A method for preparing glycerol-type phospholipid derivatives

Abstract

The present invention belongs to the technical field of enzyme genetic engineering, and specifically relates to a method for synthesizing and preparing a glycerol derivative. The present application co-expresses polyphosphate kinase and glycerol kinase, thereby improving the ability of polyphosphate kinase to regenerate ATP. The co-expressed polyphosphate kinase and glycerol kinase are immobilized, and the immobilized enzyme is applied to the batch preparation of GPA, using glycerol and ADP as substrates, and using inexpensive PolyP to provide phosphate groups, thereby solving the problem of expensive ATP and realizing low-cost preparation of GPA. The co-expressed polyphosphate kinase glycerol kinase is synthetically coupled with GPCMP, and inexpensive PolyP is used to provide phosphate groups, using glycerol, adenosine diphosphate (ADP), and cytidine monophosphate (CMP) as substrates, thereby solving the problem of expensive ATP and cytidine triphosphate (CTP), thereby realizing low-cost preparation of GPCMP.

Description

Technical field:

[0001] The invention belongs to the technical field of enzyme genetic engineering, and specifically relates to a method for synthesizing and preparing a glycerol derivative. Background technology:

[0002] Glycerol derivatives are important metabolic intermediates in cell functions and are also the starting compounds of many biologically active phospholipids. The enzymatic synthesis of glycerol derivatives is different from the existing chemical synthesis method and has milder synthesis conditions. The enzymes involved in the enzymatic synthesis of glycerol derivatives include glycerol kinase (GK), glycerol-3-phosphate cytidyl transferase (G3PCT), etc. Glycerol kinase (EC2.7.1.30) can specifically catalyze the phosphorylation of glycerol to produce glycerophosphatidic acid (GPA). Glycerol-3-phosphate cytidyl transferase (2.7.7.39) can catalyze the reaction of cytidine-5'-triphosphate (CTP) and glycerophosphatidic acid (GPA) to produce glycerophosphatidyl monophosphate cytidine (GPCMP), which is an intermediate required for the synthesis of many biologically active phospholipids.

[0003] In vitro enzyme cascades have emerged as an environmentally friendly and economical alternative for the production of valuable chemicals. The construction of in vitro biological systems often presents the problem of expensive cofactors required for cofactor-dependent reactions. One of the important cofactors, adenosine triphosphate (ATP), acts as an energy carrier in many biocatalytic cascades. It is a multifunctional nucleotide that plays an important role in cell biology.

[0004] ATP-dependent enzymes form new bonds, such as ester, thioester or amide bonds, while coupling with the ATP hydrolysis reaction. Many useful industrial materials are synthesized by ATP-dependent enzymes. The application of in vitro energy-dependent enzyme cascades in industrial production is limited by the expensive substrate ATP and is often problematic due to the inhibitory effects of the formed adenosine diphosphate (ADP) and adenosine monophosphate (AMP). In ATP-dependent multienzymatic reactions, ATP regeneration systems help reduce the cost of direct ATP use and promote the development of industrial processes.

[0005] Enzymatic systems for ATP regeneration are of great significance in the industrial applications of ATP-dependent enzymes. Several types of ATP regeneration reactions have been studied, such as phosphoenolpyruvate / pyruvate kinase, phosphoacetyl / acetate kinase, and polyphosphate (PolyP) / polyphosphate kinase (PPK)-based systems. Compared with phosphate donors such as phosphoenolpyruvate and acetyl phosphate, PolyP has the advantages of low cost, high stability, and easy availability, and is widely used as a substrate for ATP regeneration. PolyP is an inorganic polymer composed of phosphate residues connected by anhydride bonds, which plays an important role in cellular biological processes. It is stable under a wide range of temperature, pH, and oxidant conditions. ATP-dependent enzymes usually consume ATP and release inorganic phosphate and ADP. PPK can synthesize ATP using ADP and PolyP as substrates, which is suitable for these coupled reaction systems that require ATP-consuming enzymes.

[0006] Therefore, we established a synthetic route for the catalytic preparation of GPA and GPCMP based on an ATP regeneration system. Summary of the invention:

[0007] The present application co-expresses polyphosphate kinase and glycerol kinase, thereby improving the ability of polyphosphate kinase to regenerate ATP. The co-expressed polyphosphate kinase and glycerol kinase are immobilized, and the immobilized enzymes are applied to the batch preparation of GPA, using glycerol and ADP as substrates, and using inexpensive PolyP to provide phosphate groups, thereby solving the problem of expensive ATP and achieving low-cost preparation of GPA. The co-expressed polyphosphate kinase and glycerol kinase are synthetically coupled with GPCMP, and inexpensive PolyP is used to provide phosphate groups, using glycerol, adenosine diphosphate (ADP), and cytidine monophosphate (CMP) as substrates, thereby solving the problem of expensive ATP and cytidine triphosphate (CTP) and achieving low-cost preparation of GPCMP.

[0008] In order to achieve the above object, one of the technical solutions provided by the present invention is a method for improving the ability of polyphosphate kinase to regenerate ATP, wherein the method is to co-express polyphosphate kinase and glycerol kinase;

[0009] Furthermore, the co-expression is to connect the polyphosphate kinase encoding gene and the glycerol kinase encoding gene through a linker, clone them into an expression vector, and then express them through a host;

[0010] Furthermore, the polyphosphate kinase is derived from Cytophaga hutchinsonii ATCC 33406, and the GenBank number is: ABG57400.1;

[0011] Furthermore, the glycerol kinase is derived from Escherichia coli 113290, and the GenBank number is: ESA60983.1;

[0012] Furthermore, the expression vector is a pET-28a plasmid;

[0013] Furthermore, the linker includes but is not limited to: GGTGGSGGGS, GGTGGGS, SGGSGGSG;

[0014] Preferably, the linker is SGGSGGSG;

[0015] Furthermore, the host includes but is not limited to: Escherichia coli BL21, Bacillus subtilis, Bacillus amyloliquefaciens;

[0016] Preferably, the host is Escherichia coli BL21.

[0017] More preferably, the method for improving the ability of polyphosphate kinase to regenerate ATP is to connect the polyphosphate kinase encoding gene ppk with GenBank number: ABG57400.1 and the glycerol kinase encoding gene gk with GenBank number: ESA60983.1 through a linker (SGGSGGSG), clone them into the pET-28a expression vector, and then express them in Escherichia coli BL21.

[0018] The second technical solution provided by the present invention is the co-expression product of polyphosphate kinase and glycerol kinase prepared by the above method - the connecting protein PPK-GK; the ability of the connecting protein PPK-GK to regenerate ATP is improved by about 17% compared with the single expression of polyphosphate kinase PPK.

[0019] The third technical solution provided by the present invention is the application of the above-mentioned connecting protein PPK-GK, especially in the preparation of glycerol phospholipid derivatives, more particularly in the preparation of glycerophosphatidic acid (GPA) or glycerophosphatidyl monocytidine phosphate (GPCMP).

[0020] The fourth technical solution provided by the present invention is a method for producing glycerol-type phosphatidyl monocytidine phosphate (GPCMP), wherein the method uses the connecting protein PPK-GK and glycerol-3-phosphocytidyl transferase (G3PCT) as catalysts, and uses ADP, glycerol, PolyP, and CMP as substrates to synthesize GPCMP;

[0021] Furthermore, the reaction conditions were 37 °C water bath for 4 h;

[0022] Furthermore, the glycerol-3-phosphate cytidyl transferase is derived from Bacillus pumilus, and the GenBank number is: QKN77290.1;

[0023] Further, the catalysts PPK-GK and G3PCT are free enzymes or immobilized enzymes;

[0024] Furthermore, the preparation method of the immobilized enzyme is as follows:

[0025] Carboxyl carbon nanotubes were added to the PPK-GK and G3PCT enzyme solutions respectively, and after mixing and combining for a period of time, the combined mixture was centrifuged at a low speed to remove the supernatant, and the precipitate was resuspended and washed with Tris-HCl and then centrifuged at a low speed to remove the supernatant. The obtained precipitate was the immobilized enzyme;

[0026] Further, the enzyme solution was mixed with carboxyl carbon nanotubes and combined on a shaker at 16 °C for 1.5 h;

[0027] Furthermore, the mass ratios of PPK-GK, G3PCT and carboxyl carbon nanotubes were all 1:1;

[0028] Furthermore, the combined mixture was centrifuged at 3000 rpm for 1 min at 4 °C;

[0029] Furthermore, the low-speed centrifugation conditions after the Tris-HCl washing were 4° C. and 3000 rpm.

[0030] Furthermore, in 20 mL of the GPCMP synthesis system, PPK-GK with a polyphosphate kinase activity of 60 U was added, G3PCT with an enzyme activity of 60 U was added, the concentration of the substrate glycerol was 35 mM, the concentration of the substrate CMP was 40 mM, the concentration of the substrate ADP was 1 mM, the concentration of the substrate polyphosphate (PolyP) was 60 mM, and the mixture was incubated in a water bath at 37°C for 4 h.

[0031] Beneficial effects:

[0032] The present invention uses a linker to connect glycerol kinase and polyphosphate kinase to co-express and obtain a connecting protein PPK-GK. Compared with the original polyphosphate kinase monomer, the polyphosphate kinase enzyme activity of the connecting protein is increased by about 17%. The connecting protein is used with glycerol and ADP as substrates, and low-priced PolyP is used to provide phosphate groups, thereby solving the problem of expensive ATP and realizing low-cost preparation of GPA. The co-expressed polyphosphate kinase glycerol kinase is synthetically coupled with GPCMP, and low-priced PolyP is used to provide phosphate groups. Glycerol, adenosine diphosphate (ADP), and cytidine monophosphate (CMP) are used as substrates to solve the problem of expensive ATP and cytidine triphosphate (CTP) and realize low-cost preparation of GPCMP. For the immobilization of the above-mentioned PPK-GK and G3PCT, by recycling the immobilized enzyme used for each catalysis, the amount of each enzyme is saved when preparing GPA and GPCMP, further reducing the cost. Description of the drawings:

[0033] Figure 1 These are double enzyme cutting verification diagrams of the coding genes of PPK, GK, and G3PCT of the present invention and the vector recombinant plasmids, wherein 1 is the verification diagram of the ppk gene and the vector, 2 is the verification diagram of the gk gene and the vector, and 3 is the verification diagram of the g3pct gene and the vector.

[0034] Figure 2 This is the electrophoresis diagram of the ppk and gk genes of the present invention, wherein 1 is the ppk gene and 2 is the gk gene.

[0035] Figure 3 This is a schematic diagram of the immobilization of the present invention. Specific implementation method:

[0036] The technical content of the present invention is further described below in conjunction with the embodiments, but the present invention is not limited to these embodiments, and the protection scope of the present invention cannot be limited by the following embodiments.

[0037] The experimental scheme of the present invention is as follows:

[0038] 1. Preparation of free PPK, GK, PPK-GK and G3PCT:

[0039] (1) The coding genes of PPK, GK and G3PCT were submitted to Jinweizhi for synthesis and cloned on the plasmid pET-28a. The recombinant plasmids pET-28a-ppk, pET-28a-gk and pET-28a-g3pct were obtained. Then each recombinant plasmid was transformed into Escherichia coli BL21 competent cells. The recombinant strains BL21 (pET-28a-ppk), BL21 (pET-28a-gk) and BL21 (pET-28a-g3pct) were obtained. Each recombinant strain was cultured by shake flask fermentation, and the PPK, GK and G3PCT obtained by fermentation were purified by Ni resin to obtain pure free PPK, GK and G3PCT.

[0040] (2) Using molecular biology techniques, PCR primers were designed, and PCR was performed using pET-28a-ppk and pET-28a-gk as templates. The PCR products were purified and then connected. The connection products were cloned into the pET-28a plasmid vector using restriction endonucleases NcoI and XhoI, and then the plasmid was transformed into Escherichia coli BL21 competent cells. The recombinant strain BL21 (pET-28a-ppk-gk) was cultured by shake flask fermentation, and the PPK-GK obtained by fermentation was purified using Ni resin to obtain pure free PPK-GK.

[0041] 2. After comparing the regeneration ability of PPK and PPK-GK for ATP, it was found that the connection protein PPK-GK has a stronger regeneration ability for ATP. Free PPK-GK was added to the substrates ADP, glycerol, and PolyP, and the reaction was kept at 37°C to prepare GPA. Free PPK-GK and G3PCT were added to the substrates CMP, ADP, glycerol, and PolyP, and the reaction was kept at 37°C to prepare GPCMP.

[0042] 3. Add carboxyl carbon nanotubes to the purified PPK-GK and G3PCT enzyme solutions, respectively, and perform adsorption reaction on a shaker at 16°C for 1.5 hours to obtain immobilized PPK-GK and immobilized G3PCT. Add immobilized PPK-GK to substrates ADP, glycerol, and PolyP, and keep them warm at 37°C to prepare GPA. Add immobilized PPK-GK and G3PCT together to substrates CMP, ADP, glycerol, and PolyP, and keep them warm at 37°C to prepare GPCMP. The immobilized enzymes used in the preparation of GPA and GPCMP are recovered by centrifugation after the preparation is completed, and used for the next batch of catalytic preparation.

[0043] The culture medium used in the present invention and the embodiments is as follows:

[0044] LB medium (g / L): yeast extract 5.0, tryptone 10.0, NaCl 10.0.

[0045] The solid medium of the above culture medium was supplemented with 2% agar.

[0046] The solutions used in the present invention and embodiments are as follows:

[0047] Wash Buffer (mM): imidazole 100, Tris-HCl 20, dithiothreitol 1, NaCl 200;

[0048] Elution Buffer (mM): imidazole 500, Tris-HCl 20, dithiothreitol 1, NaCl 200;

[0049] Lysis Buffer (mM): imidazole 20, Tris-HCl 20, dithiothreitol 1, NaCl 200.

[0050] The polyphosphate kinase (PPK) involved in the present invention is derived from Cytophaga hutchinsonii ATCC 33406, and its GenBank number is ABG57400.1; the glycerol kinase (GK) involved in the present invention is derived from Escherichia coli 113290, and its GenBank number is ESA60983.1; the glycerol-3-phosphate cytidyl transferase (G3PCT) involved in the present invention is derived from Bacillus pumilus, and its GenBank number is QKN77290.1. When constructing a recombinant strain for producing the above-mentioned polyphosphate kinase (PPK), glycerol kinase (GK) or glycerol-3-phosphate cytidyl transferase (G3PCT), the corresponding gene sequence can be obtained according to the amino acid sequence provided above and then synthesized, or the gene synthesis can be performed according to the nucleotide sequence source shown below:

[0051] The coding gene of PPK corresponds to positions 130069-130986 of the full gene sequence of GenBank: CP000383.1;

[0052] The coding gene of GK corresponds to positions 53001-54530 of the full gene sequence of GenBank: AXUF01000246.1;

[0053] The coding gene of G3PCT corresponds to positions 1015126-1015515 of the full gene sequence of GenBank: CP054310.1.

[0054] Some of the sequences used in the embodiments of the present invention are shown in the following table:

[0055]

[0056] The present invention is further explained below by means of specific examples.

[0057] Example 1: Obtaining recombinant strains

[0058] 1. According to the amino acid sequence number, the coding gene ppk of PPK, the coding gene gk of GK, and the coding gene g3pct of G3PCT (PPK (GenBank: ABG57400.1), GK (GenBank: ESA60983.1), and G3PCT (GenBank: QKN77290.1)) were submitted to GENEWISE for synthesis and cloned between the restriction endonucleases NcoI and XhoI of the plasmid pET-28a. The recombinant plasmids pET-28a-ppk, pET-28a-gk, and pET-28a-g3pct were obtained. The recombinant plasmids were double-digested with restriction endonucleases NcoI and XhoI for verification (see Figure 1 ).

[0059] Then, each recombinant plasmid was transformed into Escherichia coli BL21 competent cells to obtain recombinant strains BL21 (pET-28a-ppk), BL21 (pET-28a-gk), and BL21 (pET-28a-g3pct).

[0060] 2. Using the recombinant plasmid pET-28a-ppk as a template, design primers (upstream primer P1, downstream primer P2) to PCR amplify the polyphosphate kinase gene ppk fragment with linker. The amplification reaction system is:

[0061]

[0062]

[0063] The amplification program was as follows: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 53°C for 20 s, extension at 72°C for 8 s, 30 cycles; and extension at 72°C for 10 min.

[0064] 3. Using the extracted pET-28a-gk as a template, design primers (upstream primer P3, downstream primer P4) to PCR amplify the gk fragment of the glycerol kinase gene with a linker. The amplification reaction system is:

[0065] Upstream primer P3 2.0μL Downstream primer P4 2.0μL pET-28a-gk 2.0μL Primer Star Max Enzyme 25μL <![CDATA[ddH2O]]> 19μL

[0066] The amplification program was as follows: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 53°C for 20 s, extension at 72°C for 10 s, 30 cycles; and extension at 72°C for 10 min.

[0067] 4. Use a small amount of DNA recovery kit to recover the PCR product and purify the product (see Figure 2 ). The pET-28a plasmid was double-digested with restriction endonucleases NcoI and XhoI, and the linearized plasmid pET-28a was recovered using a small amount of DNA recovery kit. The two PCR products were connected to the linearized plasmid pET-28a in a 50°C water bath for 15 minutes using a homologous recombinase. The ligation product was transformed into Escherichia coli BL21 competent cells, spread on a solid culture medium plate containing kanamycin sulfate resistance, and cultured in a 37°C incubator for 12 hours. The transformants grown on the plate were transferred to a test tube and the plasmid was extracted. The extracted plasmid was sent to Jinweizhi for sequencing. The sequencing results showed that the ppk and gk fragments had been successfully constructed into the pET-28a plasmid, and the recombinant plasmid pET-28a-ppk-gk and the recombinant strain BL21 (pET-28a-ppk-gk) were obtained.

[0068] Example 2: Shake flask fermentation and purification of PPK, GK, G3PCT, and PPK-GK

[0069] (1) Recombinant Escherichia coli BL21(pET-28a-ppk), BL21(pET-28a-gk), BL21(pET-28a-g3pct), and BL21(pET-28a-ppk-gk) were activated and cultured at 37°C for 12 h;

[0070] (2) Pick a single colony with good growth on each of the above strain plates and inoculate it into a 5 mL LB liquid test tube with resistance added, and culture it overnight in a shaking incubator at 220 r / min and 37°C;

[0071] (3) Transfer the seed solution in the test tube to 250 mL LB liquid medium containing resistance at a 2% inoculum volume, and culture at 220 rpm and 37°C for 2-3 h until the OD600 of the bacterial solution reaches 0.6-0.8;

[0072] (4) The culture medium was removed from the shaker and placed on ice for 5 min to cool down. In a clean bench, IPTG inducer was added for induction (final concentration of IPTG = 0.5 mmol / L), and cultured in a shaker at 110 r / min and 16°C for 16-20 h.

[0073] (5) Resin pretreatment: Add 1 mL of new Ni 2+ Resin, add two column volumes of ddH2O to wash Ni 2+Add two column volumes of Lysis Buffer to clean the Ni 2+ The resin is used to balance the pH of the resin and to transfer Ni 2+ The residual ethanol during the resin storage process is washed away;

[0074] (6) Collecting bacteria: The E. coli culture solution induced and cultured for 16-20 h in a shaker at 16°C was recovered using a 500 mL centrifuge cup, and centrifuged at 4°C, 8000 rpm for 10 min, and the supernatant was discarded;

[0075] (7) Cell disruption: Resuspend the collected bacteria in 40 mL of Lysis Buffer (pH 7.0) by shaking. Then, transfer the bacterial solution into a 100 mL beaker and place the beaker in an ice-water mixture for ultrasonic disruption of the bacterial cells.

[0076] (8) Protein binding to resin: The recovered cells were crushed and the supernatant was sterile filtered and then mixed with the above Ni 2+ The resin mixture was placed in a beaker and stirred on a magnetic stirrer at 100 r / min to allow the protein and Ni to react. 2+ The resin binding takes 50-70 minutes, and the binding process should be carried out at low temperature (0-4°C);

[0077] (9) Will be with Ni 2+ The protein solution after resin binding is poured into the purification chromatography column;

[0078] (10) Elute impurities: All the solution after binding is drained away, Ni 2+ After all the resin is concentrated in the column, add 10 mL of pre-cooled Wash Buffer to wash away the unwanted proteins bound to the resin.

[0079] (11) Elution of target protein: After the Wash Buffer has flowed out, block the outlet of the column and add 5 mL of pre-cooled Elution Buffer to the column to resuspend the Ni 2+ The resin is then opened to allow the solution to flow out, and the effluent is recovered and repeated once. At this time, the target protein is eluted by the Elution Buffer and dissolved in it;

[0080] (12) Ultrafiltration replacement: Since the target protein is dissolved in the Elution Buffer, the main component of the solution is imidazole. The presence of imidazole may affect the subsequent test results of the enzyme protein, so the imidazole must be removed with a replacement buffer. An ultrafiltration tube with a pore size of 10 kDa was selected to ultrafilter the Elution Buffer eluate containing the target protein. Centrifugation was performed at a centrifugal force of 2500 g. When the Elution Buffer eluate in the ultrafiltration tube was centrifuged to about 1.5 mL, the filtrate in the collection tube was poured out and 10 mL of pre-cooled Tris-HCl (pH 7.0, 50 mM) was added to the ultrafiltration tube. After repeating this step twice, when the supernatant in the ultrafiltration tube was about 1 mL, the supernatant was blown out and sucked into a pre-cooled EP tube to prepare pure PPK, GK, G3PCT, and PPK-GK, which were stored at 4°C for short-term use.

[0081] Example 3: Comparison of the regeneration activity of PPK and PPK-GK on ATP

[0082] The PPK and PPK-GK purified in Example 2 were diluted to 1 mg / ml respectively, and the specific enzyme activities of PPK and PPK-GK were compared by measuring their respective ATP regeneration rates.

[0083]

[0084] React at 37°C for 5 min, take out 20 μL of the reaction solution and add it to the detection system.

[0085]

[0086] After reacting at 37℃ for 3 min, dilute 2-fold and measure OD 340 Substitute the OD value into the y in the ATP standard curve y=0.20982x+0.31948 to obtain the concentration of ATP produced by the reaction. The amount of PPK / PPK-GK required to catalyze the generation of 1μM ATP within 1min is defined as 1U. The enzymatic activity of polyphosphate kinase is calculated as follows:

[0087]

[0088] Among them, 0.2 is the reaction system of 200μL, 5 is the reaction time of 5min, 10 is the enzyme volume of 10μL, and 1000 represents 1000μL, that is, 1ml, so as to calculate the enzyme activity of 1ml enzyme solution. Through calculation, we obtained that the polyphosphate kinase activity of PPK is 40U / ml, and the calculated specific activity is 40U / mg. The polyphosphate kinase activity shown by PPK-GK is 47U / ml, and the calculated specific activity is 47U / mg. The results show that the polyphosphate kinase activity shown by the co-expressed connexin PPK-GK is 117.5% of the original PPK, and the connexin has better catalytic efficiency for generating ATP.

[0089] Example 3: Preparation of GPA and GPCMP

[0090] 1. Immobilization of PPK-GK and G3PCT (process as Figure 3 )

[0091] The initial enzyme concentrations of PPK-GK and G3PCT purified in Example 2 were diluted to 5 mg / mL, and 4 mg of carboxyl carbon nanotubes were added to every 800 μL of diluted enzyme solution. The PPK-GK and G3PCT enzyme solutions were placed in a 16°C shaker for 1.5 hours with the carboxyl carbon nanotube mixture. The combined mixture was centrifuged at 4°C and 3000rpm for 1min, and the supernatant was taken out to measure the unimmobilized PPK-GK protein concentration of 1.268 mg / ml and the G3PCT protein concentration of 1.098 mg / mL. It was calculated that the immobilization rates of PPK-GK and G3PCT were 74.64% and 78.04%, respectively. After the protein was bound, each carboxyl carbon nanotube was resuspended and washed with 500 μL of Tris-HCl and centrifuged at low speed to remove the supernatant. The resulting precipitate was the immobilized enzyme material. Stored in a 4°C refrigerator for the preparation of GPA and GPCMP.

[0092] 2. PPK-GK is used to prepare GPA

[0093] GPA was prepared using the free PPK-GK obtained in Example 2. Using Tris-HCl at pH = 7 as the reaction buffer, in a 20 ml reaction system, the polyphosphate kinase activity of PPK-GK was 60 U, the enzyme activity of glycerol kinase was 52 U, 1 mM ADP was added for ATP regeneration, 35 mM glycerol and 60 mM PolyP were used as substrates, and the reaction was carried out at 37°C for 4 h, and the yield of GPA could reach 95%.

[0094] In order to save the amount of enzyme, immobilized PPK-GK was used to prepare GPA in batches, with Tris-HCl at pH = 7 as the reaction buffer, the polyphosphate kinase activity of immobilized PPK-GK added was 60U, and the enzyme activity of glycerol kinase was 52U at this time. 1mM ADP was added for ATP regeneration, 35mM glycerol and 60mM PolyP were used as substrates, and the reaction was carried out at 37°C for 4h. The yield of the first batch of GPA was measured to be 93%. After the catalysis was completed, the immobilized enzyme PPK-GK was separated and put into the same reaction system as the first batch reaction system. GPA was prepared in batches in this way. When the fifth batch of GPA was prepared, the obtained GPA yield (the amount of substance of the product GPA divided by the amount of substance of the substrate glycerol) was still maintained at more than 70%.

[0095] 3. PPK-GK and G3PCT for GPCMP preparation

[0096] GPCMP was prepared using free PPK-GK and G3PCT. Using Tris-HCl at pH = 7 as the reaction buffer, PPK-GK with a polyphosphate kinase activity of 60U was added to a 20ml reaction system, the activity of glycerol kinase was 52U, 60U of G3PCT was added, 1mM of ADP was added for ATP regeneration, 35mM glycerol, 60mM PolyP, and 40mM CMP were used as substrates, and the reaction was carried out at 37°C for 4h. The yield of GPCMP obtained (the amount of substance of the product GPCMP divided by the amount of substance of the substrate glycerol) can reach 85%.

[0097] In order to save the amount of enzyme, immobilized PPK-GK and immobilized G3PCT were used to prepare GPCMP in batches. Tris-HCl at pH = 7 was used as the reaction buffer, and immobilized PPK-GK with an enzyme activity of 60U of polyphosphate kinase was added. At this time, the enzyme activity of glycerol kinase was 52U, 60U of immobilized G3PCT was added, and 1mM ADP was added for ATP regeneration. 35mM glycerol, 60mM PolyP, and 40mM CMP were used as substrates. The reaction was carried out at 37°C for 4h, and the yield of the first batch of GPCMP was measured to be 82%. After the catalysis was completed, the immobilized enzymes PPK-GK and G3PCT were separated and put into the same reaction system as the first batch reaction system. GPCMP was prepared in batches in this way. When the fifth batch of GPCMP was prepared, the yield was still maintained above 55%.

Claims

1. A method for improving the ability of polyphosphate kinase to regenerate ATP, characterized in that: The method is to co-express polyphosphate kinase and glycerol kinase; The polyphosphate kinase, GenBank number: ABG57400.1; The glycerol kinase, GenBank number: ESA60983.1; The co-expression is to connect the polyphosphate kinase encoding gene and the glycerol kinase encoding gene through a linker, clone them into an expression vector, and then express them through a host.

2. A method for improving the ability of polyphosphokinase to regenerate ATP as claimed in claim 1, characterized in that: The expression vector is pET-28a plasmid; The linker includes: GGTGGSGGGS, GGTGGGS or SGGSGGSG; The host includes: Escherichia coli BL21, Bacillus subtilis or Bacillus amyloliquefaciens.

3. A method for improving the ability of polyphosphokinase to regenerate ATP as claimed in claim 1, characterized in that: The method for improving the ability of polyphosphate kinase to regenerate ATP is to modify the coding gene of polyphosphate kinase with GenBank number: ABG57400.1 ppk , and the gene encoding glycerol kinase with GenBank accession number ESA60983.1 gk After linker connection, it was cloned into the pET-28a expression vector and then expressed in Escherichia coli BL21; The linker is SGGSGGSG.

4. Use of the connecting protein PPK-GK, a co-expression product of polyphosphate kinase and glycerol kinase according to claim 1, in the preparation of glycerol phospholipid derivatives, characterized in that: The method for producing glycerol-type phosphatidyl monocytidine is as follows: using the connecting protein PPK-GK and glycerol-3-phosphocytidyl transferase G3PCT as catalysts and ADP, glycerol, PolyP and CMP as substrates to synthesize GPCMP.

5. The use according to claim 4, characterized in that The glycerol-3-phosphate cytidyl transferase has a GenBank number of: QKN77290.1; The catalysts PPK-GK and G3PCT are free enzymes or immobilized enzymes.

6. The use according to claim 5, characterized in that The preparation method of the immobilized enzyme is as follows: Carboxyl carbon nanotubes were added to the PPK-GK and G3PCT enzyme solutions respectively, and after mixing and combining for a period of time, the combined mixture was centrifuged at a low speed to remove the supernatant, the precipitate was resuspended and washed with Tris-HCl, and then centrifuged at a low speed to remove the supernatant, and the obtained precipitate was the immobilized enzyme.

Images