Application of polyphosphate kinase BsPPK in ATP (adenosine triphosphate) synthesis

Abstract

The invention discloses application of polyphosphate kinase BsPPK in synthesis of ATP (adenosine triphosphate). The amino acid sequence of the polyphosphate kinase BsPPK is as shown in SEQ ID NO. 1. In specific application, adenosine diphosphate or adenosine monophosphate is used as a phosphate receptor, polyphosphate is used as a phosphoric acid donor, and ATP is synthesized under the action of polyphosphate kinase BsPPK. The polyphosphate is selected from tripolyphosphate or hexametaphosphate. The invention also discloses an application of the polyphosphate kinase BsPPK in the synthesis of uridine diphosphate-galactose, and an application of the polyphosphate kinase BsPPK in the synthesis of lactosyl-N-tetrasaccharide. According to the method, the ATP is synthesized and regenerated by utilizing the polyphosphate kinase BsPPK, so that the reaction cost of UDP-Gal and LNT in-vitro synthesis routes is greatly reduced. The method has important significance on ATP regeneration and synthesis of UDP-Gal and galactosyl lactose derivatives, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the application of polyphosphate kinase BsPPK in ATP synthesis, belonging to the field of ATP in vitro synthesis technology. Background Technology

[0002] Adenosine-5'-triphosphate (ATP) is a high-energy phosphate compound that plays a crucial role in metabolic processes, enzyme catalysis, biosynthesis, cofactor synthesis, and cell-free protein synthesis. In industrial production, the application of in vitro ATP-dependent enzyme cascades is limited by the addition of the expensive substrate ATP and the accumulation of the byproduct adenosine diphosphate (ADP). Therefore, developing ATP regeneration systems is of great significance for designing efficient and economical cascade reactions.

[0003] ATP regeneration systems based on polyphosphates (polyPs) / polyphosphokinases (PPKs) are a subject of considerable interest. Existing technologies for ATP regeneration systems have been widely reported; for example, RpPPK from *Ruegeria pomeroyi* is used to produce LacNAc, and DrPPK from *Deinococcus radiodurans* is used to produce D-enulose. However, the polyphosphokinases reported for ATP regeneration suffer from poor stability and poor tolerance to polyPs. To reduce the limitations of ATP regeneration systems, developing polyphosphokinases with high stability and tolerance to high concentrations of polyP is of great significance for ATP regeneration systems. Summary of the Invention

[0004] In response to the aforementioned existing technologies, this invention has discovered a novel polyphosphate kinase—BsPPK—that exhibits high stability and tolerance to high concentrations of polyP, providing its application in ATP synthesis.

[0005] This invention is achieved through the following technical solution: The application of polyphosphate kinase BsPPK in ATP synthesis, wherein the amino acid sequence of polyphosphate kinase BsPPK is shown in SEQ ID NO.1.

[0006] Furthermore, in specific applications, ATP is synthesized using adenosine diphosphate (ADP) or adenosine monophosphate (AMP) as phosphate acceptors and polyphosphates as phosphate donors, under the action of polyphosphate kinase BsPPK.

[0007] Furthermore, the polyphosphate is selected from tripolyphosphate or hexametaphosphate (polyP6).

[0008] Application of polyphosphate kinase BsPPK in the synthesis of uridine diphosphate galactose (UDP-Gal).

[0009] Furthermore, in specific applications, the reaction system includes the following components: UDP-Gal synthesis-related enzymes, polyphosphate kinase BsPPK, galactose, polyphosphate, uridine triphosphate (UTP), and adenosine diphosphate or adenosine monophosphate.

[0010] Furthermore, the UDP-Gal synthesis-related enzymes include: galactokinase (SpGalK) derived from *Streptococcus pneumoniae*, UDP-pyrophosphorylase (BLUSP) derived from *Bifidobacterium longum*, and inorganic pyrophosphorylase (PmPpA) derived from *Pasteurella multocida*. All of these are enzymes already reported in the prior art.

[0011] Furthermore, the polyphosphate is selected from tripolyphosphate or hexametaphosphate.

[0012] Application of polyphosphate kinase BsPPK in the synthesis of lactosyl-N-tetrasaccharide (LNT).

[0013] Furthermore, in specific applications, the reaction system for synthesizing lactosyl-N-tetrasaccharide includes the following components: UDP-Gal synthesis-related enzymes, galactosyltransferase, polyphosphokinase BsPPK, galactose, lactose-N-trisaccharide, polyphosphate, and adenosine diphosphate or adenosine monophosphate. The principle is as follows: first, UDP-Gal is synthesized using UDP-Gal synthesis-related enzymes, and then galactosyltransferase catalyzes the synthesis of lactosyl-N-tetrasaccharide from UDP-Gal and lactose-N-trisaccharide.

[0014] Furthermore, the UDP-Gal synthesis-related enzymes include: galactokinase (SpGalK) derived from Streptococcus pneumoniae, UDP-pyrophosphorylase (BLUSP) derived from Bifidobacterium longum, and inorganic pyrophosphorylase (PmPpA) derived from Pasteurella multocida.

[0015] The galactosyltransferase is a galactosyltransferase (Cvβ3GalT) derived from Chromobacterium violaceum. This is an enzyme already reported in the prior art.

[0016] Furthermore, the polyphosphate is selected from tripolyphosphate or hexametaphosphate.

[0017] The polyphosphate kinase BsPPK discovered in this invention belongs to the PPK2-III family. This enzyme can catalyze the conversion of AMP or ADP into ATP and exhibits high polyP tolerance and stability (temperature stability, pH stability, and stability over long-term reactions). This invention successfully synthesized UDP-Gal and galactosyl-N-tetrasaccharide via an in vitro cascade of UDP-Gal synthesis-related enzymes and galactosyltransferase. Using 22.5 mM (15.9 mg / mL) lactose-N-trisaccharide and 30 mM AMP in a one-pot in vitro reaction for 30 h, the LNT yield reached (14.42 ± 0.21) g / L.

[0018] This invention utilizes polyphosphoric acid kinase BsPPK to synthesize and regenerate ATP, enabling AMP or ADP to replace ATP, significantly reducing the reaction cost of in vitro synthesis pathways for UDP-Gal and LNT. This invention is of great significance for ATP regeneration, the synthesis of UDP-Gal and galactosyllactose derivatives, and has broad application prospects.

[0019] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0020] Figure 1 : Schematic diagram of SDS-PAGE results of polyphosphate kinase BsPPK, where M represents standard protein, 1 is crude enzyme solution, and 2 is pure enzyme solution.

[0021] Figure 2 Schematic diagram of HPLC analysis results.

[0022] Figure 3 Schematic diagram of the effect of reaction temperature on the relative enzyme activity of polyphosphokinase BsPPK.

[0023] Figure 4 : Schematic diagram of the effect of reaction pH on the relative enzyme activity of polyphosphokinase BsPPK.

[0024] Figure 5 Schematic diagram of the relative enzyme activity of polyphosphoric acid kinase BsPPK after incubation at different temperatures for 24 h.

[0025] Figure 6 Schematic diagram of the relative enzyme activity of polyphosphoric acid kinase BsPPK after incubation for 24 h under different pH conditions.

[0026] Figure 7 Schematic diagram of the effect of polyphosphate concentration on ATP conversion rate. Detailed Implementation

[0027] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0028] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0029] Example 1: Discovery of polyphosphokinase BsPPK This invention has mined a polyphosphoric acid kinase sequence (MEE1410690.1) from the NCBI database, which consists of 292 amino acid residues and is shown in SEQ ID No.1. This invention names it polyphosphoric acid kinase BsPPK.

[0030] The amino acid sequence of polyphosphate kinase BsPPK is shown below, as indicated in SEQ ID NO.1: MKSEYTYDGSRKFNIKKVKADETSLCNNREIAEKRMQENEQELDELQQKLYAEKKEGLIIVFQAMDAAGKDGTITHVLQCLSPHGVYEAAFKSPTSTELAHDFLWRVVQKVPAKGEIAIFNRSHYEDVLIGKVKELYTSQAHAD RIDTDKVIDRRYTDIRNFEEYLYQNNVRIIKIFLNVSKEEQAKRFLSRIEEPEKNWKFSSSDVEERTYWDKYQNAFEDAVNATATSHCPWYVVPADHKWYMRYVVSEIILKTLKEMNPEYPVVSEERIQQFQSLKEQLEKELQK.

[0031] Phylogenetic analysis showed that polyphosphate kinase BsPPK belongs to the PPK2-III family. Using ExPaSy (https: / / web.expasy.org / protparam / ), the molecular weight and isoelectric point of BsPPK were predicted. The predictions showed that the molecular weight of BsPPK is approximately 34.0 kDa, and the isoelectric point is 5.78.

[0032] Sequence alignment of polyphosphoric acid kinase BsPPK with EbPPK (derived from Erysipelotrichaceae bacterium), ChPPK (derived from Cytophaga hutchinsonii), MrPPK (derived from Meiothermus ruber), and DrPPK (derived from Deinococcus radiodurans) was performed. The results showed that the amino acid sequence homology of polyphosphoric acid kinase BsPPK with the above-mentioned PPKs was 67.72%, 40.97%, 40.44%, and 37.47%, respectively.

[0033] Analysis of existing technologies only reveals that polyphosphokinase BsPPK has polyphosphokinase activity, but its specific enzymatic properties (such as polyP tolerance) are still unknown. Therefore, this invention has conducted heterologous expression and research on it, as detailed in the following examples.

[0034] Example 2: Heterologous expression and purification of polyphosphokinase BsPPK The amino acid sequence of polyphosphate kinase BsPPK was synthesized by BGI Genomics, and the corresponding gene sequence was used as the expression vector by pET-28a(+). The nucleotide sequence of the gene encoding polyphosphate kinase BsPPK is shown in SEQ ID NO.2, as shown below (direction 5'-3'): 。

[0035] The synthesized pET-28a(+) plasmid containing the BsPPK polyphosphate kinase encoding gene was introduced into competent *Escherichia coli* BL21 (DE3), and single colonies were obtained by plating. The obtained single colonies were inoculated into 5 mL of LB medium (containing 50 mg / L Kan resistance) and cultured at 37°C for 12 h; then, 1% (v / v) was inoculated into 100 mL of LB medium (containing 50 mg / L Kan resistance) and cultured at 37°C and 200 rpm. The colonies were cultured until the cell density reached OD200. 600 When the concentration was 0.6–0.8, isopropyl-β-D-thiogalactoside (IPTG) was added to a concentration of 0.1 mM, and the mixture was cultured at 18°C ​​and 200 rpm for 18 h to induce protein expression.

[0036] Collect the fermented bacterial culture, centrifuge at 8000 rpm for 10 min at 4°C to collect the cells, and resuspend in 25 mM Tris-HCl (pH 8.0) buffer. Sonicate the resuspended solution under ice-water bath conditions (sonication conditions: 400 W, on for 3 s, off for 5 s). Centrifuge the cell lysate at 8000 rpm for 10 min at 4°C to remove cell debris, obtaining the crude enzyme solution.

[0037] The crude enzyme solution was purified using a Ni-NTA affinity chromatography column, and the target protein with the His tag was collected. The protein was eluted sequentially with imidazole eluents at concentrations of 20 mM, 50 mM, 100 mM, and 200 mM, and the 200 mM eluent was collected. The solution was desalted and concentrated using an ultrafiltration tube (Millipore, 10 kDa) to obtain a pure enzyme solution. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed, and the results are shown below. Figure 1 As shown, the protein size is close to the predicted molecular weight, indicating that the purified protein is polyphosphate kinase BsPPK.

[0038] Example 3 Enzyme activity assay The reaction system 1 is as follows: 5 mM MgSO4, 1 mM polyP6, 1 mM ADP, 50 mM Tris-HCl (pH 8.0), 1 μg / mL polyphosphoric acid kinase BsPPK; react at 37℃ for 30 min, and then add 0.1% trichloroacetic acid to terminate the reaction.

[0039] The reaction system 2 is as follows: 5 mM MgSO4, 1 mM polyP6, 1 mM AMP, 50 mM Tris-HCl (pH 8.0), 1 μg / mL polyphosphoric acid kinase BsPPK; react at 37℃ for 30 min, and then add 0.1% trichloroacetic acid to terminate the reaction.

[0040] The reaction solutions of reaction system 1 and reaction system 2 were analyzed by HPLC, with ATP, ADP, and AMP standards as controls. The HPLC analysis conditions were as follows: 20 mmol / L potassium dihydrogen phosphate-dipoxatium hydrogen phosphate (pH 6.0) as the mobile phase, flow rate 1.0 mL / min, column temperature 30℃, UV detection wavelength 254 nm, and column: AQ-C18 (250 mm × 4.6 mm, 5 μm).

[0041] A schematic diagram of the HPLC analysis results is shown below. Figure 2 As shown, the reaction solution contains ATP, indicating that polyphosphokinase BsPPK can catalyze the phosphorylation of AMP or ADP into ATP.

[0042] Enzyme activity is defined as the amount of enzyme required to generate 1 μmol of ATP per minute, which is defined as 1 enzyme activity unit (U).

[0043] Enzyme activity was determined by the ratio of enzyme activity (U) to the amount of enzyme used (mg). All data were performed in triplicate. Data are presented as mean ± standard deviation.

[0044] Results: Polyphosphate kinase BsPPK catalyzed the conversion of ADP to ATP, with a specific activity of (24.58±1.62) U / mg. Polyphosphate kinase BsPPK catalyzed the conversion of AMP to ATP, with a specific activity of (19.17±0.19) U / mg.

[0045] Example 4: Determination of thermal stability and pH stability (1) Optimal temperature: The reaction system was the same as reaction system 1 in Example 3 (except for the reaction temperature), and was placed under different temperature conditions (25℃, 30℃, 35℃, 37℃, 40℃, 45℃, 50℃, 55℃, 60℃) to investigate the effect of temperature on relative enzyme activity. The results are as follows: Figure 3 As shown, the optimal temperature is 40℃.

[0046] (2) Optimal pH: The reaction system was the same as reaction system 1 in Example 3 (except for the pH buffer), and the reaction was carried out under different pH conditions. The buffers used were: citrate buffer (pH 3.0, 4.0, 5.0, 6.0), phosphate buffer (pH 6.0, 7.0, 8.0), Tris-HCl buffer (pH 7.0, 8.0, 9.0), and NaOH-glycine buffer (pH 9.0, 10.0, 11.0). The effect of pH on relative enzyme activity was investigated, and the results are as follows. Figure 4 As shown, the optimal pH is 9.0 (NaOH-glycine buffer).

[0047] (3) Temperature stability: Polyphosphoric acid kinase BsPPK was incubated at different temperatures (4℃, 25℃, 30℃, 35℃, 37℃, 40℃, 45℃, 50℃, 55℃, 60℃) for 24 h; then the enzyme activity was measured, and the reaction system was the same as reaction system 1 in Example 3. The temperature stability was examined, and the results are as follows. Figure 5 As shown, polyphosphate kinase BsPPK exhibits good thermal stability, with no significant decrease in enzyme activity after incubation at 4℃, 30℃, and 35℃ for 24 h; and a remaining enzyme activity greater than 40% after incubation at 37℃ for 24 h.

[0048] (4) pH stability: Polyphosphoric acid kinase BsPPK was incubated for 24 h at different pH conditions (4℃, 25℃, 30℃, 35℃, 37℃, 40℃, 45℃, 50℃, 55℃, 60℃) using the following buffers: citrate buffer (pH 3.0, 4.0, 5.0, 6.0), phosphate buffer (pH 6.0, 7.0, 8.0), Tris-HCl buffer (pH 7.0, 8.0, 9.0), and NaOH-glycine buffer (pH 9.0, 10.0, 11.0). Enzyme activity was then measured using the same reaction system as in reaction system 1 of Example 3. pH stability was investigated, and the results are as follows: Figure 6 As shown, after incubation in buffer (pH 7.0–10) for 72 h, the residual enzyme activity of polyphosphate kinase BsPPK was greater than 70%, and it exhibited good pH stability.

[0049] Example 5: Effect of polyphosphate concentration on enzyme activity In a 250 μL reaction system, 1 μg / mL polyphosphoric acid kinase BsPPK, 1 mM ADP, (0.05 mM–200 mM) polyP6, 5 mM MgSO4, and 50 mM Tris-HCl buffer (pH 8.0) were added. The reaction was carried out at 37 °C for 30 min. ATP content was analyzed by HPLC to determine enzyme activity. EbPPK derived from Erysipelotrichaceae bacterium was used as a control. All data were performed in duplicate or at least repeatedly. Data are expressed as mean ± standard deviation. Some data were analyzed using the SPSS t-test. *: p < 0.05; **: p < 0.01; ***: p < 0.001, with a significance level set at 0.05.

[0050] The effect of polyphosphate concentration on ATP conversion rate is as follows: Figure 7 As shown, polyphosphate kinase BsPPK exhibits good polyP tolerance. When the donor concentration is 200 mM, the ATP conversion rate is (16.21±0.21)%, which is significantly higher than that of EbPPK (3.38±0.13)%.

[0051] Example 6: Application of polyphosphokinase BsPPK in the in vitro synthesis of UDP-Gal The de novo synthesis of UDP-Gal-related enzymes was obtained through heterologous expression and purification using conventional methods. These enzymes included: galactokinase (SpGalK) from *Streptococcus pneumoniae*, UDP-pyrophosphorylase (BLUSP) from *Bifidobacterium longum*, and inorganic pyrophosphorylase from *Pasteurella multocida*. A galactosyltransferase, specifically Cvβ3GalT, derived from *Chromobacterium violaceum*, was also obtained through heterologous expression and purification using conventional methods.

[0052] Reaction system 1: 0.5 mg / mL UDP-Gal de novo synthesis of related enzymes (i.e., the concentrations of SpGalK, BLUSP, and inorganic pyrophosphatase are all 0.5 mg / L), 30 mM galactose, 30 mM ATP, 30 mM UTP, reaction pH 8.0, reaction temperature 37℃, reaction time ≥20 h.

[0053] Reaction system 2: 0.5 mg / mL UDP-Gal de novo synthesis of related enzymes (i.e., SpGalK, BLUSP, and inorganic pyrophosphatase, all at a concentration of 0.5 mg / L), 0.5 mg / mL polyphosphokinase BsPPK, 30 mM galactose, 30 mM polyP6, 30 mM ADP, and 30 mM UTP; reaction pH 8.0; reaction temperature 37℃; reaction time ≥20 h.

[0054] After 20 h of reaction, the reaction solution was analyzed by HPLC (Agilent 1260 II). The analytical conditions were as follows: mobile phase: 10 mmol / L potassium dihydrogen phosphate-dipoxatium hydrogen phosphate (pH 6.0); flow rate: 0.4 mL / min; column temperature: 35℃; UV detection wavelength: 276 nm; column: AQ-C18 (250 mm × 4.6 mm, 5 μm). Different concentrations of UDP-Gal standards were prepared, and standard curves were plotted.

[0055] Results: After 20 h of reaction, the yield of UDP-Gal in reaction system 2 reached (6.46±0.01) g / L, which was significantly higher than the yield of UDP-Gal in reaction system 1 (3.61±0.61) g / L, and the reaction continued even after 20 h.

[0056] Example 7: Application of polyphosphokinase BsPPK in the in vitro synthesis of LNT Reaction system 1: 0.5 mg / mL UDP-Gal de novo synthesis of related enzymes (i.e., SpGalK, BLUSP, and inorganic pyrophosphatase, all at concentrations of 0.5 mg / L), 0.5 mg / mL galactosyltransferase (Cvβ3GalT), 22.5 mM (15.9 mg / mL) lactose-N-trisaccharide, 30 mM galactose, 30 mM polyP6, 30 mM ATP, and 30 mM UTP. The reaction pH was 8.0, the reaction temperature was 37℃, and the reaction time was more than 30 h.

[0057] Reaction system 2: 0.5 mg / mL UDP-Gal de novo synthesis of related enzymes (i.e., SpGalK, BLUSP, and inorganic pyrophosphatase, all at 0.5 mg / L), 0.5 mg / mL polyphosphokinase BsPPK, 0.5 mg / mL galactosyltransferase (Cvβ3GalT), 22.5 mM (15.9 mg / mL) lactose-N-trisaccharide, 30 mM galactose, 30 mM polyP6, 30 mM AMP, and 30 mM UTP. The reaction was carried out at pH 8.0, at a temperature of 37℃, and for a reaction time of more than 30 h.

[0058] After 30 h of reaction, the reaction solution was analyzed by HPLC. The analytical conditions were as follows: mobile phase: acetonitrile-water mixture (70:30, v / v); flow rate: 0.5 mL / min; column temperature: 45℃; UV detection wavelength: 200 nm; column: BEH Xbridge Amide (250 mm × 4.6 mm, 5 μm). Different concentrations of LNT standards were prepared, and standard curves were plotted.

[0059] Results: After 30 h of reaction, the yield of LNT in reaction system 2 was as high as (14.42±0.21) g / L, which was significantly higher than the yield of LNT in reaction system 1 (11.70±0.14) g / L, and the reaction could continue after 30 h.

[0060] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. Application of polyphosphate kinase BsPPK in ATP synthesis, characterized by: The amino acid sequence of the polyphosphate kinase BsPPK is shown in SEQ ID NO.

1.

2. The use of the polyphosphate kinase BsPPK in synthesizing ATP according to claim 1, characterized in that: In specific applications, ATP is synthesized using adenosine diphosphate or adenosine monophosphate as a phosphate acceptor and polyphosphate as a phosphate donor under the action of polyphosphate kinase BsPPK.

3. The use of the polyphosphate kinase BsPPK in synthesizing ATP according to claim 1, characterized in that: The polyphosphate is selected from tripolyphosphate or hexametaphosphate.

4. The use of polyphosphate kinase BsPPK in the synthesis of uridine diphosphate-galactose, characterized in that: The amino acid sequence of the polyphosphate kinase BsPPK is shown in SEQ ID NO.

1.

5. The use of the polyphosphate kinase BsPPK according to claim 4 in the synthesis of uridine diphosphate-galactose, characterized in that: In specific applications, the reaction system includes the following components: UDP-Gal synthesis-related enzymes, polyphosphate kinase BsPPK, galactose, polyphosphate, uridine triphosphate, and adenosine diphosphate or adenosine monophosphate.

6. Use of the polyphosphate kinase BsPPK according to claim 5 in the synthesis of uridine diphosphate-galactose, characterized in that: The UDP-Gal synthesis-related enzymes include: galactokinase SpGalK, UDP-pyrophosphorylase BLUSP, inorganic pyrophosphatase PmPpA; And / or: the polyphosphate is selected from tripolyphosphate or hexametaphosphate.

7. Use of polyphosphate kinase BsPPK in the synthesis of lactosyl-N-tetraose, characterized in that: The amino acid sequence of the polyphosphate kinase BsPPK is shown in SEQ ID NO.

1.

8. Use of the polyphosphate kinase BsPPK in the synthesis of lactosyl-N-tetraose according to claim 7, characterized in that: In specific applications, the reaction system for synthesizing lactosyl-N-tetraose includes the following components: UDP-Gal synthesis-related enzymes, galactosyltransferase, polyphosphate kinase BsPPK, galactose, lactose-N-triose, polyphosphate, and adenosine diphosphate or adenosine monophosphate.

9. Use of the polyphosphate kinase BsPPK according to claim 8 in the synthesis of lactosyl-N-tetraose, characterized in that: The UDP-Gal synthesis-related enzymes include: galactokinase SpGalK, UDP-pyrophosphorylase BLUSP, inorganic pyrophosphatase PmPpA; the galactosyltransferase is galactosyltransferase Cvβ3GalT.

10. Use of the polyphosphate kinase BsPPK according to claim 8 in synthesizing lactosyl-N-tetraose, characterized in that: The polyphosphate is selected from tripolyphosphate or hexametaphosphate.

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