn Val Asp Arg Ile Trp Ala Val
225 230 235 240
Trp Gln Ile Ile His Arg Asn Gln Asn Tyr Gln Pro Met Lys Asn Gly
245 250 255
Pro Phe Gly Gln Asn Phe Arg Asp Pro Met Tyr Pro Trp Asn Thr Thr
260 265 270
Pro Glu Asp Val Met Asn His Arg Lys Leu Gly Tyr Ile Tyr Asp Ile
275 280 285
Glu Leu Arg Lys Ser Lys Arg Ser Ser Leu Glu
290 295
<210> 11
<211> 299
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Met Ser Asn Lys Tyr Arg Val Arg Lys Asn Val Leu His Leu Thr Asp
1 5 10 15
Thr Glu Lys Arg Asp Phe Val Arg Thr Val Leu Ile Leu Lys Glu Lys
20 25 30
Gly Ile Tyr Asp Arg Tyr Ile Ala Trp His Gly Ala Ala Gly Lys Phe
35 40 45
His Thr Pro Pro Gly Ser Asp Arg Asn Ala Ala His Met Ser Ser Ala
50 55 60
Phe Leu Pro Trp His Arg Glu Tyr Leu Leu Arg Phe Glu Arg Asp Leu
65 70 75 80
Gln Ser Ile Asn Pro Glu Val Thr Leu Pro Tyr Trp Glu Trp Glu Thr
85 90 95
Asp Ala Gln Met Gln Asp Pro Ser Gln Ser Gln Ile Trp Ser Ala Asp
100 105 110
Phe Met Gly Gly Asn Gly Asn Pro Ile Lys Asp Phe Ile Val Asp Thr
115 120 125
Gly Pro Phe Ala Ala Gly Arg Trp Thr Thr Ile Asp Glu Gln Gly Asn
130 135 140
Pro Ser Gly Gly Leu Lys Arg Asn Phe Gly Ala Thr Lys Glu Ala Pro
145 150 155 160
Thr Leu Pro Thr Arg Glu Gly Val Leu Asn Ala Leu Lys Ile Thr Gln
165 170 175
Tyr Asp Thr Pro Pro Trp Asp Met Thr Ser Gln Asn Ser Phe Arg Asn
180 185 190
Gln Leu Glu Gly Phe Ile Asn Gly Pro Gln Leu His Asp Arg Val His
195 200 205
Arg Trp Val Gly Gly Gln Met Gly Val Val Pro Thr Ala Pro Asn Asp
210 215 220
Pro Val Phe Phe Leu His His Ala Asn Val Asp Arg Ile Trp Ala Val
225 230 235 240
Trp Gln Ile Ile His Arg Asn Gln Asn Tyr Gln Pro Met Lys Asn Gly
245 250 255
Pro Phe Gly Gln Asn Phe Arg Asp Pro Met Tyr Pro Trp Asn Thr Thr
260 265 270
Pro Glu Asp Val Met Asn His Arg Lys Leu Gly Tyr Ile Tyr Asp Ile
275 280 285
Glu Leu Arg Lys Ser Lys Arg Ser Ser Leu Glu
290 295
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
taatacgact cactataggg 20
<210> 13
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gctagttatt gctcagcgg 19
Claims (10)
1. A tyrosinase mutant, characterized in that: the amino acid sequence shown in SEQ ID NO.1 is mutated, and the mutated amino acid position comprises one or more of N205D, D166E, D167G and V285I.
2. The mutant according to claim 1, characterized in that: the mutation mode comprises any one of the following 10 types:
N205D; D166E; D167G; V285I; N205D and D166E; N205D and D167G; N205D and V285I; N205D and D166E and D167G; N205D and D166E and V285I; N205D and D166E and D167G and V285I.
3. The mutant according to claim 2, characterized in that: the mutation mode comprises any one of the following 7 types:
N205D; N205D and D166E; N205D and D167G; N205D and V285I; N205D and D166E and D167G; N205D and D166E and V285I; N205D and D166E and D167G and V285I.
4. The mutant according to claim 2, characterized in that: the sequence is shown in SEQ ID NO.2-11 in sequence.
5. A nucleotide encoding the tyrosinase mutant of any one of claims 1-4.
6. Use of the tyrosinase mutant according to any one of claims 1-4, wherein the theaflavins are catalytically synthesized under aerobic conditions using catechins as substrates.
7. Use according to claim 6, the catechin primary substrates comprising: epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate, wherein the reaction substrate and the synthesis product are any one of the following substances:
epicatechin + epigallocatechin producing theaflavin TF 1;
epicatechin + epigallocatechin gallate to theaflavin-3-gallate TF2 a;
epicatechin gallate + epigallocatechin to produce theaflavin-3' -gallate TF2 b;
the epicatechin gallate and the epigallocatechin gallate form theaflavin digallate TF 3.
8. Use according to claim 7, characterized in that: in the reaction system, the activity of the tyrosinase mutant is 200-500U/L, the EC, the EGC and the EGCG are all 10-100mM, and the ECG is 2-10 mM.
9. Use according to claim 7, characterized in that: the reaction temperature is 25-35 ℃, the reaction pH is 4.0-6.0, the reaction stirring speed is 150-.
10. Use according to claim 7, characterized in that: the tyrosinase mutant is an immobilized tyrosinase mutant.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113584008A (en) * | 2021-07-30 | 2021-11-02 | 湖南福来格生物技术有限公司 | Penicillin G acylase mutant and application thereof |
| CN115029326A (en) * | 2022-05-27 | 2022-09-09 | 湖南福来格生物技术有限公司 | Enzyme cross-linked aggregate and preparation method and application thereof |
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| WO2012027282A2 (en) * | 2010-08-23 | 2012-03-01 | Codexis, Inc. | Recombinant lignocellulose degradation enzymes for the production of soluble sugars from cellulosic biomass |
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| CN111004785A (en) * | 2019-12-16 | 2020-04-14 | 华中科技大学 | Tyrosinase protein sequence and application thereof in preparation of tyrosinase |
| CN111218432A (en) * | 2018-11-27 | 2020-06-02 | 中国科学院大连化学物理研究所 | Tyrosinase precursor, encoding gene, preparation and application thereof |
| CN111235126A (en) * | 2020-04-01 | 2020-06-05 | 湖南福来格生物技术有限公司 | S-adenosylmethionine synthetase mutant and preparation method using same |
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| WO1991014698A1 (en) * | 1990-03-21 | 1991-10-03 | University Of Maryland At College Park | Marine tyrosinase gene |
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