A recombinant escherichia coli engineering bacteria and a method for preparing theaflavin by using whole cell catalysis

Abstract

The application belongs to the technical field of biotechnology, and relates to a kind of recombinant escherichia coli engineering bacteria and a method for preparing theaflavin by whole cell catalysis thereof.The engineering bacteria realizes the co-expression of double polyphenol oxidase Bmtyrc24 and Bmtyrc25 mutants in a vector, and a whole cell catalyst is prepared based on the engineering strain, and theaflavin digallate (TFDG) is catalytically synthesized using epicatechin gallate (ECG) and epigallocatechin gallate (EGCG) as substrates.The whole cell catalyst prepared by the escherichia coli engineering strain of the application is applied to the catalytic synthesis of theaflavin TFDG, and compared with the prior art, has the advantages of simpler operation, higher catalytic efficiency on substrates, higher product TFDG yield, good multi-batch reaction stability and lower production cost.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the construction of a recombinant engineered Escherichia coli strain E. coli BL21 / pETDuet-Bmtyrc-24-Bmtyrc-25, the preparation of a whole-cell E. coli catalyst, and a method for catalyzing the production of theaflavins using it. Background Technology

[0002] Black tea is the most consumed type of tea in the world, accounting for 70-80% of total tea consumption. Theaflavins are key active ingredients in the quality of black tea. Their formation process involves the oxidation and self-polymerization of tea polyphenols under the catalysis of polyphenol oxidases and peroxidases within cells, resulting in a class of substances that typically transform into theaflavins, thearubigins, and theabrownins. Studies have shown that theaflavins possess a range of health benefits, such as antioxidant, anti-inflammatory, blood sugar-lowering, lipid-lowering, weight-loss, and antiviral functions. Therefore, the development of theaflavin-based products has broad market prospects.

[0003] Currently, theaflavins are mainly obtained through three routes: (1) direct extraction from black tea; (2) chemical oxidation synthesis; and (3) biocatalytic synthesis. Since theaflavins are present in relatively small amounts in black tea, accounting for only 2-3% of its dry weight, extraction from black tea requires organic solvents, which easily causes environmental pollution and involves cumbersome purification processes, thus not conforming to the concept of low-carbon and green development. Similarly, chemical oxidation methods also have the same drawbacks. Chemical oxidation processes require chemical oxidizing agents such as potassium ferricyanide, ferric oxide, oxidases, and acids and bases, resulting in severe environmental pollution. Furthermore, due to the lack of specificity for the substrate, chemical oxidation methods easily form byproducts, limiting the control, separation, and purification of subsequent product quality. Biocatalytic synthesis can overcome these drawbacks. Biocatalytic oxidation uses tea polyphenols as a substrate, exhibiting strong specificity and high specificity. The reaction conditions are mild, do not require organic solvents, and are highly controllable, conforming to the green and low-carbon development direction and concept, making it the most promising production method for the future. Current reports primarily focus on extracting polyphenol oxidase from plant cell lysates to catalyze the synthesis of theaflavins from catechin substrates. However, this method is limited by raw material availability, susceptible to seasonal variations, and suffers from low yields of polyphenol oxidase extracted from plants, hindering large-scale control and application. Therefore, employing modern genetic engineering and protein engineering techniques to prepare exogenous polyphenol oxidases for efficient, multi-batch reuse in the synthesis of theaflavins aligns with green and low-carbon development principles, offering high production efficiency and low cost, and possessing broad market prospects.

[0004] There are already reports in relevant literature or patents on the in vitro enzymatic catalytic synthesis of theaflavins. For example, in the literature (Wang Bin, Jiang Heyuan, et al. Continuous preparation of theaflavins by immobilized polyphenol oxidase packed bed reactor [J]. Food and Fermentation Industries, 2011, 37(05):40-44), polyphenol oxidase was immobilized and the continuous preparation of theaflavins was achieved. The reaction conditions for the continuous preparation of theaflavins were explored, showing that the conversion rate of catechins can reach 20.99% when the reaction pH is 5.6 and the temperature is 28℃, which provides a new idea for the industrial preparation of theaflavins. In patent CN201580034729.3, the inventors used metal nanoparticles to anchor the theaflavin catalyst and achieved the preparation of theaflavins. In patent CN202110868216.5, the inventors screened and modified the catalyst for theaflavin synthesis and obtained a catalyst that can efficiently catalyze the synthesis of theaflavins. To achieve the in vitro enzymatic targeted synthesis of theaflavins, enzyme efficiency and cost are key. This invention addresses these two key issues by, on the one hand, constructing a co-expression model of polyphenol oxidase to solve the problem of low enzyme catalytic efficiency for different polyphenol substrates; on the other hand, constructing a whole-cell catalyst for the preparation of theaflavins, solving the problem of stable use of the enzyme in multiple batches and reducing costs.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] The primary objective of this invention is to provide a genetically engineered bacterial strain for the catalytic synthesis of theaflavins and a method for catalytic synthesis of theaflavins using a whole-cell catalyst prepared from this strain. Compared with existing reported techniques, this invention offers the following advantages: co-expression of two polyphenol oxidases results in higher catalytic efficiency for different substrates EGCG and ECG, leading to a higher yield of theaflavins (TFDG); secondly, compared to traditional immobilized enzyme preparation methods, the whole-cell catalyst eliminates the need for carriers, saving raw material costs; and the preparation of the bacterial whole-cell catalyst is simpler, faster, lower in cost, and allows for multiple batches to be recycled.

[0007] To achieve this objective, the technical solution adopted by the present invention is as follows:

[0008] A recombinant engineered Escherichia coli strain containing an expression vector for an enzyme mutant gene Bmtyrc24 with an amino acid sequence as shown in SEQ ID NO.1 and / or an enzyme mutant gene Bmtyrc25 with an amino acid sequence as shown in SEQ ID NO.2.

[0009] The enzyme mutant Bmtyrc24 of this invention is a polyphenol oxidase with high catalytic activity toward the substrate EGCG; the enzyme mutant Bmtyrc25 is also a polyphenol oxidase with high catalytic activity toward the substrate ECG.

[0010] This invention provides the application of the recombinant Escherichia coli engineered strain in the whole-cell catalytic preparation of theaflavins. Specifically, it includes the following steps:

[0011] (1) Preparation of whole-cell catalyst: The recombinant Escherichia coli engineered bacteria were activated and fermented to induce the expression of the target enzyme. The bacterial cells were then collected. The bacterial cells were resuspended with a permeability reagent, centrifuged, the supernatant was removed, and the cells were resuspended with deionized water and centrifuged again for later use.

[0012] (2) Synthesis of theaflavins TFDG: Prepare a substrate solution, add an appropriate amount of bacterial cells for resuspending, place under appropriate conditions to catalyze the synthesis of theaflavins TFDG, and centrifuge to obtain a solution containing the product from the supernatant.

[0013] The permeation reagent used in step (1) includes at least one of the following: cetyltrimethylammonium bromide (CTAB), polyethylene glycol octyl ether Triton X-100, toluene, and ethanol solution. Preferably, it is 0.5-1% CTAB, 0.5-2% polyethylene glycol octyl ether Triton X-100, 0.5-1% toluene, and 5% ethanol solution.

[0014] The reaction concentration of the permeation reagent used in step (1) is 0.5%-4%, the treatment time is 10-120 min, preferably 30-40 min; the pH is 3.0-5.0, and the temperature is 28-32 ℃.

[0015] The reaction parameters for the catalytic synthesis of theaflavins (TFDG) in step (2) are: whole-cell catalyst 50-100 g / L, substrate solution pH 4.0-6.0, reaction temperature 25-35℃, Cu 2+ The concentration was 0.1-0.2 mM, the reaction time was 30-60 min, the substrate EGCG concentration was 1-8 mg / mL, the substrate ECG concentration was 1-4 mg / mL, and the stirring speed was 180-200 r / min.

[0016] Step (2) The substrate solution includes citrate-phosphate buffer.

[0017] After the reaction in step (2) is completed, the bacterial precipitate obtained by centrifugation can be reused for the next batch of catalytic reaction.

[0018] The present invention also provides an enzyme mutant Bmtyrc24 with an amino acid sequence as shown in SEQ ID NO.1 and / or an enzyme mutant Bmtyrc25 with an amino acid sequence as shown in SEQ ID NO.2.

[0019] The present invention also provides expression vectors containing enzyme mutant gene Bmtyrc24 with amino acid sequence as shown in SEQ ID NO.1 and / or enzyme mutant gene Bmtyrc25 with amino acid sequence as shown in SEQ ID NO.2.

[0020] Taking TFDG oxidation products as an example, the preparation method steps are as follows:

[0021] (1) Construction of genetically engineered strains: The polyphenol oxidase gene Bmtyrc24 was constructed into the multiple cloning site MCS1 of the vector pETDuet-1. The gene has an NcoI restriction site at the 5' end and a HindIII restriction site at the 3' end. The polyphenol oxidase gene Bmtyrc25 was constructed into the multiple cloning site MCS2 of pETDuet-1. The gene has an NdeI restriction site at the 5' end and an XhoI restriction site at the 3' end.

[0022] (2) Preparation of whole-cell catalyst: The constructed engineered strain was activated, and the activated strain was fermented and cultured to induce the expression of the target enzyme. The bacterial cells were collected by centrifugation. The bacterial cells were resuspended in a solution containing 0.5-4% chemical reagent and treated at 30℃ for 60 min. Then, the supernatant was removed by centrifugation, and the cells were resuspended in deionized water and centrifuged again for later use.

[0023] (3) Synthesis of theaflavins TFDG. The substrate solution was prepared with citrate-phosphate buffer, and an appropriate amount of bacterial cells was added for resuspension. The solution was then placed under appropriate conditions to catalyze the synthesis of theaflavins TFDG. After centrifugation, the supernatant was obtained as a solution containing the product. The bacterial cell precipitate could be reused for the next batch of catalytic reactions.

[0024] Compared with existing conventional methods, the advantages of the method of the present invention are as follows:

[0025] (1) Two polyphenol oxidases were co-expressed, and their catalytic efficiency against different substrates EGCG and ECG was higher, and the yield of theaflavin TFDG was higher.

[0026] (2) Compared with the traditional preparation of immobilized enzymes, whole-cell catalyst preparation does not require the addition of carriers, eliminates the cost of raw materials, is simpler to operate, has lower cost, and the generated products will not bind to the cells (carriers) and cause enzyme activity loss. It can be recycled and reused in multiple batches, significantly reducing production and application costs and has industrial application attributes. Detailed Implementation

[0027] The specific embodiments of the present invention will be further described below with reference to the examples and accompanying drawings.

[0028] The method for determining polyphenol oxidase activity is as follows (using polyphenols EGCG or ECG as substrates):

[0029] Substrate composition: 10 mM EGCG or ECG solution, dissolved in 90 mL of 0.1 mol / L pH 5.0 citrate-phosphate buffer, preheated to 30 °C, then diluted to 100 mL with buffer. 0.01 mol / L copper sulfate solution. Accurately weigh 0.249 g CuSO4·5H2O and dissolve in 100 mL of water.

[0030] In a 1 cm quartz cuvette, add 4 mL of 0.01 mol / L substrate solution preheated to 30 °C, followed by 0.1 mL of 0.01 mol / L copper sulfate solution. Mix thoroughly and zero the sample at 420 nm. Then add 100 μL of crude enzyme solution, mix quickly, and begin the measurement. Record the absorbance (A420) every 10 seconds. Enzyme activity units: at 25 °C and pH 5.0, the absorbance at OD0.05 per minute... 420 nm A change of 0.001 is defined as one unit (1U).

[0031] HPLC detection method for theaflavins:

[0032] HPLC analysis was performed using an Agilent liquid chromatograph. Column: 5C18-AR-II (250 mm × 4.6 mm, 5 μm); Column temperature: 40 ℃; Injection volume: 20 μL; Detection wavelength: 280 nm; Mobile phase: A: 50 mmol / L phosphoric acid; B: acetonitrile-ethyl acetate (7:1, V / V); Flow rate: 1.0 mL / min; Elution program: 0–25 min, 82%–68% A phase.

[0033] TFDG yield calculation, TFDG yield (%) = a: Molar amount of TFDG produced in the reaction, b: Molar amount of ECG added in the reaction.

[0034] Example 1: Screening for polyphenol oxidase genes with high activity against substrates EGCG and ECG

[0035] Polyphenol oxidases and their mutant strains from different sources were selected from a polyphenol oxidase gene library constructed in the laboratory. The activities of EGCG and ECG as substrates were measured, and strains with high activity (polyphenol oxidase genes) were selected for subsequent co-expression vector construction. The activity of EGCG and ECG under the same bacterial cell volume was analyzed through screening enzyme genes from different sources as follows:

[0036] Table 1: Activity of polyphenol oxidases from different sources on EGCG and ECG

[0037]

[0038] As shown in the table above, through screening polyphenol oxidases from different sources, the polyphenol oxidase mutant Bmtyrc-24 from Bacillus megaterium exhibits the highest catalytic activity for the substrate ECG, while the polyphenol oxidase mutant Bmtyrc-25 exhibits the highest catalytic activity for the substrate EGCG. Therefore, the two can be co-expressed to construct a co-expression strain of Bmtyrc-24 and Bmtyrc-25, thereby further improving the catalytic efficiency for the substrates EGCG and ECG, and further enhancing the catalytic synthesis performance of the product TFDG.

[0039] Example 2: Construction of a genetically engineered strain of polyphenol oxidase with high activity against substrates EGCG and ECG

[0040] The polyphenol oxidase gene Bmtyrc24 was constructed into the multiple cloning site MCS1 of the pETDuet-1 vector (Novagen). The gene contains an NcoI restriction site at its 5' end and a HindIII restriction site at its 3' end. The polyphenol oxidase gene Bmtyrc-25 was constructed into the multiple cloning site MCS2 of pETDuet-1. The gene contains an NdeI restriction site at its 5' end and an XhoI restriction site at its 3' end. After verification by restriction enzyme digestion and sequencing, the co-expression plasmid was constructed into the *E. coli* expression strain BL21(DE3) using CaCl2 heat shock transformation. An appropriate amount was plated onto LB agar plates containing ampicillin resistance and incubated overnight at 37°C for 12–16 h. The resulting positive engineered clones of polyphenol oxidase were obtained from the culture.

[0041] Example 3: Preparation of whole-cell catalyst

[0042] Single colonies were picked from LB solid medium and inoculated into LB liquid medium, incubated overnight at 37°C with shaking at 200 rpm. The next day, 2% colonies were inoculated into TB fermentation medium and incubated at 37°C with shaking at 200 rpm for 5-6 hours, waiting for OD. 600nm When the enzyme concentration reached 0.6, 1% lactose was added, and the cells were induced to mature at 200 rpm and 25°C for 8-12 h. Enzyme activity was then measured according to the above method, and whole-cell catalysts were prepared. The main steps are as follows:

[0043] (1) Collect the bacterial cells. Transfer the fermentation cells to a centrifuge tube, centrifuge at 12000 r / min for 5 min, and discard the supernatant;

[0044] (2) Resuspend in deionized water. Add an appropriate amount of deionized water to the centrifuge tube and resuspend thoroughly with a pipette tip. Repeat 1-2 times.

[0045] (3) Selection of the optimal permeability reagent. The selection of the optimal permeability reagent mainly considers the following factors: simple processing; able to ensure the integrity of bacterial cells; fully exhibiting catalytic activity; and able to be used repeatedly in multiple batches. After collecting the bacterial cells, they were resuspended in appropriate deionized water and treated with different permeability reagents for 60 min under the same conditions (temperature 30℃, pH 7.0). After treatment, the activity was measured using polyphenol EGCG as a substrate. The results are as follows:

[0046] Table 2: Effects of different permeability reagents

[0047]

[0048] As can be seen from the above, the system treated with 0.5% polyethylene glycol octyl ether Triton X-100 showed the highest enzyme activity recovery rate and can be used as a permeability reagent for the preparation of whole-cell catalysts.

[0049] (4) Optimization of whole-cell catalyst preparation conditions. Under the conditions of optimal permeability reagent and concentration (permeability reagent: polyethylene glycol octyl ether Triton X-100, concentration 0.5%), different treatment times and pH factors were optimized. The catalytic synthesis performance of the treated whole-cell catalyst for TFDG was evaluated (reaction conditions for TFDG synthesis by whole-cell catalyst: pH 5.0, EGCG: 4 mg / mL, ECG: 2 mg / mL), thereby determining the optimal conditions for the preparation of the whole-cell catalyst.

[0050] Table 3: Effects of different pH treatments on whole-cell catalysts

[0051]

[0052] Table 3 shows that the enzyme activity varies under different pH conditions. The activity is highest at pH 5.0, and the yield of TFDG is also the highest. As the pH increases, the yield decreases sharply, and the product binds and aggregates with the whole-cell catalyst, making the product impossible to separate. Therefore, pH 5.0 can be considered the optimal condition for preparing permeability reagents.

[0053] The effect of different treatment times on the catalytic performance of the whole-cell catalyst was investigated at pH 5.0.

[0054] Table 4: Effects of different treatment times on whole-cell catalysts

[0055]

[0056] As can be seen from Table 4, different treatment times have little effect on the activity of whole-cell catalysts, but have a significant impact on the yield of TFDG. As shown in the table, the TFDG yield is the highest when treated with 0.5% permeability reagent polyethylene glycol octyl ether Triton X-100 for 40 min, reaching 39.23%, which is the optimal time for permeability reagent treatment.

[0057] Example 4: Performance evaluation of whole-cell catalyst for the synthesis of theaflavins

[0058] Performance evaluation of whole-cell catalyst in the synthesis of theaflavins. A suitable amount of permeability-treated whole-cell catalyst cells were prepared into a bacterial suspension with a concentration of 50-100 g / L (wet weight) using citrate-phosphate buffer (pH 5.0). Substrate was added to achieve a final EGCG concentration of 4 mg / mL and a final ECG concentration of 2 mg / mL. After thorough mixing, the mixture was incubated at 30℃ for 60 min with a shaking speed of 180-200 r / min. After the reaction, a suitable amount of supernatant was collected for HPLC analysis of the product TFDG. The collected cells after centrifugation were used for the next batch of repeat reactions. Cells with equivalent activity but without permeability treatment and resin-fixed immobilized enzymes were used as controls.

[0059] Table 5: Whole-cell catalyst-catalyzed synthesis of TFDG

[0060]

[0061] As can be seen from Table 5, the whole-cell catalyst for the preparation of theaflavins TFDG has significant advantages over resin-immobilized enzymes and untreated bacterial control in catalytic TFDG synthesis. The TFDG yield can reach more than 35% in 12 consecutive batches of reaction. Compared with existing methods, this technology is simpler to operate, has a higher yield, and the whole-cell catalyst can be reused for multiple batches, resulting in lower cost.

[0062] The above embodiments or implementation methods are merely illustrative of the present invention. The present invention may also be implemented in other specific ways or forms without departing from the spirit or essential characteristics of the invention. Therefore, the described embodiments should be considered illustrative rather than limiting in any respect. The scope of the present invention should be defined by the appended claims, and any variations equivalent to the intent and scope of the claims should also be included within the scope of the present invention.

[0063] SEQ ID NO. 1

[0064] Bmtyrc-24 amino acid sequence

[0065] MSNKYRVRKNVLHLTDTEKRDFVRTVLILKEKGIYDRYIAWHGAAGKFHTPPGSDRNAAHMSSAFLPWHREYLLRFERDLQSINPEVTLPYWEWETDAQMQDPSQSQIWSADFMGGNGNPIKDFIVDTGPFAAGRWTTIDEQGNPSGGLKRNFGATKEAPTLPTREGVLNALKITQYDTPPWDMTSQNSFRNQLEGFINGPQLHDRVHRWVGGQMGVVPTAPNDPVFFLHHANVDRIWAVWQIIHRNQNYQPMKNGPFGQNFRDPMYPWNTTPEDVTNHRKLGYIYDIELRKSKRSSLEHHHHHH

[0066] SEQ ID NO. 2

[0067] Bmtyrc-25 amino acid sequence

[0068] MSNKYRVRKNVLHLTDTEKRDFVRTVLILKEKGIYDRYIAWHGAAGKFHTPPGSDRNAAHMSSAFLPWHREYLLRFERDLQSINPEVTLPYWEWETDAQMQDPSQSQIWSADFMGGNGNPIKDFIVDTGPFAAGRWTTIDEQGNPSGGLKRNFGATKEAPTLPTREGVLNALKITQYDTPPWDMTSQNSFRNQLEGFINGPQLHDRVHRWVGGQMGVVPTAPNDPVFFLHHANVDRIWAVWQIIHRNQNYQPMKNGPFGQNFRDPMYPWNTTPEDVMNHRKLDYIYDIELRKSKRSSLEHHHHHH

Claims

1. A recombinant engineered Escherichia coli strain, characterized in that, Expression vectors containing the enzyme mutant gene Bmtyrc24 with the amino acid sequence shown in SEQ ID NO. 1 and / or the enzyme mutant gene Bmtyrc25 with the amino acid sequence shown in SEQ ID NO.

2.

2. The application of the recombinant Escherichia coli engineered strain according to claim 1 in the whole-cell catalytic preparation of theaflavins, characterized in that, Includes the following steps: (1) Preparation of whole-cell catalyst: The recombinant Escherichia coli engineered bacteria were activated and fermented, and the target enzyme was induced to express. The bacterial cells were collected. The bacterial cells were resuspended with a permeation reagent, centrifuged, the supernatant was removed, and the cells were resuspended with deionized water and centrifuged for later use. (2) Synthesis of theaflavins TFDG: The substrate solution was prepared, an appropriate amount of bacterial cells were added and resuspended, and the theaflavins TFDG were catalyzed under appropriate conditions. After centrifugation, the supernatant was the solution containing the product. The permeation reagent used in step (1) included at least one of cetyltrimethylammonium bromide (CTAB), polyethylene glycol octyl ether Triton X-100, toluene, and ethanol solution. The reaction concentrations of cetyltrimethylammonium bromide (CTAB) and toluene were 0.5%-3%, the reaction concentrations of polyethylene glycol octyl ether Triton X-100 were 0.5-4%, and the reaction concentrations of ethanol were 5-20%. The treatment time was 10-60 min.

3. The application according to claim 2, characterized in that, Step (1) Add permeability reagent to react at pH 3.0-5.0 and temperature 28-32 ℃.

4. The application according to claim 2, characterized in that, The reaction parameters for the catalytic synthesis of theaflavins (TFDG) in step (2) are: whole-cell catalyst 50-100 g / L, substrate solution pH 4.0-6.0, reaction temperature 25-35℃, Cu 2+ The concentration was 0.1-0.2 mM, the reaction time was 30-60 min, the substrate EGCG concentration was 1-8 mg / mL, the substrate ECG concentration was 1-4 mg / mL, and the stirring speed was 180-200 r / min.

5. The application according to claim 2, characterized in that, Step (2) The substrate solution includes citrate-phosphate buffer.

6. The application according to claim 2, characterized in that, After the reaction in step (2) is completed, the bacterial precipitate obtained by centrifugation can be reused for the next batch of catalytic reaction.

7. The enzyme mutant Bmtyrc24 with the amino acid sequence shown in SEQ ID NO.1 or the enzyme mutant Bmtyrc25 with the amino acid sequence shown in SEQ ID NO.

2.

8. An expression vector containing the enzyme mutant gene Bmtyrc24 with the amino acid sequence shown in SEQ ID NO. 1 and / or the enzyme mutant gene Bmtyrc25 with the amino acid sequence shown in SEQ ID NO. 2.

Images