Method for synthesizing theaflavins by a polyphenol oxidase of microbial origin and use thereof

Abstract

This invention belongs to the field of theaflavin enzymatic synthesis technology, and discloses a method for synthesizing theaflavin using microbial polyphenol oxidase and its application. Recombinant Escherichia coli engineered strains of polyphenol oxidase from Bacillus megaterium and Streptomyces spp. are used to synthesize theaflavin by converting crude enzymes in cultures; theaflavin monomers are synthesized by whole-cell catalysis using permeabilization reagent-treated cells; the polyphenol oxidase encoding gene of Streptomyces spp. is fused with a fusion gene of Escherichia coli outer membrane protein and its secretory signal peptide to construct a polyphenol oxidase Escherichia coli surface display vector, and theaflavin is synthesized by whole-cell catalysis, thus obtaining the optimal synthesis method.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology, specifically relating to a method for the enzymatic synthesis of theaflavins using polyphenol oxidases derived from microorganisms and its application. Background Technology

[0002] Theaflavins (TFs) are key compounds that determine the quality of black tea and are known as "soft gold" in tea. They have been widely reported to have significant biological activities in protecting the human cardiovascular system, resisting inflammation and oxidative stress, regulating glucose and lipid metabolism, protecting skeletal muscle health, and anti-tumor effects, thus becoming a research hotspot in the fields of food nutrition, medicine, and health.

[0003] Theaflavins, a key product in the fermentation process of black tea, are chemically classified as benzophenone compounds. They are natural products formed from catechin monomers through enzymatic oxidative polymerization. Their core structural feature lies in a seven-membered benzophenone ring formed by the oxidative coupling of two catechin units. This unique benzophenone skeleton endows theaflavins with specific physicochemical properties and physiological functions that distinguish them from other polyphenolic compounds (see [link to article]). Figure 1 Within the existing research framework, over twenty monomeric compounds of this family have been isolated and identified from the black tea matrix. It is noteworthy that, despite the large number of species, the four components that have received the most focused attention, constitute a significant proportion of samples, and have been most thoroughly studied are: theaflavin (TF1), theaflavin-3-gallate (TF-3-G, TF2a), theaflavin-3′-gallate (TF-3'-G, TF2b), and theaflavin-3,3′-digallate (TFDG, TF3). Minor components such as theaflavic acid and theaflavic acid-3-gallate have also been identified and utilized. From a biosynthetic perspective, theaflavin formation originates from different types of catechins (see...). Figure 2 Oxidative coupling of precursors. Specifically, TF1 is generated by the oxidative polymerization of epigallocatechin (EGC) and epicatechin (EC); TF2a and TF2b are products of the reaction of epigallocatechin gallate (EGCG) with EC, and EGC with epicatechin gallate (ECG), respectively; while TF3 is formed by the coupling of EGCG and ECG. Different combinations of catechins form four major theaflavins (see...). Figure 3 ).

[0004] The preparation methods of theaflavins can be summarized into three major technical routes: extraction and purification from natural black tea, chemical oxidation synthesis, and enzymatic oxidation synthesis. Enzymatic oxidation synthesis, using polyphenol oxidase (PPO) as a biocatalyst, catalyzes the oxidation and polymerization of catechin substrates into theaflavins under mild conditions, and is considered the most promising technical route for achieving green preparation of theaflavins.

[0005] Currently, the PPO enzyme sources reported in the literature mainly include two categories: plant-based (pear, apple, tea, etc.) and microbial-based (bacteria, fungi), but the depth and breadth of research are extremely unbalanced.

[0006] Plant-derived PPO has insufficient enzyme activity stability, high purification costs, and often involves the formation of a large amount of insoluble non-target oxidized polymers during the catalytic reaction, reducing the yield of the target product.

[0007] In theory, microbial resources have outstanding advantages such as rapid growth and reproduction, controllable culture conditions, and mature genetic manipulation systems, making them an ideal source for the industrialization of enzyme preparations. Zhou Jinghui (Study on the Screening of Polyphenol Oxidase and Catalytic Synthesis of Theaflavins TFDG [D], Doctoral Dissertation, Hunan Agricultural University, 2024) successfully screened a highly efficient microbial polyphenol oxidase, PPO (Bmtyrc), through screening. Its ability to catalyze the synthesis of TFDG was significantly improved through directed evolution. Two stable and reusable enzyme catalytic systems, enzyme cross-linked aggregates and whole-cell catalysis, were developed, providing key technical support for the green and low-cost industrial production of theaflavins TFDG.

[0008] Furthermore, polyphenol oxidases (PPOs) have unique theoretical value as target enzymes for surface display. However, the phenolic compounds that serve as substrates for PPOs often exhibit high membrane permeability and chemical reactivity, posing two theoretical challenges: first, after PPOs are displayed on the cell surface, their reaction intermediates (such as quinones) may be toxic to the host cell, involving a trade-off between catalytic function and host adaptability; second, the catalytically active site of PPOs contains copper ions, and their proper assembly requires the participation of the intracellular copper transport system, while whether cell surface display affects the integration of copper cofactors remains unclear. Summary of the Invention

[0009] Based on the current status of theaflavin enzymatic synthesis technology, this invention systematically carries out screening of microbial PPO-producing strains, isolation, purification and catalytic characterization of enzymes, and optimization of reaction conditions for enzymatic synthesis of theaflavin, providing a method for the enzymatic synthesis of theaflavin monomer TF1 using polyphenol oxidase from microorganisms.

[0010] This application aims to establish a highly efficient, controllable, and reusable microbial-derived PPO catalytic synthesis system for theaflavin monomer TF1, particularly addressing the problems of limited transmembrane mass transfer of substrates and decreased catalytic efficiency in conventional permeable whole-cell catalysis.

[0011] To achieve the above objectives, the present invention employs the following technical solution:

[0012] (I) Activation and induction of expression of recombinant strains: Recombinant Escherichia coli engineered strains (pET30a-Bmtyrc-24) derived from Bacillus megatherium PPO and recombinant Escherichia coli strains (pETDuet-1-ORF378 and pETDuet-1-ORF378-TYRC) derived from Streptomyces castaneoglobisporus PPO were activated by streak plating and expression was induced.

[0013] (ii) PPO enzyme activity assay: using levodopa standard and four tea polyphenols (EC, ECG, EGC and EGCG) as substrates, the enzyme activity of PPO from different sources was determined to identify the optimal enzyme source.

[0014] (III) Crude enzyme conversion to theaflavin synthesis: In order to evaluate the performance of PPO from different sources in catalyzing the synthesis of theaflavin from different substrates, this invention uses TF1, TF2a, TF2b and TF3 as target products and evaluates the catalytic performance of each PPO under the same conditions.

[0015] (iv) Optimization of fermentation conditions for PPO enzymatic synthesis of theaflavins: The substrate concentration ratio, time, temperature and pH value were selected, and the catalytic conditions were optimized through single-factor experiments to determine the optimal process parameters.

[0016] (V) Preparation of whole-cell catalyst: The theaflavin monomer TF1 was synthesized by whole-cell catalysis after being treated with 0.5% Triton X-100 permeation reagent at pH 5 and temperature 30℃ for 40 min.

[0017] (vi) Construction of cell surface display system: The polyphenol oxidase ORF378-TYRC derived from Streptomyces was fused with the fusion gene Lpp-OmpA of Escherichia coli outer membrane protein and its secretory signal peptide using restriction-free cloning (RF-clone) to construct a polyphenol oxidase Escherichia coli surface display vector, and theaflavin was synthesized by whole-cell catalysis.

[0018] See the technical route of this invention. Figure 4 .

[0019] Specifically, this invention provides a method for the enzymatic synthesis of theaflavins using polyphenol oxidase from microorganisms and its application. The method uses Bm-TYRC from Bacillus megaterium and ORF378 and ORF378-TYRC from Streptomyces as candidate enzyme sources. After heterologous expression in Escherichia coli, the preferred enzyme source ORF378-TYRC is obtained through screening. This enzyme can catalyze the oxidative coupling of tea polyphenol substrates such as EC, ECG, EGC, and EGCG to generate theaflavins, and is particularly suitable for catalyzing the synthesis of the theaflavin monomer TF1 from EC and EGC.

[0020] This invention also provides crude enzyme catalysis, permeabilized whole-cell catalysis, and Lpp-OmpA cell surface display catalytic systems based on ORF378-TYRC. The preferred synthesis conditions for TF1 are pH 5.0, 30°C, 60 min, and EGC:EC = 1:1. The permeabilized whole-cell catalyst is preferably prepared by treating with 0.5% Triton X-100 at pH 5.0 and 30°C for 40 min. The cell surface display system is achieved by fusing ORF378-TYRC with Lpp-OmpA, allowing PPO to be displayed on the outer membrane surface of *E. coli* for whole-cell catalytic synthesis of TF1.

[0021] This invention provides a method for the enzymatic synthesis of theaflavins using polyphenol oxidase, wherein polyphenol oxidase catalyzes substrates EC+EGC, EC+EGCG, ECG+EGC, or ECG+EGCG to generate the target products theaflavins, theaflavins-3-gallate, theaflavins-3′-gallate, and theaflavins-3,3′-bisgallate.

[0022] The method for the enzymatic synthesis of theaflavins using polyphenol oxidase as described above, wherein the polyphenol oxidase is derived from microorganisms capable of producing polyphenol oxidase, preferably from Streptomyces genus or from Bacillus megaterium, and more preferably from Streptomyces pETDuet-1-ORF378-TYRC.

[0023] In the method for the enzymatic synthesis of theaflavins using polyphenol oxidase as described above, the polyphenol oxidase is a crude enzyme, which is the supernatant obtained by centrifuging the fermentation broth of a microorganism capable of producing polyphenol oxidase. Preferably, the method for collecting the supernatant is to collect the fermentation broth, centrifuge and discard the supernatant, wash with PBS, resuspend, lyse, and then centrifuge again to collect the supernatant.

[0024] The method for the enzymatic synthesis of theaflavins using polyphenol oxidase as described above, wherein the polyphenol oxidase is a whole-cell catalyst, preferably, in the preparation of the whole-cell catalyst, hexadecyltrimethylammonium bromide, Triton X-100 or toluene is used as a permeation reagent, the concentration of the permeation reagent is 0.25%-2%, preferably 0.3%-1.0%, more preferably 0.5%; the permeation treatment time is 10-60 min, preferably 40 min.

[0025] In the method described above for the enzymatic synthesis of theaflavins using polyphenol oxidase, the polyphenol oxidase is anchored on the surface of host cells via a cell surface display system and performs whole-cell catalytic synthesis of theaflavins.

[0026] The method for synthesizing theaflavins using polyphenol oxidase as described above, and the method for anchoring the polyphenol oxidase on the surface of host cells via a cell surface display system, includes fusing the encoding gene of polyphenol oxidase ORF378-TYRC from Streptomyces with the fusion gene Lpp-OmpA of Escherichia coli outer membrane protein and its secretory signal peptide to construct a polyphenol oxidase Escherichia coli surface display vector.

[0027] The method for the enzymatic synthesis of theaflavins using polyphenol oxidase as described above, wherein the method for constructing the polyphenol oxidase Escherichia coli surface display vector includes using the genomic DNA of an Escherichia coli clone strain as a cloning template, and performing PCR amplification using primers to obtain the ORF378-TYRC fragment, preferably, the nucleotide sequence of the primers is:

[0028] ORF378-TYRC-F:

[0029] 5'-GGATCCATGCCGGAAATTACCCGTCGTCGCGCCCTGACCGCCGCCGCAGCAGTGG-3';

[0030] ORF378-TYRC-R:

[0031] 5'-GAATTCTGCATCAAAGGTATAATATGCGGTATGATCC-3';

[0032] The amplified ORF378-TYRC gene fragment was purified and recovered. Using pLpp-OmpA plasmid as a template, PCR was performed using the purified and recovered ORF378-TYRC gene fragment as a large primer to insert and fuse ORF378-TYRC into the pLpp-OmpA vector, thereby obtaining the pLpp-OmpA-PPO display expression vector.

[0033] The method for the enzymatic synthesis of theaflavins using polyphenol oxidase as described above, wherein the substrate for enzymatic synthesis is EC+EGC, with an EGC:EC ratio of 0.2:1-4:1, more preferably 0.5:1-3:1, more preferably 1:1-2:1, and most preferably 1:1; pH 4.5-6.5, preferably pH 4.8-6.0, more preferably pH 5.0; temperature 10-60℃, preferably 20℃-50℃, more preferably 25℃-40℃, and even more preferably 30℃; reaction time 5 min-120 min, preferably 30-90 min, even more preferably 40-80 min, and most preferably 60 min.

[0034] The present invention also provides the application of the method described above in the preparation of theaflavins.

[0035] The present invention also provides the application of theaflavins prepared by the method described above in the production of food, biological agents, health products and pharmaceuticals.

[0036] In summary, the present invention has the following technical effects.

[0037] (1) The present invention screened and obtained microbial PPOs suitable for the enzymatic synthesis of theaflavins. Among them, ORF378-TYRC has high catalytic activity for EC, ECG, EGC and EGCG, and is especially suitable for catalyzing the synthesis of TF1 from EC and EGC.

[0038] (2) This invention establishes a mild enzymatic synthesis process for TF1. Under the conditions of pH 5.0, 30℃, 60 min, and EGC:EC = 1:1, the concentration of TF1 can reach approximately 2.65 mg / mL. -1 This improves the controllability of the synthesis of the target product.

[0039] (3) The present invention constructs an ORF378-TYRC permeabilized whole-cell catalytic system, which eliminates the step of purifying free enzymes and can continuously catalyze the synthesis of TF1 in multiple batches. The yield of TF1 in nine batches of reaction was maintained at 0.6 mg / mL. -1 above.

[0040] (4) This invention further constructed an Lpp-OmpA-PPO cell surface display system, enabling PPO to be successfully displayed on the outer membrane surface of E. coli. The first four batches of TF1 production using this system maintained a yield of 2.5 mg / mL. -1 In summary, the catalytic effect is superior to that of ordinary permeable whole-cell systems. Attached Figure Description

[0041] Figure 1 Chemical structural formulas of the four main components of theaflavins.

[0042] Figure 2 Catechins: structure and types.

[0043] Figure 3 The mechanism by which polyphenol oxidase (PPO) catalyzes the synthesis of theaflavins from catechins.

[0044] Figure 4 The main technical approach of this invention.

[0045] Figure 5 Validation of recombinant strain plasmid DNA. M: DNA standard molecular weight marker; Lane 1: pETDuet-1-ORF378 plasmid electrophoresis; Lane 2: pETDuet-1-ORF378-TYRC plasmid electrophoresis.

[0046] Figure 6 The activity of PPO enzymes from different sources was detected using four tea polyphenols EC, ECG, EGC, and EGCG as substrates.

[0047] Figure 7 Color changes of four theaflavin monomers synthesized by PPO from different sources.

[0048] Figure 8 Catalytic synthesis of TF1 monomer under different fermentation conditions. (A) Different substrate concentration ratios EGC:EC; (B) Different reaction temperatures; (C) Different reaction pH; (D) Different reaction times.

[0049] Figure 9 HPLC determination of the content of theaflavin monomer TF1 under different process conditions. A. Different substrate concentration ratios EGC:EC; B. Different reaction pH; C. Different reaction temperature; D. Different reaction time.

[0050] Figure 10 Enzymatic activities of whole-cell catalysts against four tea polyphenols EC, ECG, EGC and EGCG substrates.

[0051] Figure 11 Color changes during multi-batch catalytic synthesis of TF1 in whole cells.

[0052] Figure 12 TF1 concentration was synthesized through multiple batches of whole-cell catalysis.

[0053] Figure 13 pLpp-OmpA vector spectrum.

[0054] Figure 14 PPO expression vector double enzyme digestion verification. M. DNA standard molecular weight marker; Lane 1: PPO plasmid electrophoresis; Lane 2: PPO plasmid double enzyme digestion (EcoRI-BamHI).

[0055] Figure 15PPO colony PCR verification. M. DNA standard molecular weight marker; Lane 1: Single colony 1 PCR electrophoresis; Lane 2: Single colony 2 PCR electrophoresis; Lane 3: Single colony 3 PCR electrophoresis; Lane 4: Single colony 4 PCR electrophoresis; Lane 5: Single colony 5 PCR electrophoresis.

[0056] Figure 16 PPO protein SDS-PAGE electrophoresis detection. M. Protein molecular weight marker; Lane 1: Uninduced supernatant; Lane 2: Induced supernatant; Lane 3: Uninduced whole cells; Lane 4: Induced whole cells.

[0057] Figure 17 Whole-cell immunofluorescence assay. A. Induced whole-cell FITC labeling; B. Induced whole-cell DAPI labeling; C. No induced whole-cell FITC labeling; D. Induced whole-cell DAPI labeling.

[0058] Figure 18 Western blotting detection of PPO. M. Protein molecular weight marker; 1. Induction supernatant; 2. Induction precipitate; 3. Inner membrane component; 4. Periplasmic component; 5. Outer membrane component.

[0059] Figure 19 The cell surface display system showed the enzyme activity of four tea polyphenols EC, ECG, EGC and EGCG substrates.

[0060] Figure 20 Color changes during multi-batch catalytic synthesis of TF1 in whole cells.

[0061] Figure 21 The cell surface display system catalyzed the synthesis of TF1 concentrations in multiple batches. Detailed Implementation

[0062] The present invention will be further described below through specific embodiments in order to better understand the present invention, but this does not constitute a limitation on the present invention.

[0063] The reagents, culture media, and instruments used in this invention are all commercially available. Specifically, epigallocatechin gallate (EGCG, CAS No.: 985-51-5), epicatechin gallate (ECG, CAS No.: 1257-08-5), epicatechin (EC, CAS No.: 490-46-0), epigallocatechin (EGC, CAS No.: 970-74-1), and theaflavins standard (TF1, CAS No.: 4670-05-7) were all provided by Hunan Fulaige Biotechnology Co., Ltd. The plasmid extraction kit was purchased from Omega. Copper sulfate pentahydrate, sodium chloride, potassium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, anhydrous ethanol, dipotassium hydrogen phosphate, formic acid, acetonitrile, methanol, citric acid, and sodium hydroxide were all purchased from China National Pharmaceutical Group Co., Ltd. Yeast extract and tryptone used in the culture media were purchased from Oxoid (Shanghai Company). Agar powder, ampicillin, kanamycin sulfate, and agarose were all from Tiangen Biotech. Reagents for agarose gel electrophoresis were purchased from Shanghai Sangon Biotech. The strains: recombinant polyphenol oxidase strains pET30a-Bmtyrc, pETDuet-1-ORF378, and pETDuet-1-ORF378-TYRC (these recombinant strains are also referred to as Bmtyrc, ORF378, and ORF378-TYRC in this invention), and the E. coli expression host cell E. coli BL21(DE3) were all provided by Hunan Fulaige Biotechnology Co., Ltd.

[0064] Example 1 Screening of PPO from Microbial Sources

[0065] 1. Activation culture of recombinant strains

[0066] The origin of the PPO gene and related information are shown in Table 1.

[0067] Table 1 PPO Information

[0068]

[0069] (1) Streak plate method: Take out the recombinant Escherichia coli strains ORF378, ORF378-TYRC and BmTYRC and thaw them. Inoculate the strains onto LB solid medium containing the corresponding resistance using the streak plate method and incubate overnight at 37°C.

[0070] (2) Seed culture of PPO recombinant strain: Single colonies with rounded surfaces, no discoloration, and relatively large size were picked from three LB solid medium plates and inoculated into Erlenmeyer flasks containing 10 mL of LB liquid medium (Kan concentration of 50 μg / mL). -1 The final concentration of Amp is 100 μg / mL. -1 ), 37℃, 200 r•min -1Incubate for 14-16 hours.

[0071] (3) Plasmids were extracted and heat-shocked transformed from the fermentation broth of two recombinant strains of the Top 10 competent cells from the genus Streptomyces.

[0072] ① Plasmid extraction: Take 1.5 mL of bacterial culture into an EP tube and rotate at 11000 r•min. -1 Centrifuge for 1 min and discard the supernatant; add 250 μL of solution 1 resuspension buffer and mix well; add 250 μL of solution 2 lysis buffer and mix well; add 350 μL of solution 3 neutralization precipitate buffer and mix well; centrifuge for 10 min; add the supernatant to the column and centrifuge for 1 min and discard the waste liquid; add 500 μL of HBC high-salt binding buffer and centrifuge for 1 min and discard the waste liquid; add 700 μL of DNA wash buffer and centrifuge for 1 min (wash twice) and discard the waste liquid; centrifuge for 2 min and then let stand for about 10 min to evaporate the residual ethanol; transfer the column to a new microcentrifuge tube, add 30-100 μL of elution buffer, let stand for 1 min and then centrifuge for 1 min to obtain the plasmid solution.

[0073] ② The extracted plasmids were verified by agarose gel electrophoresis. Since the pETDuet-1-ORF378 and pETDuet-1-ORF378-TYRC vectors are 5.6 kb and 6.5 kb respectively, a 0.8% agarose gel was prepared. 0.4 g of agarose was weighed and dissolved in 50 mL of 1×Tris-acetic acid-EDTA (1×TAE) buffer by heating. After slight cooling, 4 μL of staining solution was added, mixed well, and the gel was poured. The gel running parameters were set as follows: constant voltage 120 V, time 30 min.

[0074] The results of plasmid DNA verification of recombinant strains are as follows: Figure 5 As shown, both plasmid DNA from channel 1 (ORF378) and channel 2 (ORF378-TYRC) contained clear main bands greater than 5000 bp, with sizes M (5000 bp) < 1 < 2, consistent with the expected size. Therefore, the extracted plasmid DNA can be used for subsequent transformations.

[0075] ③ Heat shock transformation: Take one BL21(DE3) competent cell, thaw it on ice, add 2-5 μL of plasmid DNA extracted in ①, let it stand on ice for 30 min, heat shock it in a 42℃ water bath for 90 s, then quickly remove the Ep tube and place it on ice for 5 min, add 1 ml of recovery medium, and shake and incubate at 37℃ for 1 h. Spread it on an Amp-resistant plate and incubate overnight. A large number of regular, uniform white single colonies grow on the plate, with no obvious satellite colonies or contamination by other bacteria, indicating that the recombinant plasmid transformation is successful and can be used for subsequent experiments. Repeat (2) to pick positive strains for seed culture.

[0076] (4) Induction of expression: Take the bacterial cell concentration OD from (2) 600 2 mL of seed culture medium (0.6-0.8 g) was transferred to an Erlenmeyer flask containing 100 mL of TB liquid medium. The flask was then placed on a shaker for scale-up culture. The culture conditions were set as follows: temperature 37℃, shaker speed 200 rpm. -1 The fermentation time is 4-6 hours. When the OD of the fermentation broth... 600 When the expression reaches 0.8-1.2, lactose is added to induce expression, so that the final mass-to-volume ratio is 1% (m / v), and the culture conditions are adjusted to a temperature of 25℃ and a rotation speed of 180 r•min-1, and the induction culture is continued for 12-16 h.

[0077] 2. PPO activity assay

[0078] Referring to the following references: Zhou Jinghui, Screening of polyphenol oxidase and study on catalytic synthesis of theaflavins TFDG [D], Hunan Agricultural University (Doctoral Dissertation), 2024c; Shevchuk, A., Jayasinghe, L., & Kuhnert, N. (2018). Differentiation of black tea infusions according to origin, processing and botanical varieties using multivariate statistical analysis of LC-MS data. Food Research International, 109, 387-402. The activity of PPO was detected and analyzed.

[0079] Substrate preparation: Accurately weigh 0.3 g of each of the four tea polyphenol substrates (EC, EGC, ECG, and EGCG), dissolve them in 100 mL of 0.1 M citrate-phosphate buffer (pH 5.0), and stir thoroughly until completely dissolved to obtain a concentration of 3 mg / mL. -1 The substrate solution (containing 0.1 mL Cu) 2+ Preheat the substrate solution in a 30°C water bath for 10 minutes.

[0080] Cell collection and lysis: The fermentation broth after induction was collected using 50 mL centrifuge tubes. Each tube contained 50 mL of the induced broth and was centrifuged at 10,000 rpm. -1 Centrifuge for 5 minutes under the specified conditions to precipitate the bacterial cells. After discarding the supernatant, wash the bacterial pellet twice with PBS buffer, and finally resuspend in 10 mL of PBS buffer. Lyse the resuspended bacterial culture by sonication, followed by centrifugation again at 10000 rpm.-1 Centrifuge for 5 minutes, and the resulting supernatant is the crude enzyme solution containing PPO.

[0081] Enzyme activity assay: Add 450 μL of preheated (30℃) polyphenol substrate (EC, ECG, EGC, or EGCG) solution and 0.5 mL of crude enzyme solution sequentially to a 1.5 mL Eppendorf tube. Mix well and incubate at 30℃ in a shaker for 5 minutes. Then, quickly add an equal volume of 20% (w / v) trichloroacetic acid solution and mix thoroughly to terminate the reaction. After centrifugation, collect the supernatant and measure the absorbance at 420 nm using a microplate reader (as shown in Table 2). The reaction system without added enzyme solution serves as a negative control. All samples were measured three times, and the arithmetic mean was used. The enzyme activity unit is defined as follows: Under the above reaction conditions (temperature 30℃, pH 5.0), the amount of enzyme required to cause a 0.001 change in absorbance at 420 nm per minute is defined as one enzyme activity unit, denoted as U•min. -1 •mL -1 .

[0082] The formula for calculating enzyme activity is: In D, U represents enzyme activity. This indicates the change in absorbance over the reaction time, where t represents the reaction time in minutes, and D represents the dilution factor, i.e., the ratio of the total extracted enzyme solution to the enzyme solution in the reaction system.

[0083] Based on the enzyme activity calculation formula above, the activities of PPO enzymes from different sources were calculated (as shown in Table 3).

[0084] Table 2. Average absorbance values ​​of PPO bacterial solutions from different sources, using EC, ECG, EGC, and EGCG as substrates for the detection of four types of tea polyphenols.

[0085]

[0086] Table 3. Activity of PPO enzymes from different sources detected using EC, ECG, EGC, and EGCG as substrates for four types of tea polyphenols.

[0087]

[0088] Table 3 shows the activity of PPO enzymes from different sources, using four tea polyphenols (EC, ECG, EGC, and EGCG) as substrates. Figure 6 As shown:

[0089] For the same substrate EC, the catalytic performance of PPO from different sources is as follows: ORF378-TYRC②>ORF378-TYRC①>Bm-TYRC②>ORF378②>ORF378①>Bm-TYRC①;

[0090] For the same substrate ECG, the catalytic performance of PPO from different sources is as follows: ORF378-TYRC②>ORF378-TYRC①>Bm-TYRC①>Bm-TYRC②>ORF378①>ORF378②;

[0091] For the same substrate EGC, the catalytic performance of PPO from different sources is as follows: ORF378-TYRC②>ORF378①>ORF378-TYRC①>Bm-TYRC②>Bm-TYRC①>ORF378②;

[0092] For the same substrate EGCG, the catalytic performance of PPO from different sources is as follows: ORF378-TYRC①>ORF378-TYRC②>ORF378①>Bm-TYRC①>Bm-TYRC②>ORF378②.

[0093] In summary, the PPO derived from ORF378-TYRC exhibited the best enzyme activity, therefore it was chosen as the research subject for subsequent experiments.

[0094] 3. Crude enzyme catalyzes the synthesis of theaflavins.

[0095] To evaluate the performance of heterologous expression products of the microbial PPO gene (ORF378, ORF378-TYRC, Bmtyrc) in catalyzing the synthesis of theaflavins from different substrates, this invention used TF1, TF2a, TF2b, and TF3 as target products and evaluated the catalytic performance of each recombinant protein under the same conditions. The specific steps are as follows:

[0096] (1) Use 0.1M pH 5.0 citrate-phosphate buffer solution (containing 0.2mM Cu) 2+ Prepare 15 mL solutions of different substrates containing EC+EGC, EC+EGCG, ECG+EGC, and ECG+EGCG, with each substrate concentration of 3 mg / mL. -1 The prepared substrate solution was preheated in a 30°C water bath for 10 minutes.

[0097] (2) Pipette 450 μL of the preheated substrate solution into a 96-well plate. Add 50 μL of crude enzyme solution obtained from fermentation and lysis of different PPO recombinant strains to the reaction system and mix gently. Then place the mixture in a shaker for catalytic shaking. The reaction parameters are set as follows: pH 5.0, reaction temperature 25℃, and shaker speed 180 r•min. -1 After 25 minutes, immediately add an equal volume (50 μL) of 20% (w / v) trichloroacetic acid to the reaction system and mix thoroughly to terminate the enzymatic reaction.

[0098] (3) Centrifuge the reaction solution after termination in the above 96-well plate at high speed for 1 min and observe the color of the four monomers.

[0099] Color changes during the synthesis of four theaflavin monomers catalyzed by PPO from different sources, as shown below. Figure 7 As shown. From Figure 7 It can be clearly seen that in the same reaction system, different shades of color are presented. PPO from different enzyme sources is used to catalyze the synthesis of TFs. Among them, EC and EGC are used as substrates to catalyze the synthesis of theaflavin monomer TF1 with the best effect. The corresponding color is significantly darker, which means that the concentration of product TF1 is higher than that of the other three theaflavin monomers.

[0100] 4. Optimization of PPO-catalyzed synthesis of TF1 monomer process

[0101] Considering the key influencing factors in the synthesis of theaflavins, this experiment selected four key parameters—pH, reaction temperature, reaction time, and substrate concentration ratio—and optimized them using a single-factor experimental method. During the experiment, three of these parameters were kept constant, while the fourth parameter was tested with varying gradient levels to investigate the effects of pH, temperature, reaction time, and substrate concentration ratio on the experimental results, thus identifying the optimal process conditions for each parameter. In this invention, the parameters for the theaflavin monomer TF1 synthesis reaction system were uniformly set as follows: pH 5.0, substrate concentration 3 mg / mL. -1 The reaction temperature was 30℃ and the reaction time was 30 min (Cu2+ content in the system: 0.1 mM). After the reaction was completed, an appropriate amount of the reaction solution was taken for HPLC detection and analysis of TF1 content.

[0102] Optimal substrate concentration ratio (EGC:EC): The reaction system was set at pH 5.0, reaction temperature 30℃, and reaction time 30 min. The effect of different substrate concentration ratios (EGC:EC of 1:1, 2:1, 3:1, and 4:1) on the yield of TF1 was studied.

[0103] Optimal pH: The optimal substrate concentration ratio was determined, and the effect of different pH values ​​on the yield of TF1 was investigated.

[0104] Optimal reaction temperature: The optimal substrate concentration ratio and pH of the reaction system were determined, and the effect of different reaction temperatures on the yield of TF1 was studied.

[0105] Optimal reaction time: The optimal substrate concentration ratio, reaction system pH, and reaction temperature were determined, and the effects of different reaction times on the yield of TF1 were studied.

[0106] The results of catalytic synthesis of TF1 monomers under different fermentation conditions are as follows: Figure 8As shown in (A), (B), (C), and (D), different reaction conditions significantly affect the amount of TF1 generated. Regarding the substrate concentration ratio, a 1:1 EGC:EC ratio results in better TF1 generation. Regarding pH, pH 5.0 yields the highest TF1 generation; excessive acidity or increased pH are detrimental to TF1 accumulation. Regarding temperature, 30℃ provides the best catalytic effect. Regarding reaction time, the TF1 content initially increases and then decreases with increasing reaction time, reaching a relatively high level at 60 min; further extending the reaction time leads to further oxidation or degradation of TF1. In summary, the optimal conditions for the synthesis of TF1 from EC and EGC catalyzed by ORF378-TYRC are: pH 5.0, 30℃, 60 min, and EGC:EC = 1:1.

[0107] The substrate concentration ratio (EGC:EC) directly affects the competitive binding and electron transfer efficiency of the two substrate molecules at the PPO active site. Table 4 shows that when the EGC:EC ratio is 1:1, the TF1 concentration is the highest (2.5376 mg / mL). -1 As the EGC ratio increased to 2:1, 3:1, and 4:1, the TF1 concentration decreased to 2.1303, 1.3441, and 0.8615 mg / mL, respectively. -1 The yield shows a monotonically decreasing trend. Stoichiometrically, TF1 is formed by the ring coupling of one molecule of EGC and one molecule of EC through benzo[a]phenone. Theoretically, an equimolar ratio should be most favorable for product formation. When EGC is in excess, the competitive advantage leads to a relative deficiency of EC, and some EGC molecules undergo self-coupling to form non-target dimers, thereby inhibiting the synthesis of TF1. From the perspective of substrate economy, an equimolar ratio (1:1) avoids the waste of expensive substrates and achieves the highest TF1 yield, making it the optimal choice for this process.

[0108] Table 4. Concentration of TF1 catalyzed by different substrate concentrations (EGC:EC)

[0109]

[0110] pH, as a core parameter affecting the conformational stability of PPO and the substrate dissociation state, directly alters the microenvironment of the chemical reaction. Table 5 shows that at pH 3.0, the TF1 concentration is zero, indicating that PPO activity is completely inhibited under strongly acidic conditions, possibly due to protonation of the copper ion at the enzyme's active site or irreversible denaturation of the enzyme protein. As the pH rises to 5.0, the TF1 concentration reaches a peak of 1.9905 mg / mL. -1 This indicates that the pH conditions are most favorable for the oxidative coupling reaction of PPO with EGC and EC. Upon further increasing the pH to 6.0 and 7.0, the TF1 concentration decreased sharply to 0.5401 mg / mL. -1 and 0.0233 mg•mL -1The trend shows a clear inflection point. This asymmetric change suggests that the optimal pH for PPO-catalyzed synthesis of TF1 is not a broad range, but rather concentrated in a narrow window of acidic to neutral pH (around 5.0). Compared to the optimal pH of conventional PPO (mostly between 5.5 and 6.5), the advantage of pH 5.0 in this system may stem from the higher matching degree of phenolic hydroxyl dissociation states between EGC and EC at this pH, which is conducive to the formation of a stable quinone intermediate and its directional condensation into TF1, rather than other byproducts.

[0111] Table 5. Concentration of TF1 catalytically synthesized at different pH values.

[0112]

[0113] The effect of temperature on the enzymatic reaction exhibits a typical bell-shaped curve. Table 6 shows that within the temperature range of 20℃ to 50℃, the TF1 concentration first increases and then decreases, reaching a maximum of 2.6459 mg / mL at 30℃. -1 It is worth noting that the TF1 concentration at 20℃ was 2.3206 mg / mL. -1 ) and 40℃ (2.4912 mg•mL) -1 ) and 50℃ (2.2341 mg•mL) -1 The TF1 concentration did not decrease significantly compared to the previous temperature, indicating that PPO maintains high catalytic activity at lower temperatures. This differs from the simplistic model that "high temperatures accelerate the reaction but also accelerate enzyme inactivation": in this system, heating from 30°C to 40°C only reduced the TF1 concentration by about 5.8%, while retaining approximately 84.5% of the optimal yield at 50°C. This expanded temperature tolerance window may be related to the thermophilic nature of this PPO source or the thermoprotective effect of the substrate EGC / EC on the enzyme. From a process economy perspective, 30°C avoids the high energy consumption of low-temperature cooling and mitigates the risk of enzyme inactivation at high temperatures, making it the optimal choice for this system.

[0114] Table 6. Concentration of TF1 catalytically synthesized at different reaction temperatures.

[0115]

[0116] The reaction time curves (Table 7) revealed a complex dynamic equilibrium between the formation and transformation of TF1. During the 10–30 min period, the TF1 concentration decreased from 2.2344 mg / mL. -1 It rapidly rose to 2.6399 mg / mL. -1 The net growth rate was 18.1%. The concentration remained between 2.62 and 2.65 mg / mL during the 30-60 minute period. -1 The plateau region, with the maximum value occurring at 60 min (2.6486 mg•mL). -1However, after 60 minutes, the TF1 concentration began to decrease: at 90 minutes and 120 minutes, it dropped to 2.3896 mg / mL, respectively. -1 and 2.3225 mg•mL -1 The decrease was approximately 12.3%. This "accumulation followed by decay" curve indicates that TF1 is not the final product, but rather an intermediate that can further participate in subsequent oxidative polymerization reactions. Excessive reaction time can lead to the transformation of TF1 into theaflavins dimers or polymers, or ring-opening degradation. Therefore, the process design should select 60 minutes as the termination point to achieve maximum TF1 accumulation, rather than simply extending the reaction time to pursue a higher conversion rate.

[0117] Table 7. Concentration of TF1 in catalytic synthesis at different reaction times.

[0118]

[0119] Based on Tables 2, 3, 4, 5, and 6, a comparative HPLC analysis of the content of theaflavin monomer TF1 under different parameter variations was plotted. Figure 9 As shown in A, B, C, and D: The optimal catalytic process conditions were finally determined to be: pH 5.0, reaction temperature 30℃, reaction time 60 min, and substrate concentration ratio EGC:EC (1:1).

[0120] This invention uses *Escherichia coli* BL21(DE3) as a heterologous prokaryotic expression host. Compared with existing eukaryotic expression systems (such as *Pichia pastoris*), the prokaryotic system has advantages such as shorter culture cycle, higher protein yield, and easier operation. In this invention, *Streptomyces*-derived ORF378-TYRC exhibited higher enzyme activity under the same prokaryotic conditions, especially in catalyzing EC and EGC, where its activity was significantly superior to that of single ORF378 and BmTYRC. This may be due to intramolecular or intermolecular synergistic effects, enhancing the catalytic ability of recombinant PPO. This result suggests that the TYRC gene fragment may be involved in the conformational stability of the enzyme active site or the correct folding of the copper ion binding site, thereby improving the functional integrity of the prokaryotic expression product.

[0121] Example 2: Synthesis of theaflavins TF1 by ORF378-TYRC whole-cell catalysis

[0122] 1. Preparation of whole-cell catalysts

[0123] The preparation of the ORF378-TYRC whole-cell catalyst should be based on the premise of not losing enzyme activity, and the effect on TF1 yield also needs to be investigated. Based on this, this invention refers to the method of Zhou Jinghui, Study on Screening of Polyphenol Oxidase and Catalytic Synthesis of Theaflavins TFDG [D], Hunan Agricultural University (Doctoral Dissertation), 2024c, to prepare the ORF378-TYRC-based whole-cell catalyst. The preparation conditions were systematically optimized, and the factors investigated included the type and concentration of permeabilizing reagent, pH value of the treatment system, treatment time and temperature.

[0124] (1) Screening of permeation reagents and their concentrations: Three reagents were selected, namely hexadecyltrimethylammonium bromide (CTAB), polyethylene glycol octyl ether (Triton X-100) and toluene, and four concentration gradients of 0.5%, 1%, 1.5% and 2% were set to evaluate the effects of different reagents and concentrations on enzyme activity.

[0125] (2) Determination of optimal pH: After determining the optimal permeability reagent and its concentration, the treatment effects under pH conditions of 3, 4, 5, 6, 7 and 8 were compared, with enzyme activity and theaflavin TF1 production as evaluation indicators.

[0126] (3) Determination of optimal treatment time: Based on the selected reagent concentrations and pH conditions, the effects of different treatment times (10, 20, 30, 40, 50, 60 min) on enzyme activity and TF1 yield were investigated.

[0127] (4) Determination of optimal treatment temperature: Under the premise that the reagent concentration, pH and treatment time have been optimized, the effects of different treatment temperatures (25, 30, 35 and 40℃) on enzyme activity and TF1 yield were further compared.

[0128] The final parameters obtained were consistent with the process parameters under the optimal catalytic conditions of the ORF378-TYRC strain used in this invention. Therefore, this invention selected 0.5% Triton X-100 as the permeation reagent, pH 5, temperature 30℃, and permeation treatment time 40 min. Using unpermeation-treated cells as a negative control, the enzyme activities of the whole-cell catalyst were measured using EC, ECG, EGC, and EGCG as substrates (the method is the same as the PPO activity determination in Example 1). The results are as follows... Figure 10 As shown, whole cells treated with 0.5% Triton X-100 permeabilizing agent at pH 5 and 30℃ for 40 min exhibited enzymatic activity against all four substrates. This demonstrates the feasibility of preparing whole-cell catalysts using the recombinant strain ORF378-TYRC.

[0129] 2. Whole-cell catalyst-catalyzed synthesis of TF1

[0130] Based on the optimal reaction parameters determined in Example 1, the catalytic efficiency of the whole-cell catalyst in the synthesis of TF1 was evaluated, with cells of the same activity but without permeabilization treatment serving as a control group. An appropriate amount of permeabilized whole-cell catalyst cells were collected and prepared into a bacterial suspension using the optimal buffer solution, followed by the addition of substrate to initiate the reaction. After the reaction, a certain amount of supernatant was aspirated, and the target product TF1 was quantitatively analyzed using HPLC. The control group (CK) consisted of cells without permeabilization treatment.

[0131] Take appropriate amounts of permeabilized whole-cell catalyst cells and untreated control cells with equal activity, and prepare a cell concentration of 100 g / L using citrate-phosphate buffer at pH 5.0. -1 The bacterial suspension was prepared. Substrate was added to the system to achieve final concentrations of 3 mg / mL for both EC and EGC. -1 After thorough mixing, place at 30℃ and incubate at 180 r•min. -1 The conversion reaction was carried out at a rotation speed of [speed value missing]. After 60 minutes of reaction, the cells were centrifuged, and the supernatant was analyzed by HPLC. The remaining bacterial cells were reused for the next batch of conversion reaction.

[0132] The color of theaflavins produced in multiple batches of reactions is as follows: Figure 11 As shown: from the first batch to the ninth batch, there is color and it gradually becomes lighter, which is initially estimated to be a gradual decrease in the amount of TF1 generated.

[0133] HPLC results of TF1 concentration in multiple batches of reactions are as follows Figure 12 As shown, the amount of theaflavins synthesized by the whole-cell catalyst was significantly higher than that of the control cells, while the TF1 production in the untreated control group was extremely low. The concentration of TF1 synthesized by the permeabilized strain ORF378-TYRC decreased sequentially in multiple batches, consistent with the above predictions. In nine consecutive batches of reactions, the theaflavin TF1 production remained at 0.6 mg / mL. -1 In the first reaction, the theaflavins concentration was higher than 1 mg / mL. -1 As the number of reaction batches increases, the bacteria may lose some enzyme activity during the reaction and repeated recycling process, resulting in a decreasing trend in the production of theaflavins.

[0134] Example 3: Construction and Catalytic Performance Study of PPO Escherichia coli Cell Surface Display System

[0135] 1. Biomaterials

[0136] Strains: The cloned strain E. coli JM109 (K-12 line) and the E. coli BL21 (DE3) expression host cell were both obtained from Hunan Fulaige Biotechnology Co., Ltd. The expression vector pLpp-OmpA was purchased from Wuhan Miaoling Biotechnology Co., Ltd. In this invention, primer synthesis and DNA sequencing services were completed by Sangon Biotech (Shanghai) Co., Ltd. The expression vector map used is shown below. Figure 13 As shown in Table 8, the primers used are listed below.

[0137] Table 8. Primers for Polyphenol Oxidase

[0138]

[0139] 2. Methods

[0140] 2.1 Construction of the PPO cell surface display system

[0141] Primer-specific amplification: Using the DNA sequence of Escherichia coli clone strain JM109 as a template, the PPO gene was cloned using the primers in Table 8 and the reaction system in Table 9, and the ORF378-TYRC fragment was amplified.

[0142] Table 9. PCR reaction system for PPO gene cloning

[0143]

[0144] Perform the PCR reaction program as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 1.5 min, skip to step 2 for 25 cycles, 72℃ final extension for 5 min, and terminate the program.

[0145] Purification of PCR products: Add the PCR product to a HiBind DNA Mini Column and place the column inside a 2ml collection tube. Add 400μL Binding Buffer to the column, centrifuge at 13000×g for 1 min, and discard the waste liquid; add 500μL spw Buffer for two elutions, centrifuge and discard the waste liquid; allow to stand empty; insert the column into a new 1.5mL EP tube, add 50μL elution buffer, let stand for 2 min, centrifuge for 1 min, pour the waste liquid back into the column, centrifuge for 1 min to obtain the purified PCR product.

[0146] The ORF378-TYRC fragment obtained from the above amplification was constructed into the expression vector pLpp-OmpA: using pLpp-OmpA plasmid as a template, the recovered fragment was used for large primer PCR, and the reaction system is shown in Table 10.

[0147] Table 10 Primer PCR Reaction Systems

[0148]

[0149] Perform the PCR reaction program as follows: 94℃ pre-denaturation for 5 min, 98℃ denaturation for 30 s, 60℃ annealing for 30 s, 68℃ extension for 4 min, skip to step 2 for 25 cycles, 68℃ final extension for 5 min, and terminate the program.

[0150] The PCR products were purified and recovered, and then digested with EcoRI and BamHI: 5 μL of 10xbufffer, 1 μL of EcoRI and 1 μL of BamHI were added to 50 μL of the recovered whole plasmid PCR product. The digestion was carried out in a PCR instrument at 37℃ for 2 h. The digested products were detected by agarose gel electrophoresis.

[0151] Heat shock transformation plating: Escherichia coli BL21(DE3) competent cells were used as expression host cells to perform heat shock transformation of the enzyme digestion products (method as described in Example 1) and then plating them onto plates with Amp resistance.

[0152] Colony PCR verification: Five plump, round single colonies were randomly selected from the plate and dissolved in 20 μL of sterile water to prepare bacterial suspensions. The colony PCR reaction system shown in Table 11 was used to verify whether the bacteria carried the target gene fragment. The colony PCR products were detected by agarose gel electrophoresis. The bacterial suspensions that were verified were sent for sequencing and inoculated for fermentation to induce expression (method as described in Example 1).

[0153] Table 11 Colony PCR Reaction System

[0154]

[0155] 2.2 SDS-PAGE identification of PPO surface

[0156] SDS-PAGE was used to identify the successful expression of polyphenol oxidase. 50 mL of induced and uninduced ORF378-TYRC fermentation broth were centrifuged at 4°C and 8000×g for 5 min to collect cell cells and fermentation supernatant. The cells were washed with 30 mL of PBS buffer, centrifuged again under the same conditions, and the supernatant was discarded. This process was repeated twice. The precipitated cells were then resuspended in 10 mL of PBS buffer to prepare whole-cell samples. Finally, the fermentation supernatant and the whole-cell fraction were collected separately, and protein expression was identified using 12% SDS-PAGE.

[0157] 2.3. Wester blotting identification of PPO surface display

[0158] In the *E. coli*-based surface display system used in this invention, the Lpp-OmpA fusion protein was selected as the anchoring unit. The Lpp domain in this anchoring system primarily functions as a directional guide, promoting the migration of the recombinant protein towards the inner region of the outer membrane of the bacteria; while the OmpA component undertakes the final localization of the exogenous protein sequence to the outer surface of the outer membrane. Based on this mechanism, PPO was successfully displayed on the outer membrane protein framework of *E. coli*. To clarify the specific distribution site of this display enzyme on the outer membrane, this invention further employed Western blotting technology for its localization and identification.

[0159] To clarify the subcellular localization of the target protein in the display strain, the following reference was used: Zhang Qian et al., display of xylanase on the cell surface of Escherichia coli [J]. Microbiology Bulletin, 2025, 52(10):4527-4541. This invention fractionally extracts proteins from the periplasmic space, inner membrane, and outer membrane of the bacterial cells. The specific operation procedure is as follows: Take 50 mL of the induced fermentation bacterial solution, centrifuge at 6000×g for 10 min at 4℃, and collect the bacterial precipitate. The precipitate is treated with 20 mL of 20 mmol•L... -1 After washing twice with Tris-HCl buffer (pH 8.0), add 5 mL of pre-chilled TES buffer (composition: Tris-HCl 0.2 mol•L). -1 EDTA 0.5 mol•L -1 0.5 mmol / L sucrose -1 The bacterial cells were resuspended at pH 8.0 and stirred at room temperature for 20 min. Then, 7.5 mL of double-distilled water cooled in an ice bath was added, and the mixture was incubated on ice for another 30 min. The mixture was then centrifuged again at 4 °C and 12000 × g for 30 min, and the supernatant and precipitate were collected separately. The resulting supernatant was filtered through a 0.22 µm filter membrane to obtain the periplasmic space protein fraction.

[0160] The precipitate obtained after centrifugation was treated with 12.5 mL of 10 mmol·L⁻¹ solution. -1 Resuspend thoroughly in Tris-HCl (pH 8.0) and incubate on ice for 1 hour. Then, sonicate at 550 W (5 seconds on, 2 seconds off, total 30 min). Centrifuge the lysate at 5000×g for 10 min at 4 °C to separate the supernatant from the precipitate. Resuspend the precipitate in 5 mL of double-distilled water and add Triton X-100 to a final concentration of 2%. Incubate at room temperature for 10 min, then centrifuge at 12000×g for 40 min. The resulting supernatant is the inner membrane protein component.

[0161] For the extraction of outer membrane proteins, the above centrifuged precipitate is redispersed in 5 mL of buffer containing 2% SDS, and the resulting solution represents the outer membrane protein component.

[0162] To identify the target proteins in the outer membrane, inner membrane, periplasmic space, and whole-cell components of the aforementioned extracted ORF378-TYRC strain, this invention employs Western blotting technology for analysis. The specific operating procedure is as follows.

[0163] First, cut a gel block containing the desired molecular weight band from the SDS-PAGE gel. Simultaneously, prepare one PVDF membrane and ten sheets of filter paper of the same size as the gel; the PVDF membrane must be pre-activated with methanol. Immerse the gel, activated PVDF membrane, and filter paper together in the electroporation buffer for 10 to 30 minutes to equilibrate. After equilibration, stack five sheets of filter paper, the PVDF membrane, the SDS-PAGE gel, and the remaining five sheets of filter paper on the anode plate of the electroporator in a bottom-to-top order. Set the transfer parameters: the current intensity is calculated by multiplying the product of the gel's length and width by 0.8, and the transfer duration is 40 minutes.

[0164] After transfer, the PVDF membrane was removed, washed three times with TBS buffer, and then transferred to blocking buffer. Blocking was performed for 2 hours under low-speed shaking. After blocking, 3 mL of fresh blocking buffer was thoroughly mixed with 1 μL of mouse anti-6×His tag monoclonal antibody (primary antibody) and added to the PVDF membrane. The membrane was incubated overnight at 4°C. The next day, the primary antibody reaction solution was discarded, and the membrane was washed three times with TBST buffer. Subsequently, 3 mL of blocking buffer was mixed with 1 μL of goat anti-mouse IgG antibody (secondary antibody) and added to the membrane. Incubation was continued at 4°C for 2 hours. After secondary antibody incubation, the membrane was washed three more times with TBST buffer.

[0165] Finally, perform the colorimetric reaction: take 800 μL of TBS buffer, 100 μL of solution A from the ECL protein blot substrate kit, and 100 μL of solution B, mix them well, and then drop them evenly onto the surface of the PVDF membrane. The colorimetric signal of the target protein can then be observed.

[0166] 2.4 Immunofluorescence identification of PPO on surface

[0167] To verify whether PPO was successfully displayed on the surface of host cells, this invention uses immunofluorescence technology for detection. The experimental procedure consists of four stages: cell pretreatment, primary antibody labeling, secondary antibody labeling, and fluorescence observation. The specific steps are as follows.

[0168] Cell pretreatment: After centrifugation to obtain bacteria, the cell culture medium was discarded, and the bacterial pellet was resuspended in 1×PBS buffer (0.01M, pH 7.4). The suspension was centrifuged at 5000×g for 5 min at 4°C, and the supernatant was removed. This washing operation was performed twice. After washing, the cell pellet was resuspended in a solution containing 1 mg / mL PBS. -1 Non-immune goat serum in PBS buffer (containing 0.2% Triton X-100).

[0169] Primary antibody labeling: Add 50 μL of mouse anti-6×His-tagged monoclonal antibody (primary antibody) to the cell suspension above, mix well, and incubate on ice for 4 to 5 hours. After incubation, centrifuge the sample at 4°C and 10000×g for 5 min, and discard the supernatant. Then add 0.1 mol•L... -1 Resuspend the bacterial cells in PBS buffer at 5000 rpm. -1 Centrifuge at 4°C for 5 minutes, remove the supernatant, and repeat this washing step twice.

[0170] Secondary antibody labeling: Cells labeled with the primary antibody were resuspended in PBS buffer containing non-immune goat serum, and 50 μL of FITC-labeled goat anti-mouse IgG antibody (secondary antibody) was added. The samples were incubated at 4°C in the dark for 1 hour, followed by centrifugation at 10000×g for 5 min (4°C), and the supernatant was discarded. 0.1 mol•L... -1 Resuspend the bacterial cells in PBS buffer, centrifuge at 5000×g and 4℃ for 5 min, discard the supernatant, and repeat this washing step twice for later use.

[0171] Fluorescence observation: Take 5 to 10 μL of the cell suspension prepared above, add DAPI, and protect from light for 5 minutes. Shake off the excess liquid, prepare a slide specimen, and observe under a fluorescence microscope.

[0172] 2.5 PPO Activity Assay

[0173] The method is slightly modified from Example 1: after the bacterial cells are collected and resuspended, there is no need to perform ultrasonic disruption; the resuspended solution is directly used for enzyme activity determination.

[0174] 2.6 Cell surface display system catalyzes the synthesis of theaflavins

[0175] The method is slightly modified from Example 1. To evaluate the catalytic efficiency of the constructed whole-cell surface display system in TF1 synthesis, this invention uses a reaction system without added bacterial suspension as a reference. A certain amount of whole-cell catalyst bacterial cells are sampled and suspended in the optimal reaction buffer to form a homogeneous bacterial suspension, followed by the addition of substrate to initiate the transformation reaction. The reaction time is set to 180 rpm. -1 The substrate concentration ratio was 1:1, pH 5.0, temperature 30℃, and reaction time 60 min. After each reaction, centrifugation was performed, and an appropriate amount of reaction supernatant was taken. The target product TF1 was quantitatively analyzed by high performance liquid chromatography. Multiple batches of the bacterial cells were repeatedly recycled to examine the batch recycling performance of the whole-cell catalyst.

[0176] 3 Results and Analysis

[0177] 3.1 Construction and Analysis of PPO Cell Surface Display System

[0178] Following the method described in Example 1, ORF378-TYRC from *Streptomyces castaneoglobisporus* was used. Its encoding gene was fused with the fusion gene Lpp-OmpA, representing an outer membrane protein and its secretory signal peptide, using unrestricted cloning to construct a polyphenol oxidase surface display vector for *E. coli*. The obtained recombinant plasmid DNA was double-digested and analyzed by agarose gel electrophoresis. The results are as follows:

[0179] DNA agarose gel electrophoresis images from double enzyme digestion verification and PPO colony PCR verification of PPO expression vector (see [link]). Figure 14 , Figure 15 The test results showed that the PPO fragment size of Streptomyces castaneoglobisporus was correct. Sequencing analysis of the recombinant plasmid showed results consistent with expectations. Therefore, subsequent fermentation of the strain and expression analysis of the PPO protein can be performed for verification.

[0180] 3.2 SDS-PAGE Identification Analysis of PPO Surface Display

[0181] The correctly identified PPO surface-displaying strain ORF378-TYRC was inoculated into an Erlenmeyer flask containing 10 mL of LB liquid medium (final Amp concentration 100 μg / mL). -1 The culture medium was placed on a shaker and cultured under the following conditions: temperature 37℃, rotation speed 200 rpm, and culture time 14-16 h. When the cell concentration reached OD600 0.6-0.8, 2 mL of the seed culture was inoculated into 100 mL of TB medium. The Erlenmeyer flask was then placed on a shaker for further culture. The culture conditions were set as follows: temperature 37℃, rotation speed 200 rpm. -1 The fermentation time is 4-6 hours. When the OD600 of the fermentation broth reaches 0.8-1.2, 0.5 mM IPTG is added to induce expression, and the culture conditions are adjusted to a temperature of 25℃ and a rotation speed of 180 r•min. -1 Continue induction culture for 12-16 hours. Use uninduced cells as a control. Collect fermentation broth supernatant and whole cell fractions from ORF378-TYRC induced for 16 hours and uninduced cells respectively, and perform SDS-PAGE identification. The results are as follows. Figure 16 As shown, the whole-cell fraction of ORF378-TYRC after induction has a clear and distinct target band, and the size is consistent with the theoretical size of the target protein (45.4 kDa), indicating that PPO was successfully expressed.

[0182] 3.3 Immunofluorescence Identification and Analysis of PPO Displayed on Surface

[0183] The constructed PPO surface display strain has a 6×His tag at its C-terminus. Using an antigen-antibody reaction, the PPO is fluorescein-labeled. If the PPO protein is successfully displayed on the surface of the *E. coli* cell wall, the displayed strain exhibits green fluorescence under a fluorescence microscope, with the green fluorescence distributed across the cell surface. DAPI was used to counterstain the cell nucleus and mark cell localization; the DAPI channel appears blue. Fluorescence microscopy identification results are as follows: Figure 17 As shown: Induced ( Figure 17 (B) and uninduced ( Figure 17 Blue localization fluorescence was observed in all cells of the ORF378-TYRC strain (D), but only the induced surface-displaying strain ORF378-TYRC showed green fluorescence on the cell surface. Figure 17 (A), uninduced almost no green fluorescence ( Figure 17 (C) This indicates that the PPO protein was successfully displayed on the surface of E. coli cells.

[0184] 3.4 Wester blotting analysis of PPO surface display

[0185] To precisely locate the position of PPO displayed in ORF378-TYRC cells, proteins from different components, including outer membrane proteins, inner membrane proteins, and periplasmic space, were extracted from induced whole-cell ORF378-TYRC cells and analyzed using Western blotting. The results are as follows: Figure 18 As shown, the target protein was detected in both whole-cell and outer membrane protein fractions of ORF378-TYRC, and its size was consistent with the theoretical size of the target protein (45.4 kDa), indicating that PPO was successfully displayed on the outer membrane of E. coli.

[0186] 3.5 PPO Activity Assay PPO Analysis

[0187] Using uninduced bacterial cells as a negative control, the enzyme activity assays of the whole-cell surface display system using EC, ECG, EGC, and EGCG as substrates were performed as follows: Figure 19 As shown, the induced whole cells exhibited enzyme activity against all four substrates. This demonstrates the feasibility of the surface display system of the recombinant strain ORF378-TYRC.

[0188] 3.6 Analysis of the catalysis and theaflavin formation of the cell surface display system

[0189] Based on the above enzyme activity assay, an experimental study was conducted on the synthesis of TF1 by whole-cell catalysts, with uninduced cells used as the control group.

[0190] The bacterial cells were collected and prepared into a concentration of 100 g / L using citrate-phosphate buffer at pH 5.0. -1The bacterial suspension was prepared. Substrate was added to the system to achieve a final concentration of 3 mg / mL for both EC and EGC. -1 After thorough mixing, place at 30℃ and incubate at 180 r•min. -1 The conversion reaction was carried out at a rotation speed of [speed value missing]. After 60 minutes of reaction, the cells were centrifuged, and the supernatant was analyzed by HPLC. The remaining bacterial cells were reused for the next batch of conversion reaction.

[0191] The color of theaflavins produced in multiple batches of reactions is as follows: Figure 20 As shown: from the first batch to the eighth batch, there is color and it gradually becomes lighter, which is initially estimated to be a gradual decrease in the amount of TF1 generated.

[0192] HPLC results of TF1 concentration in multiple batches of reactions are as follows Figure 21 As shown, the amount of theaflavins synthesized by the cell surface display system was significantly higher than that of the control cells, while the TF1 production in the uninduced control group was extremely low. The concentration of TF1 synthesized by the whole-cell surface display strain ORF378-TYRC decreased sequentially across multiple batches, consistent with the above predictions. From the first to the fourth batch, the theaflavin TF1 production remained at 2.5 mg / mL. -1 As the number of reaction batches increases, the bacteria may lose some enzyme activity during the reaction and repeated recycling process, resulting in a decreasing trend in the production of theaflavins.

Claims

1. A method for the enzymatic synthesis of theaflavins using polyphenol oxidase, characterized in that, Polyphenol oxidase catalyzes substrates EC+EGC, EC+EGCG, ECG+EGC, or ECG+EGCG to generate target products theaflavins, theaflavins-3-gallate, theaflavins-3′-gallate, and theaflavins-3,3′-bisgallate.

2. The method for the enzymatic synthesis of theaflavins using polyphenol oxidase according to claim 1, characterized in that, The polyphenol oxidase is derived from microorganisms capable of producing polyphenol oxidase. Preferably, the polyphenol oxidase is derived from Streptomyces or Bacillus megaterium, and more preferably from Streptomyces pETDuet-1-ORF378-TYRC.

3. The method for the enzymatic synthesis of theaflavins using polyphenol oxidase according to claim 1 or 2, characterized in that, The polyphenol oxidase is a crude enzyme, which is the supernatant obtained by centrifuging the fermentation broth of a microorganism capable of producing polyphenol oxidase. Preferably, the method for collecting the supernatant is to collect the fermentation broth, centrifuge and discard the supernatant, wash with PBS, resuspend, lyse, and then centrifuge again to collect the supernatant.

4. The method for the enzymatic synthesis of theaflavins using polyphenol oxidase according to claim 1 or 2, characterized in that, The polyphenol oxidase is a whole-cell catalyst. Preferably, in the preparation of the whole-cell catalyst, cetyltrimethylammonium bromide, Triton X-100 or toluene is used as a permeation reagent, and the concentration of the permeation reagent is 0.25%-2%, preferably 0.3%-1.0%, more preferably 0.5%; the permeation treatment time is 10-60 min, preferably 40 min.

5. The method for the enzymatic synthesis of theaflavins using polyphenol oxidase according to claim 1 or 2, characterized in that, The polyphenol oxidase is anchored on the surface of host cells via a cell surface display system and performs whole-cell catalytic synthesis of theaflavins.

6. The method for the enzymatic synthesis of theaflavins using polyphenol oxidase according to claim 5, characterized in that, The method of anchoring the polyphenol oxidase on the surface of a host cell via a cell surface display system includes fusing the encoding gene of polyphenol oxidase ORF378-TYRC derived from Streptomyces with the fusion gene Lpp-OmpA of Escherichia coli outer membrane protein and its secretory signal peptide to construct a polyphenol oxidase Escherichia coli surface display vector.

7. The method for the enzymatic synthesis of theaflavins using polyphenol oxidase according to claim 6, characterized in that, The method for constructing the polyphenol oxidase Escherichia coli surface display vector includes using the genomic DNA of an Escherichia coli clone strain as a cloning template and performing PCR amplification using primers to obtain the ORF378-TYRC fragment. Preferably, the nucleotide sequence of the primers is: ORF378-TYRC-F:5'-GGATCCATGCCGGAAATTACCCGTCGTCGCGCCCTGACCGCCGCCGCAGCAGTGG-3'; ORF378-TYRC-R:5'-GAATTCTGCATCAAAGGTATAATATGCGGTATGATCC-3'; The amplified ORF378-TYRC gene fragment was purified and recovered. Using pLpp-OmpA plasmid as a template, PCR was performed using the purified and recovered ORF378-TYRC gene fragment as a large primer to insert and fuse ORF378-TYRC into the pLpp-OmpA vector, thereby obtaining the pLpp-OmpA-PPO display expression vector.

8. The method for the enzymatic synthesis of theaflavins using polyphenol oxidase according to any one of claims 1-7, characterized in that, The substrate for the enzymatic synthesis is EC+EGC, with an EGC:EC ratio of 0.2:1-4:1, more preferably 0.5:1-3:1, even more preferably 1:1-2:1, and most preferably 1:1; pH 4.5-6.5, preferably pH 4.8-6.0, even more preferably pH 5.0; temperature 10-60℃, preferably 20℃-50℃, even more preferably 25℃-40℃, and even more preferably 30℃; reaction time 5min-120min, preferably 30-90min, even more preferably 40-80min, and most preferably 60min.

9. The application of the method as described in claims 1-8 in the preparation of theaflavins.

10. The use of the theaflavins prepared by the method according to claims 1-8 in the production of food, biological agents, health products and pharmaceuticals.

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