A recombinant tyrosinase mutant and its application

Abstract

The present invention discloses a recombinant tyrosinase mutant and its application, belonging to the field of bioengineering. The present invention obtains tyrosinase BM Tyr from the Bacillus megaterium genome by molecular biological means, and mutates one or more of the amino acids at positions 218, 215, and 209 of the recombinant tyrosinase BM Tyr to obtain a recombinant tyrosinase mutant. The activity of the recombinant tyrosinase BM Tyr mutant oxidizing epicatechin gallate is increased by 1.14 to 6.26 times relative to the recombinant tyrosinase BM Tyr, and the recombinant tyrosinase BM Tyr mutant exhibits a higher activity of epicatechin gallate. The method of the present invention is adopted to improve the production capacity of the unit catalyst and the reaction efficiency of the catalyst, reduce the cost of the reaction, and accelerate the industrialization process of producing theaflavin-3,3'-digallic acid by enzyme conversion.

Description

Technical Field

[0001] The invention relates to a recombinant tyrosinase mutant and application thereof, belonging to the technical field of bioengineering. Background Art

[0002] Theaflavins (TFs) were first isolated and discovered from black tea by Roberts EAH in 1957. They are a class of compounds that are formed by the oxidation and condensation of polyphenols, are soluble in ethyl acetate, are orange-yellow in color, and have a benzophenone structure. At present, nearly 30 TFs have been discovered and structurally identified, among which the main components are: theaflavin (TF), theaflavin-3-gallate (TF-3-G), theaflavin-3'-monogallate (TF-3'-G) and theaflavin-3,3'-digallate (TF-3,3'-G). The content of theaflavins in black tea is generally 0.3% to 1.5%. They are the main ingredients in black tea and play a decisive role in the color, aroma and quality of black tea. They can make the color of black tea "bright" and have a strong taste and freshness. They are known as the "soft gold" in tea and are highly safe. Theaflavins are not only an important quality component of black tea, but also have a variety of physiological activities that are beneficial to human health, such as anti-oxidation, anti-cancer, lowering blood lipids, preventing cardiovascular diseases, anti-diabetes, antibacterial, antiviral, etc. In addition, theaflavins are also natural colorants for cosmetics and food. Therefore, they have received widespread attention at home and abroad and have become a research hotspot.

[0003] At present, there are roughly three methods for preparing theaflavins: extraction, chemical oxidation, and enzymatic oxidation. Extraction method: Due to the low content of theaflavins in black tea, it is difficult to directly extract and purify theaflavins from black tea, and the yield is low; Chemical oxidation method: It requires the use of a large amount of potassium ferrocyanide, ferric oxide, magnesium oxide, acid and alkali and other chemical reagents, which seriously pollutes the environment and requires more sewage treatment costs. In addition, due to the lack of substrate specificity of the chemical method, the by-products are complex, and the purity and safety of the product are limited; Enzymatic oxidation method has mild reaction conditions and strong controllability, which can avoid the disadvantages of the above two methods. In vitro enzymatic oxidation is a more suitable method. However, TFs are generated during tea fermentation, which is formed by enzymatic oxidation and condensation of catechins by polyphenol oxidase (PPO) of cells. However, the amount of naturally generated enzymes is extremely low and is greatly affected by seasonal changes, so it is difficult to obtain polyphenol oxidase directly from plants on a large scale. A large number of studies have shown that industrial production of TFs by obtaining exogenous enzymes through protein engineering can not only increase the yield of TFs but also solve the problem of seasonal limitations.

[0004] However, among the polyphenol oxidases (laccase, catechol oxidase and tyrosinase), tyrosinase (EC1.14.18.1) is particularly important due to its wide substrate selectivity. Tyrosinases can widely catalyze the oxidation of polyphenols, aminophenols, methoxyphenols, aromatic amines, inorganic or organic metal compounds, etc. They are activated when combined with molecular oxygen and can catalyze the monophenolase reaction cycle or the diphenolase reaction cycle. The enzyme binds to an external diphenol molecule (such as L-dopa) and oxidizes it to o-quinone released with a water molecule, placing the enzyme in an intermediate transition state. The enzyme then binds to a second diphenol molecule and repeats the process, ending in a deoxygenated state. Theaflavins are prepared by in vitro enzymatic oxidation of tyrosinase, protein engineering of the selected parent tyrosinase is performed to improve regioselectivity and optimize conversion conditions, in order to solve the source and cost problems of polyphenol oxidase and increase the yield of theaflavins. Summary of the invention

[0005] The present invention provides a recombinant tyrosinase BM Tyr mutant, wherein the amino acid sequence of a parent enzyme of the recombinant tyrosinase BM Tyr mutant is shown as SEQ ID NO.1, and the nucleotide sequence thereof is shown as SEQ ID NO.2.

[0006] The parent enzyme of the recombinant tyrosinase BM Tyr mutant is tyrosinase BM Tyr derived from Bacillus megatherium.

[0007] The present invention also provides a tyrosinase BM Tyr variant, wherein the mutant is obtained by mutating one or more of the amino acids at positions 218, 215 and 209 of the tyrosinase BM Tyr parent enzyme having an amino acid sequence as shown in SEQ ID NO.1.

[0008] The numbering of the mutation sites at positions 218, 215 and 209 described in the present invention is based on the original source sequence of the recombinant tyrosinase BM Tyr (amino acid sequence such as SEQ ID NO.1).

[0009] In one embodiment of the present invention, the mutant is any one of the following (a) to (f):

[0010] (a) The mutant is obtained by mutating the valine at position 218 of tyrosinase BM Tyr as shown in the amino acid sequence of SEQ ID NO.1 to asparagine, and the mutant is named V218N;

[0011] (b) The mutant is obtained by mutating the methionine at position 215 of tyrosinase BM Tyr as shown in the amino acid sequence of SEQ ID NO.1 to glutamic acid, and the mutant is named M215E;

[0012] (c) The mutant is obtained by mutating the arginine at position 209 of tyrosinase BM Tyr as shown in the amino acid sequence of SEQ ID NO.1 to serine, and the mutant is named R209S;

[0013] (d) The mutant is obtained by mutating the valine at position 218 of the tyrosinase BM Tyr as shown in the amino acid sequence of SEQ ID NO.1 to asparagine, and mutating the methionine at position 215 to glutamic acid, and the mutant is named V218N / M215E;

[0014] (e) The mutant is obtained by mutating the valine at position 218 of the tyrosinase BM Tyr as shown in the amino acid sequence of SEQ ID NO.1 to asparagine, and mutating the arginine at position 209 to serine, and the mutant is named V218N / R209S;

[0015] (f) The mutant is obtained by mutating the valine at position 218 of the tyrosinase BM Tyr as shown in the amino acid sequence of SEQ ID NO.1 to asparagine, the methionine at position 215 to glutamic acid, and the arginine at position 209 to serine. The mutant is named V218N / M215E / R209S.

[0016] In one embodiment of the present invention, the nucleotide sequence of the recombinant tyrosinase BM Tyr is shown as SEQ ID NO.2.

[0017] The present invention also provides a gene encoding the tyrosinase BM Tyr mutant.

[0018] The invention also provides a recombinant vector carrying the gene.

[0019] In one embodiment of the present invention, the recombinant vector uses PET-28a as an expression vector.

[0020] The present invention also provides a recombinant cell expressing the mutant, or carrying the gene, or carrying the recombinant vector.

[0021] In one embodiment of the present invention, the recombinant cell uses bacteria or fungi as expression hosts.

[0022] In one embodiment of the present invention, the recombinant cell uses Escherichia coli BL21 (DE3) as an expression host.

[0023] The present invention also provides a recombinant Escherichia coli, wherein the recombinant Escherichia coli expresses the recombinant tyrosinase BM Tyr mutant.

[0024] In one embodiment of the present invention, the recombinant Escherichia coli uses Escherichia coli BL21 (DE3) as an expression host and pET28a as an expression vector.

[0025] The present invention also provides a method for obtaining the above-mentioned tyrosinase BM Tyr mutant, the method comprising the following steps:

[0026] (1) Determining the mutation site based on the amino acid sequence of recombinant tyrosinase BM Tyr; designing mutation primers for site-directed mutagenesis, and performing site-directed mutagenesis using a vector carrying the recombinant tyrosinase BM Tyr gene as a template; and constructing a plasmid vector containing the mutant;

[0027] (2) transforming the plasmid vector containing the mutant into a host cell;

[0028] (3) Selecting positive clones, fermenting and culturing the obtained recombinant cells, preparing tyrosinase BM Tyr mutants, and purifying tyrosinase BM Tyr mutants.

[0029] In one embodiment of the present invention, the host cell is Escherichia coli.

[0030] The present invention also provides a method for preparing theaflavin-3,3'-digallic acid, which comprises:

[0031] The above-mentioned recombinant tyrosinase BM Tyr mutant, or the above-mentioned recombinant cells are crushed and centrifuged to collect the supernatant, or the above-mentioned recombinant Escherichia coli is crushed and centrifuged to collect the supernatant, and added to a reaction system containing epigallocatechin 3-gallate and epicatechin gallate to react and prepare.

[0032] In one embodiment of the present invention, the recombinant cells, or the supernatant collected after the above-mentioned recombinant Escherichia coli is crushed and centrifuged, that is, the recombinant tyrosinase BM Tyr mutant enzyme solution, is added to the reaction system at a final concentration of 4 to 10 μg / mL.

[0033] In one embodiment of the present invention, the recombinant tyrosinase BM Tyr mutant enzyme solution is added to the reaction system at a final concentration of 5 μg / mL.

[0034] In one embodiment of the present invention, the Cu added to the recombinant Escherichia coli supernatant 2+ Final concentration: 0.1 mM.

[0035] In one embodiment of the present invention, the final concentration of epigallocatechin 3-gallate in the reaction system is 3 g / L.

[0036] In one embodiment of the present invention, the final concentration of epicatechin gallate in the reaction system is 2 g / L.

[0037] In one embodiment of the present invention, the reaction conditions are: pH 5.5-8.0, 30-50° C., and reaction time of 10-60 min.

[0038] The present invention also provides the use of the above-mentioned recombinant tyrosinase BM Tyr mutant, or the above-mentioned gene, or the above-mentioned recombinant vector, or the above-mentioned recombinant cell, or the above-mentioned recombinant Escherichia coli in the preparation of theaflavin-3,3'-digallic acid, or products containing theaflavin-3,3'-digallic acid.

[0039] In one embodiment of the present invention, the product is a chemical.

[0040] Beneficial Effects

[0041] (1) The present invention provides a recombinant tyrosinase BM Tyr and a mutant thereof for catalyzing the production of theaflavin-3,3'-digallate. The recombinant tyrosinase BM Tyr of the present invention achieves soluble expression in Escherichia coli BL21 (DE3), and the epicatechin gallate activity is increased by 5.02 times compared with the control group.

[0042] (2) The activity of the recombinant tyrosinase BM Tyr mutant in oxidizing epicatechin gallate is 1.14 to 6.26 times higher than that of the recombinant tyrosinase BM Tyr, and the recombinant tyrosinase BM Tyr mutant exhibits a higher activity of epicatechin gallate. The space-time yield of theaflavin-3,3'-digallic acid reaches 36.01 g / L / d and the conversion rate is 47.8% after reacting for 0.5 h at 30°C and pH 5.0. The method of the present invention improves the production capacity of the unit catalyst and the reaction efficiency of the catalyst, reduces the cost of the reaction, and accelerates the industrialization process of theaflavin-3,3'-digallic acid production by enzyme conversion. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 : SDS-PAGE of tyrosinase BM Tyr induced expression from Bacillus megatherium; Lane M refers to low molecular weight protein marker; Lanes 1 to 3 are the band sizes of the target protein in whole cells, precipitate and supernatant after induced expression at 0.2 mM IPTG concentration at 25°C.

[0044] Figure 2 : Conversion rate of each mutant into theaflavin-3,3'-digallic acid.

[0045] Figure 3A : Chromatograms of epigallocatechin 3-gallate and epicatechin gallate standards.

[0046] Figure 3B : Chromatogram of theaflavin-3,3'-digallic acid standard.

[0047] Figure 3C :Chromatogram of the conversion of theaflavin-3,3'-digallate by tyrosinase BM Tyr. DETAILED DESCRIPTION

[0048] The pET-28a(+) mass involved in the following examples was purchased from Novagen (Madison, WI, USA), restriction endonucleases, T4 DNA ligase, primeSTAR, etc. were purchased from TaKaRa (Dalian, China). Standard products epigallocatechin-3-gallate, epicatechin gallate, and theaflavin-3,3'-digallate were purchased from Sigma-Aldrich and Aladdin, USA, and the remaining reagents were purchased from the market.

[0049] The culture medium involved in the following examples is as follows:

[0050] LB liquid medium: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, sterilized at 121°C for 20 min.

[0051] LB solid medium: Add 2% agar to LB liquid medium.

[0052] TB liquid culture medium: KH2PO4 2.31 g / L, K2HPO4·3H2O 16.42 g / L, yeast powder 24 g / L, peptone 12 g / L, glycerol 4 g / L.

[0053] Prepare pH 5.0 sodium hydrogen phosphate-citric acid buffer: 0.2 mol / L sodium hydrogen phosphate and 0.1 mol / L citric acid mixed buffer. For the specific formula, see "Industrial Microbiology Experimental Technology Manual" (China Light Industry Press, edited by Zhuge Jian)

[0054] The detection methods involved in the following embodiments are as follows:

[0055] Detection of tyrosinase BM Tyr enzyme activity:

[0056] Epigallocatechin-3-gallate and epicatechin-3-gallate were dissolved in sodium hydrogen phosphate-citric acid buffer to prepare a 20 g / L solution. In the reaction system, the final concentrations of epigallocatechin-3-gallate and epicatechin-3-gallate were 3 g / L and 2 g / L, respectively. After mixing, 20 μL of 0.1 mM Cu 2+ The enzyme solution was shaken at 30°C and 400 rpm for 5 min, and then 50 μL of 2M trichloroacetic acid was added to terminate the reaction. The solution was centrifuged at 12000 rpm for 5-10 min and passed through a 0.22 μm organic membrane for HPLC analysis.

[0057] Enzyme activity is defined as: the amount of enzyme required to generate 1 μmol theaflavin-3,3'-digallate per minute at 30°C using epigallocatechin-3-gallate and epicatechin-3-gallate as reaction substrates is defined as one enzyme activity unit, recorded as U·mL -1 .

[0058] Detection of Epigallocatechin-3-gallate, Epigallocatechin-3-gallate, Theaflavins-3,3'-digallate:

[0059] The effect of disodium hydrogen phosphate-citrate buffer as the reaction medium on the conversion of epigallocatechin 3-gallate and epicatechin gallate to theaflavin-3,3'-digallate by tyrosinase. The final concentrations of the converted substrates epigallocatechin 3-gallate and epicatechin gallate were 3g / L and 2g / L, respectively. 20μL of enzyme solution containing 0.1mM Cu2+ was shaken at 30℃ and 400rpm for 5min. After the reaction, 50μL of 2M trichloroacetic acid was added to terminate the reaction. The reaction was centrifuged at 12000rpm for 5-10min and passed through a 0.22μm organic membrane for HPLC analysis.

[0060] The above specific high performance liquid chromatography HPLC analysis method is:

[0061] by 100DIOL (5μm, 250×4mm) was used as the chromatographic column, and 2% acetic acid-water (v / v) (A) and acetonitrile (B) that had been filtered and ultrasonically degassed were used as the mobile phases. The column temperature was 25℃; the flow rate was 1.0mL / min; the injection volume was 5μL; the detection wavelength was 278nm; the gradient elution was: 0-5min, 30% B; 5-8min, 30-34% B; 8–11min, 34–38% B; 11–13min, 38–30% B; 13-18min, 30% B, and the sample processing time was 15min. Under these detection conditions, the retention times of epigallocatechin-3-gallate, epicatechin gallate, and theaflavins-3,3'-digallate were 3.062min, 3.561min, and 5.525min, respectively.

[0062] Example 1: Expression and purification of BM Tyr

[0063] Construction of genetically engineered bacteria and protein expression:

[0064] F1: ggtcgcggatccgaattcATGAGTAACAAGTAT and R1: agcttgtcgacggagctcTTATGAGGAACG were used as primers, and the underlines are the EcoR I and Sac I restriction enzyme sites, respectively.

[0065] The nucleotide sequence of the target protein encoding gene in Bacillus megatherium disclosed in NCBI (shown in SEQ ID NO.2) was used as a template, and the amplification conditions were 95°C for 5 min, 29 cycles (98°C for 10 s, 55°C for 15 s, 72°C for 1.5 min), and 72°C for 5 min to obtain the cDNA sequence of the Tyr gene coding region. After the PCR product was recovered, it was connected with the pET-28a(+) plasmid vector that had been digested with the same double enzymes through homologous recombination to obtain the recombinant expression plasmid pET-28a(+)-BM Tyr. The recombinant plasmid pET-28a(+)-BM Tyr was transformed into E. coli BL21(DE3). After PCR identification, the positive engineering bacteria were named E. coli BL21 / pET-28a(+)-BM Tyr.

[0066] The engineered bacteria E.coli BL21 / pET-28a(+)-BM Tyr were inoculated into LB liquid medium, cultured at 37°C for 12 hours to obtain seed solution, and the seed solution was inoculated into fresh TB liquid medium at a 5% (v / v) inoculation amount. After culturing at 37°C for 2 hours, IPTG with a final concentration of 0.2mM was added and cultured at 25°C for 14 hours to induce the expression of the recombinant target protein. 150mL of the induced fermentation broth was collected by centrifugation at 6000r / min. The results are as follows: Figure 1 shown.

[0067] The results showed that the target protein was expressed in whole cells, supernatant and precipitate, and the band sizes were the same.

[0068] Example 2: Preparation of theaflavin-3,3'-digallic acid

[0069] (1) Preparation of crude enzyme solution

[0070] The strain E.coli BL21 / pET-28a(+)-BM Tyr preserved in the glycerol tube was spread on LB solid culture medium and cultured at 37°C until a single clone grew out. The single clone was picked out and transferred to fresh LB liquid culture medium. The culture was carried out at 200 rpm and 37°C for 12 h to obtain a seed solution. The seed solution was inoculated into fresh TB liquid culture medium at an inoculation rate of 5% (v / v). After culturing at 37°C for 2 h, IPTG with a final concentration of 0.2 mM was added, and the culture was induced at 25°C for 14 h to prepare a fermentation broth.

[0071] The fermentation broth was centrifuged at 4°C and 6000 rpm for 10 min to obtain the bacterial cells. 10 mL of binding solution A (20 mM sodium phosphate, 0.5 mM NaCl, 20 mM imidazole, 1% glycerol, pH adjusted to 8.5 with HCl) was added to fully resuspend the bacterial cells, and then the centrifuge tube was placed in an ice bath and placed in an ultrasonic cell disruptor. The ultrasonic disruption conditions were: working time 4 s, interval time 4 s, a total of 10 min, to obtain a disrupted solution.

[0072] The obtained broken liquid was subjected to low temperature high speed centrifugation at 4°C and 8000 rpm for 30 min to obtain crude enzyme solution. Filtered with a 0.22 μm microporous membrane, 0.1 mM Cu 2+ spare.

[0073] (2) Preparation of theaflavin-3,3'-digallic acid

[0074] Using disodium hydrogen phosphate-citrate buffer as the reaction medium, tyrosinase was used to convert epigallocatechin-3-gallate and epicatechin gallate to produce theaflavin-3,3'-digallate.

[0075] To the reaction system containing sodium hydrogen phosphate-citrate buffer, add the substrate epigallocatechin-3-gallate at a final concentration of 3 g / L and the substrate epicatechin-3-gallate at a final concentration of 2 g / L, and add 20 μL of 0.1 mM Cu 2+ The enzyme solution was shaken at 30°C and 400 rpm for 5 min. After the reaction, 50 μL of 2M trichloroacetic acid was added to terminate the reaction. The solution was centrifuged at 12000 rpm for 5-10 min and passed through a 0.22 μm organic membrane for HPLC analysis. Figure 3A ~C).

[0076] The results showed that the space-time yield of theaflavin-3,3'-digallic acid was 5.8 g / L / d, and the conversion rate was 7.7%.

[0077] Example 3: Enzyme expression, purification and enzyme activity verification

[0078] The specific steps are as follows:

[0079] (1) Preparation of crude enzyme solution

[0080] The strain E.coli BL21 / pET-28a(+)-BM Tyr preserved in the glycerol tube was spread on LB solid culture medium and cultured at 37°C until a single clone grew out. The single clone was picked out and transferred to fresh LB liquid culture medium. After cultured at 200 rpm and 37°C for 12 h, a seed solution was obtained. The seed solution was inoculated into fresh TB liquid culture medium at an inoculum amount of 5% (v / v). After cultured for 2 h, IPTG with a final concentration of 0.2 mM was added, and the culture was induced at 25°C for 14 h. After the culture was completed, the fermentation liquid was collected.

[0081] Centrifuge the fermentation broth at 4°C and 6000rpm for 10 minutes to obtain the bacteria. Add 10mL of binding solution A (20mM sodium phosphate, 0.5mM NaCl, 20mM imidazole, 1% glycerol, adjust pH to 8.5 with HCl) to fully resuspend the bacteria, then place the centrifuge tube in an ice bath and put it into an ultrasonic cell disruptor. The conditions for ultrasonic disruption are: working time 4s, interval time 4s, total 10min. Perform low-temperature high-speed centrifugation on the obtained disrupted liquid, centrifuge at 4°C and 8000rpm for 30min to obtain a crude enzyme solution. Filter with a 0.22μm microporous filter membrane and add 0.1mM Cu 2+ spare.

[0082] (2) Purification of tyrosinase BM Tyr

[0083] Prepare the nickel ion affinity chromatography column. First, use a constant flow pump at 4°C to pump ultrapure water into the column to rinse the column (about 6 to 12 times the column volume), and then balance the column environment with 10mL of binding solution A. When the effluent at the bottom of the column is consistent with the pH value of the low salt concentration buffer pumped into the column (about 5 times the column volume buffer), add the obtained membrane-passed crude enzyme solution to the column. First, rinse the impurities with binding solution A to the baseline balance, and then elute with eluent B (20mM sodium phosphate, 0.5mM NaCl, 500mM imidazole). Collect the eluate of the absorption peak, determine the enzyme activity, and obtain the target protein that is electrophoretically pure.

[0084] The enzyme activity of the purified BM Tyr pure enzyme solution was tested, and the BM Tyr enzyme activity was 13.37 U·mL -1 .

[0085] Example 4: Construction and screening of single mutants

[0086] The specific steps are as follows:

[0087] Using the pET-28a(+)-BM Tyr constructed in Example 2 as a template, BM Tyr V218N BM Tyr M215E BM Tyr R209S BM TyrR191E The primers for the mutation sites are shown in Table 1 , and mutants were constructed by whole plasmid PCR.

[0088] Table 1: Single mutant mutagenesis primer sequences

[0089]

[0090] Construct the PCR amplification system: 0.5 μL of PrimSTAR enzyme, 10 μL of 5× PrimeSTAR Buffer, 4 μL of dNTP, 1 μL of each primer for each mutation site, and 1 μL of template (Sr MBP PLD wt )4μL, water 32.5μL;

[0091] The reaction conditions are: ① 94℃ for 3 min; ② 98℃ for 10 s; ③ 55℃ for 30 s; ④ 72℃ for 3 min; ⑤ cycle the three steps ② to ④ for 29 times; ⑥ 72℃ for 5 min; ⑦ keep warm at 12℃.

[0092] The reaction system was incubated at 37°C for 3 h to digest the plasmid template (the digestion system was: DpnI 0.5 μL, PCR product 45 μL, 10×T Buffer 5 μL). The digestion product obtained after digestion was introduced into Escherichia coli BL21 competent cells by chemical transformation.

[0093] The specific steps of the chemical transformation method are as follows: (1) Introduce 10 μL of homologous recombination product into 100 μL of E. coli BL21 (DE3) competent cells; (2) Ice bath for 15-30 minutes; (3) Heat shock at 42°C water bath for 90 seconds, take out and quickly put in ice bath for 3-5 minutes; (4) Add 800 μL of non-resistant LB medium and mix well, and culture at 37°C, 200 rpm for 1 hour; (5) Centrifuge at 5000 rpm for 2 minutes to collect bacteria; (6) Remove the supernatant, and apply the remaining 100-200 μL of aspiration and mix well to the LB resistance plate containing 0.05 mg / mL kanamycin, and culture at 37°C for about 12 hours. (7) Pick a single clone and culture it in LB containing 0.05 mg / mL kanamycin resistance, 200 rpm, 37°C for 12 hours, and then send it to the company for sequencing. The one with correct sequencing is the positive transformant.

[0094] The genetically engineered bacteria E. coli BL21 / pET-28a(+)-BM Tyr V218N , E.coli BL21 / pET-28a(+)-BM Tyr M215E , E.coli BL21 / pET-28a(+)-BM Tyr R209S, E.coli BL21 / pET-28a(+)-BM Tyr R191E .

[0095] Subsequently, the obtained genetically engineered bacteria were transformed according to the method of Example 2 to prepare theaflavin-3,3'-digallic acid, and better mutants were screened. The results are shown in Table 2 and Figure 2 Shown

[0096] Table 2: Conversion rates of theaflavin-3,3'-digallate from single mutants

[0097]

[0098] The results showed that the engineered bacteria E. coli BL21 / pET-28a(+)-BM Tyr V218N , E.coli BL21 / pET-28a(+)-BM Tyr M215E , E.coli BL21 / pET-28a(+)-BM Tyr R209S The effect of generating theaflavin-3,3'-digallic acid was relatively good, which was 1.14-1.30 times that of wild-type E. coli BL21 / pET-28a(+)-BM Tyr.

[0099] Example 5: Construction and screening of double mutants and triple mutants

[0100] (1) Construction of double mutants:

[0101] The double mutants of this example were constructed by whole plasmid PCR based on the corresponding single mutants using the primers in Table 3. V218N Based on the results, the double mutant E. coli BL21 / pET-28a(+)-BMTyr was cloned by whole plasmid PCR using the mutation primers R209S-F and R209S-R (Table 3). V218N / 209S Build.

[0102] Table 3 Double mutant mutation primer sequences

[0103]

[0104] The preparation method of the genetically engineered bacteria is as shown in step (1) of Example 2. The primers used are shown in Table 3. According to the method of Example 4, the genetically engineered bacteria containing double mutants were prepared: E. coli BL21 / pET-28a(+)-BM Tyr V218N / 209S , E.coli BL21 / pET-28a(+)-BM Tyr V218N / M215E, E.coli BL21 / pET-28a(+)-BM Tyr V218N / R191E .

[0105] (2) Screening of double mutants:

[0106] The mutant strain with correct sequencing was inoculated into LB seed medium and cultured at 200 rpm and 37 °C for 10 h. The seed solution was inoculated into fresh TB liquid medium at a 5% (v / v) inoculation rate and cultured at 200 rpm and 37 °C until OD 600 = about 0.8, add IPTG with a final concentration of 0.2mM for induction, and the induction conditions are 200rpm, 25℃ for 14h.

[0107] Then, the obtained genetically engineered bacteria were transformed according to the method of Example 2 to prepare theaflavin-3,3'-digallic acid.

[0108] After the reaction was completed, the yield of theaflavin-3,3'-digallic acid was determined by HPLC. The results are shown in Table 4.

[0109] Table 4: Conversion rate of theaflavin-3,3'-digallate in double mutants

[0110]

[0111] The results showed that the mutant BM Tyr V218N / M215E Genetically engineered bacteria and mutant BM Tyr V218N / M215E The genetically engineered bacteria had the best effect, with the conversion rates of theaflavin-3,3'-digallic acid reaching 29.9% and 36.7%, respectively.

[0112] (3) Construction of triple mutants:

[0113] In mutant BM Tyr V218N / M215E On the basis of the above, the triple mutant was constructed by whole plasmid PCR using the mutation primers R209S-F and R209S-R (Table 3). The specific implementation method is shown in step (1) of Example 2. The primers used are shown in Table 3. According to the method of Example 4, a triple mutant BM Tyr V218N / M215E / R209S Genetically engineered bacteria: E.coliBL21 / pET-28a(+)-BM Tyr V218N / M215E / R209S .

[0114] According to the method of Example 2, theaflavin-3,3'-digallic acid was prepared by transformation. After the reaction, the yield of theaflavin-3,3'-digallic acid was determined by HPLC. V218N / M215E / R209SThe spatiotemporal yield of theaflavin-3,3'-digallic acid prepared by genetically engineered bacteria reached 36.01 g / L / d, and the conversion rate was 47.8%.

[0115] (4) Detection of mutant enzyme activity

[0116] The beneficial mutants obtained in Examples 4 and 5 were tested for enzyme activity. For details, see Example 3. The enzyme activity of the purified pure enzyme solution of each mutant was tested, and the results are shown in Table 5.

[0117] Table 5: Enzyme activity data of beneficial mutants

[0118]

[0119]

[0120] The results showed that the best triple mutant BM Tyr V218N / M215E / R209S The enzyme activity was 83.06 U·mL -1 , relative to wild-type BM Tyr (13.37 U·mL -1 ) increased by 6.26 times.

[0121] Example 6: Performance determination of parent enzyme and mutants

[0122] (1) Determination of kinetic parameters

[0123] In order to evaluate the beneficial mutants, the present invention measured the kinetic parameters of the mutant parent Q0 and mutants Q1 to Q6 (specific meanings are shown in Table 7) at 30°C. cat / K m The initial rate of quinones produced by oxidizing epicatechin gallate at different concentrations at 30°C was calculated. For specific implementation steps, see the literature Comparison of Spectrophotometric assay methods for theaflavins and thearubigins in black tea, [J]. Journal of Tea, 1995, 16 (1): 41-47. and Biochemistry (Higher Education Press, edited by Wang Jingyan). The results are shown in Table 7.

[0124] (2) Detection of specific enzyme activity

[0125] According to the detection method of relative enzyme activity (enzyme activity detection divided by enzyme mass), the relative enzyme activity data of BM Tyr parent enzyme and its mutant were detected respectively. The results are shown in Table 6.

[0126] Table 6: Kinetic parameters of BM Tyr parent enzyme and its mutants

[0127]

[0128] The results showed that the mutant K m The values ​​of the triple mutants were lower than those of WT. m The lowest, indicating that the triple mutant has an increased affinity for the substrate. V218N / M215E / R209S The catalytic efficiency V max / K m (18.65mM -1 ·s -1 ) than WT (3.29mM -1 ·s -1 ) increased by 4.67 times.

[0129] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A recombinant tyrosinase BM Tyr mutant, characterized in that: The mutant is any one of the following (a) to (f): (a) The mutant is obtained by mutating the valine at position 218 of tyrosinase BM Tyr as shown in the amino acid sequence of SEQ ID NO.1 to asparagine, and the mutant is named V218N; (b) The mutant is obtained by mutating the methionine at position 215 of tyrosinase BM Tyr as shown in the amino acid sequence of SEQ ID NO.1 to glutamic acid, and the mutant is named M215E; (c) The mutant is obtained by mutating the arginine at position 209 of tyrosinase BM Tyr as shown in the amino acid sequence of SEQ ID NO.1 to serine, and the mutant is named R209S; (d) The mutant is obtained by mutating the valine at position 218 of the tyrosinase BM Tyr as shown in the amino acid sequence of SEQ ID NO.1 to asparagine, and mutating the methionine at position 215 to glutamic acid, and the mutant is named V218N / M215E; (e) The mutant is obtained by mutating the valine at position 218 of the tyrosinase BM Tyr as shown in the amino acid sequence of SEQ ID NO.1 to asparagine, and mutating the arginine at position 209 to serine, and the mutant is named V218N / R209S; (f) The mutant is obtained by mutating the valine at position 218 of the tyrosinase BM Tyr as shown in the amino acid sequence of SEQ ID NO.1 to asparagine, the methionine at position 215 to glutamic acid, and the arginine at position 209 to serine. The mutant is named V218N / M215E / R209S.

2. A gene encoding the recombinant tyrosinase BM Tyr mutant according to claim 1.

3. A recombinant vector carrying the gene according to claim 2.

4. A recombinant cell expressing the mutant according to claim 1, or carrying the gene according to claim 2, or carrying the recombinant vector according to claim 3.

5. A recombinant Escherichia coli, characterized in that The recombinant tyrosinase BM Tyr mutant according to claim 1 is expressed.

6. The recombinant Escherichia coli according to claim 5, characterized in that The recombinant Escherichia coli uses Escherichia coli BL21 (DE3) as an expression host and pET28a as an expression vector.

7. A method for preparing theaflavin-3,3'-digallic acid, characterized in that: The recombinant tyrosinase BM Tyr mutant according to claim 1, or the recombinant cell according to claim 4, or the recombinant Escherichia coli according to claim 5 or 6 is crushed and centrifuged, and then added to a reaction system containing epigallocatechin 3-gallate and epicatechin gallate to react and prepare the product.

8. The method according to claim 7, characterized in that The recombinant tyrosinase BM Tyr mutant enzyme solution was added with 0.1 mM Cu 2+ In the reaction system, the mutant enzyme solution is added to a final concentration of 4 to 10 μg / mL.

9. Use of the recombinant tyrosinase BM Tyr mutant according to claim 1, or the gene according to claim 2, or the recombinant vector according to claim 3, or the recombinant cell according to claim 4, or the recombinant Escherichia coli according to claim 5 or 6 in the preparation of theaflavin-3,3'-digallic acid, or in the preparation of a product containing theaflavin-3,3'-digallic acid.

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