A 5'-phosphosulfate kinase mutant and its application

Abstract

The present invention relates to the technical field of enzyme catalysis, and in particular to a 5'-phosphosulfate kinase mutant and application, characterized in that the 5'-phosphosulfate kinase mutant is obtained by subjecting the amino acid sequence shown in SEQ ID NO:1 to single mutation or multiple mutation, including one of the following mutation modes: (1) subjecting the amino acid sequence shown in SEQ ID NO:1 to single mutation at position 66 or position 148 to obtain; (2) subjecting the amino acid sequence shown in SEQ ID NO:1 to simultaneous mutation at positions 66 and 148 to obtain. The present invention obtains a 5'-phosphosulfate kinase mutant that can catalyze AMP to generate PAP and has significantly enhanced activity based on the design of the three-dimensional structure of 5'-phosphosulfate kinase, wherein the PAP activity of the 5'-phosphosulfate kinase mutant with the highest activity is about 16 times that of the wild enzyme, laying a solid foundation for the industrial production of 3'-phosphoadenosine-5'-phosphosulfate.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme catalysis, and in particular to a 5'-phosphosulfate kinase mutant and application thereof. Background Art

[0002] 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is a sulfate group donor widely used in organisms and has the function of sulfation modification of various active substances. Precursors such as heparin and chondroitin sulfate need to be sulfation-modified to have corresponding biological activities. PAPS currently has a narrow access channel, low purity and high price in the market. In the current research on direct synthesis of PAPS by enzymatic method, there are problems such as separation difficulty, low yield and phosphorus pollution of waste liquid.

[0003] The structural difference between 3',5'-adenosine diphosphate (PAP) and PAPS is only one sulfate group, and the two can be efficiently converted by sulfotransferase. In 2000, Michael D. Burkart reported a sulfotransferase (ASST IV) from rat liver that can transfer the sulfate group on p-nitrophenolsulfate (PNPS) to PAP to generate PAPS and PNP (p-nitrophenol). APSK (5'-phosphosulfate kinase) has the ability to convert APS into PAPS and ADP, and AMP, as a structural analog of APS, should also have the potential to be catalyzed by the reaction. In 1989, C. Satishchandran reported an APSK from Escherichia coli K12, which had a weak catalytic ability to convert AMP into PAP. In 2013, Yang Yang et al. introduced an APSK from E. coli DH5α, which also had a weak ability to synthesize PAP using AMP as a substrate. After mutating its 68th amino acid from arginine to lysine, its ability to catalyze AMP to synthesize PAP increased to 5 times the original, but its conversion rate and conversion amount were still extremely low. Therefore, to date, there is still no method to use APSK enzyme to catalyze AMP to synthesize PAP. Summary of the invention

[0004] The present invention is made to solve the above problems, and aims to provide a highly active 5'-phosphosulfate kinase mutant and its application.

[0005] In a first aspect of the present invention, a 5'-phosphosulfate kinase mutant is provided, characterized in that: the 5'-phosphosulfate kinase mutant is obtained by subjecting the amino acid sequence shown in SEQ ID NO: 1 to single mutation or multiple mutations, including one of the following mutations:

[0006] (1) Single mutation at position 66 or 148 of the amino acid sequence shown in SEQ ID NO:1;

[0007] (2) The amino acid sequence shown in SEQ ID NO:1 is simultaneously mutated at positions 66 and 148.

[0008] In some embodiments, the 5'-phosphosulfate kinase mutant comprises:

[0009] (1) A mutant in which the arginine at position 66 of the N-terminus of SEQ ID NO.1 is mutated to lysine: Mu-1, wherein the amino acid sequence of Mu-1 is: SEQ ID NO: 2;

[0010] (2) A mutant in which the arginine at position 148 at the N-terminus of SEQ ID NO.1 is mutated to lysine: Mu-2, the amino acid sequence of Mu-2 is: SEQ ID NO: 3;

[0011] (3) A mutant in which the arginine at position 66 of the N-terminus of SEQ ID NO.1 is mutated to lysine and the arginine at position 148 is mutated to lysine: Mu-3, the amino acid sequence of Mu-3 is: SEQ ID NO: 4.

[0012] The present invention obtains 5'-phosphosulfate kinase mutants that can catalyze AMP to generate PAP and have significantly enhanced activity based on the rational design of the three-dimensional structure of 5'-phosphosulfate kinase, wherein the PAP activities of the 5'-phosphosulfate kinase mutants Mu-1, Mu2, and Mu-3 are 917.41%, 1273.26%, and 1616.57% of that of the wild enzyme, respectively, which are 9 to 16 times higher than the activity of the wild enzyme, making up for the defect of 5'-phosphosulfate kinase without high PAP activity in the prior art, and laying a solid foundation for the industrial production of 3'-adenosine phosphate-5'-phosphosulfate.

[0013] The second aspect of the present invention provides a coding gene, characterized in that it encodes the 5'-phosphosulfate kinase mutant described in the first aspect.

[0014] The third aspect of the present invention provides a recombinant vector, characterized in that it contains the coding gene described in the second aspect.

[0015] The fourth aspect of the present invention provides a recombinant strain, characterized in that it contains the encoding gene described in the second aspect.

[0016] The fifth aspect of the present invention provides an enzymatic synthesis method of 3',5'-adenosine diphosphate, characterized in that it comprises: using the 5'-phosphosulfate kinase mutant as described in the first aspect as a biocatalyst for synthesizing 3',5'-adenosine diphosphate.

[0017] In some embodiments, the enzymatic synthesis method of 3',5'-adenosine diphosphate comprises: using AMP as a raw material, ATP as a coenzyme, and the 5'-phosphosulfate kinase mutant as a biocatalyst to carry out a catalytic reaction in a buffer solution, and the reaction formula is as follows:

[0018] .

[0019] In some embodiments, in the catalytic reaction, the amount of AMP added is 0.1 ~ 10 mM, the amount of ATP added is 0.1 ~ 30 M, the amount of 5'-phosphosulfate kinase mutant added is 0.1 ~ 5 mg / mL, the amount of magnesium chloride added is 0.15 ~ 20 mM, and the amount of lithium chloride added is 0.15 ~ 45 M.

[0020] The invention controls the content of substrate AMP, coenzyme ATP and various additives, thereby controlling the rate of the catalytic reaction and improving the synthesis yield.

[0021] In some embodiments, in the catalytic reaction, the pH value is 5 to 10, the reaction temperature is 22 to 30° C., and the reaction time is 1 to 24 h.

[0022] The invention controls the reaction conditions to keep the 5'-phosphosulfate kinase mutant at a high activity, thereby ensuring the smooth progress of the catalytic reaction.

[0023] The sixth aspect of the present invention provides a method for purifying 3',5'-adenosine diphosphate, characterized in that the reaction solution obtained by the enzyme synthesis method of the fifth aspect is separated by liquid chromatography, and the fractions are freeze-dried to obtain 3',5'-adenosine diphosphate.

[0024] By implementing the above technical solution, the present invention has the following beneficial effects:

[0025] The present invention obtains 5'-phosphokinase mutants which can catalyze AMP to generate PAP and have significantly enhanced activity based on the rational design of the three-dimensional structure of 5'-phosphokinase, wherein the PAP activities of the 5'-phosphokinase mutants Mu-1, Mu2 and Mu-3 are 917.41%, 1273.26% and 1616.57% of the wild enzyme, respectively.

[0026] The purification method of 3',5'-adenosine diphosphate (PAP) provided by the present invention has the advantages of high yield and high purity of the purified product. The yield of the purification method can reach more than 80%, and the purity of the purified PAP is as high as more than 95%. In addition, the method does not use toxic and harmful organic solvents, is green and environmentally friendly, has a simple process and is easy to operate, and has a low production cost, so that the purified product is very competitive in the market and can be further synthesized into PAPS later.

[0027] The purification method of 3',5'-adenosine diphosphate (PAP) provided by the present invention has universal applicability for the purification of crude PAP product obtained by preparing PAP by biocatalysis, and has certain application prospects for large-scale industrial synthesis of downstream product 3'-adenosine 5'-phosphosulfate. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a schematic diagram of the reaction of 5'-phosphosulfate kinase catalyzing AMP to generate PAP;

[0029] Figure 2 It is a high performance liquid chromatogram of a mixture of ATP, ADP, AMP and PAP standards of the present invention;

[0030] Figure 3 is a high performance liquid chromatogram of the catalytic reaction solution of the wild type 5'-phosphosulfate kinase in Example 3 of the present invention;

[0031] Figure 4 is a high performance liquid chromatogram of the reaction solution catalyzed by the 5'-phosphosulfate kinase mutant (mu-3) in Example 3 of the present invention;

[0032] Figure 5 is a high performance liquid chromatogram of the reaction solution catalyzed by the 5'-phosphosulfate kinase mutant (mu-2) in Example 3 of the present invention;

[0033] Figure 6 is a high performance liquid chromatogram of the reaction solution catalyzed by the 5'-phosphosulfate kinase mutant (mu-1) in Example 3 of the present invention;

[0034] Figure 7 is a high performance liquid chromatogram of the purified catalytic reaction solution of the 5'-phosphosulfate kinase mutant (mu-3) in Example 4 of the present invention;

[0035] Figure 8 is a high performance liquid chromatogram of the production of PAPS from PAP after catalytic purification by aryl sulfotransferase IV in Example 5 of the present invention;

[0036] Fig. 9 It is a high performance liquid chromatogram of a mixture of PAP and PAPS standards in Example 5 of the present invention. DETAILED DESCRIPTION

[0037] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is described in detail below in conjunction with embodiments and drawings.

[0038] The molecular biology experiments in the embodiments, including plasmid construction, restriction digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., are mainly carried out with reference to Molecular Cloning Experiment Guide (3rd edition, edited by J. Sambrook and DW Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002).

[0039] LB medium: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract, 2 g / L agar powder (solid culture medium), solvent is water, pH natural.

[0040] Example 1: Preparation of wild-type 5'-phosphosulfate kinase

[0041] Encoding Penicillium chrysogenum ( Penicillium Chrysogenum ) 5'-phosphosulfate kinase amino acid sequence (UniProtKB / Swiss-Prot: Q12657.1), after Escherichia coli codon optimization (sequence as SEQ ID NO.5), was fully synthesized by GenScript (Nanjing) Co., Ltd. and then linked into the pET-28a (+) vector to construct APSK-pET-28a (+) plasmid. After sequencing and sequence verification, it was heat-shock transformed into E. coli BL21 (DE3) competent cells to obtain 5'-phosphosulfate kinase expression engineering bacteria. A single colony was picked from the LB plate medium containing 50 μg / mL kanamycin and inoculated into LB liquid medium containing 50 μg / mL kanamycin. After shaking culture at 37°C and 200 rpm for 12 h, it was transferred to 3 L liquid TB medium for expansion culture and continued to shake and culture at 37°C and 200 rpm for 12 h. When the optical density OD of the culture solution reached 0.04, the expression of 5'-phosphosulfate kinase was 0.04. 600 When it reaches 0.6-0.8, lower the temperature to 16-25 °C, add IPTG solution with a final concentration of 0.15-1.5 mM to induce expression for 8-20 h, centrifuge the culture solution at 4000 rpm for 25 min, discard the supernatant culture solution, and store the bacteria at -20 °C for later use.

[0042] For every 5 g of collected bacteria, 20 mL of cell lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 0.1% triton X-100, pH 8.0) was added, and the cells were ultrasonically disrupted at 4 °C for 20 min, and centrifuged at 12,000 rpm for 25 min. The supernatant was the crude enzyme solution. 3 mL of Ni-NTA filler was added to the glass chromatography column, and after the filler settled, 20 mL of lysis buffer was balanced, and the crude enzyme solution was loaded at a rate of 1 mL / min. 20 mL of washing solution (50 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0) was used to elute the impurities, and the target protein was eluted with elution buffer (500 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0). The protein concentration was determined and dialyzed in dialysate (20 mM NaH2PO4, 150 mM NaCl, pH 8.0) at 4°C for 16 h to remove imidazole. 5'-phosphosulfate kinase with higher purity was obtained and stored at 4°C for future use.

[0043] Example 2: Preparation of 5'-phosphosulfate kinase mutants

[0044] By comparing the structure and amino acid sequence, the amino acid chains of 50-100 amino acids and 130-180 amino acids at the N-terminus of wild-type 5'-phosphosulfate kinase were determined to be the key sites for substrate binding. The plasmid APSK-pET-28a (+) connected with the wild-type 5'-phosphosulfate kinase DNA was used as a template to mutate the arginine at positions 66 and 148 on the wild-type APSK sequence to lysine respectively or simultaneously. After each mutant product was verified by sequencing, it was transformed into E. coli BL21 (DE3) competent cells for expression, and 5'-phosphosulfate kinase mutant expression engineering bacteria mu-1 (R66K), mu-2 (R148K), and mu-3 (R66K, R148K) were obtained. The obtained mutants were subjected to protein extraction according to the operation described in Example 1 and stored at 4°C for future use.

[0045] Table 1 Primers used for 5'-phosphosulfate kinase mutation

[0046]

[0047] Example 3: Wild-type and mutant 5'-phosphosulfate kinase catalyzes the synthesis of PAP using AMP as substrate

[0048] To a 50 mM pH 7.5 Tris-HCl buffer containing 5 mM MgCl2, AMP and 5 mM ATP were added as substrates at a final concentration of 10 mM, and the wild type or mutant of 5'-phosphosulfate kinase obtained in Examples 1 and 2 was added to the reaction system at a concentration of 0.6-5.0 mg / ml, respectively. The reaction was carried out at 30°C with constant temperature shaking (660 rpm) for 16 h, and an equal volume of acetonitrile was added to terminate the reaction. The reaction solution was centrifuged at 12000 rpm for 30 min, and the supernatant was sampled and injected into high performance liquid chromatography (HPLC) to analyze the amount of substrate and product.

[0049] The HPLC analysis method was as follows: chromatograph Agilent high performance liquid chromatography 1260; chromatographic column HILIC Silica 250*4.6mm; column temperature 30°C; flow rate 1 mL / min; detection wavelength 260 nm;

[0050] Mobile phase: Phase A: 10 mM ammonium formate, Phase B: 10 mM ammonium formate in acetonitrile,

[0051] Gradient elution, the elution program is: 0-10 min, 60% B phase; 10-10.1 min, 90% B phase; 10.1-20min90% B phase.

[0052] The HPLC chromatogram of the wild-type 5'-phosphosulfate kinase catalytic reaction solution is shown in Figure 3 As shown, the HPLC chromatogram of the reaction solution catalyzed by 5'-phosphosulfate kinase mutants (mu-3, mu-2, mu1) is shown Figure 4 , Figure 5 , Figure 6 As shown;

[0053] The product is PAP. Using PAP standard as control, the catalytic reaction of 5'-phosphosulfate kinase wild type and mutants was analyzed, and the yield was calculated through the PAP standard concentration curve to compare the relative activities of each mutant.

[0054] Table 2 5'-phosphosulfate kinase activity detection

[0055] 5'-phosphosulfate kinase Relative activity (%) APSK wild type 100 Mu-1 917.41 Mu-2 1273.26 Mu-3 1616.57

[0056] Table 3 5'-phosphosulfate conversion rate detection

[0057] 5'-phosphosulfate kinase PAP conversion rate (%) APSK wild type 10.28 Mu-1 52.67 Mu-2 81.40 Mu-3 94.14

[0058] Example 4: Isolation and purification of 3',5'-adenosine diphosphate

[0059] The reaction solution was pretreated according to the method described in Example 3 and separated by preparative liquid chromatography. The chromatographic conditions were: chromatograph Waters Alliance e2695; chromatographic column Syncronis™ PREP HILIC 250 mm*30 mm; column temperature 30°C; flow rate 15 mL / min, detection wavelength 260 nm; mobile phase: phase A 10 mM ammonium formate, phase B 10 mM ammonium formate acetonitrile, gradient elution, elution program: 0-10 min, 60% phase B; 10-10.1 min, 90% phase B; 10.1-20 min 90% phase B; injection volume 30 mL, and the fractions were freeze-dried to obtain a high-purity 3',5'-adenosine diphosphate sample. The HPLC chromatogram of the purified catalytic reaction solution is shown in the figure below. Figure 7 shown.

[0060] Example 5: Sulfotransferase (ASST IV) catalyzes the synthesis of PAPS using PAP as substrate

[0061] To a 20 mM pH 7.5 Tris-HCl buffer containing 2 mM MgCl2, PNPS and 5 mM PAP were added as substrates at a final concentration of 50 mM, and sulfotransferase stored in the laboratory was added to the reaction system at a concentration of 1.0-5.0 mg / ml. The reaction was carried out at 30°C with constant temperature shaking (660 rpm) for 2 h, and an equal volume of acetonitrile was added to terminate the reaction. The reaction solution was centrifuged at 12000 rpm for 30 min, and the supernatant was sampled and injected into high performance liquid chromatography (HPLC) to analyze the amount of substrate and product. HPLC analysis method: chromatograph Agilent HPLC 1260; chromatographic column HILIC Silica 250*4.6mm; column temperature 30℃; flow rate 1 ml / min; detection wavelength 260 nm; mobile phase: phase A 10 mM ammonium formate, phase B 10 mM ammonium formate acetonitrile, gradient elution, elution program: 0-10 min, 60% phase B; 10-10.1 min, 90% phase B; 10.1-20 min 90% phase B. Compared with the PAPS standard, the yield was confirmed to be 27.93±0.24%.

[0062] The above-mentioned embodiments are preferred examples of the present invention and are not intended to limit the protection scope of the present invention.

Claims

1. A 5'-phosphosulfate kinase mutant, characterized in that: The 5'-phosphosulfate kinase mutants include: (1) A mutant in which the arginine at position 66 of the N-terminus of SEQ ID NO.1 is mutated to lysine: Mu-1, wherein the amino acid sequence of Mu-1 is: SEQ ID NO: 2; (2) A mutant in which the arginine at position 148 of the N-terminus of SEQ ID NO.1 is mutated to lysine: Mu-2, wherein the amino acid sequence of Mu-2 is: SEQ ID NO: 3; (3) A mutant in which the arginine at position 66 of the N-terminus of SEQ ID NO.1 is mutated to lysine and the arginine at position 148 is mutated to lysine: Mu-3, wherein the amino acid sequence of Mu-3 is: SEQ ID NO:

4.

2. A coding gene, characterized in that Encoding the 5'-phosphosulfate kinase mutant of claim 1.

3. A recombinant vector, characterized in that: Contains the coding gene according to claim 2.

4. A recombinant strain, characterized in that: Contains the coding gene according to claim 2.

5. A method for enzymatic synthesis of 3',5'-adenosine diphosphate, characterized in that: include: The 5'-phosphosulfate kinase mutant as claimed in claim 1 is used as a biocatalyst for synthesizing 3',5'-adenosine diphosphate.

6. The method for enzymatic synthesis of 3',5'-adenosine diphosphate according to claim 5, characterized in that: include: Using AMP as a raw material, ATP as a coenzyme, and the 5'-phosphosulfate kinase mutant as a biocatalyst, a catalytic reaction is carried out in a buffer solution, and the reaction formula is as follows: 。 7. The method for enzymatic synthesis of 3',5'-adenosine diphosphate according to claim 6, characterized in that: In the catalytic reaction, the added amount of AMP is 0.1 ~ 10 mM, the added amount of ATP is 0.1 ~ 30 M, the added amount of 5'-phosphosulfate kinase mutant is 0.1 ~ 5 mg / mL, the added amount of magnesium chloride is 0.15 ~ 20 mM, and the added amount of lithium chloride is 0.15 ~45 M.

8. The method for enzymatic synthesis of 3',5'-adenosine diphosphate according to claim 6, characterized in that: In the catalytic reaction, the pH value is 5 to 10, the reaction temperature is 22 to 30° C., and the reaction time is 1 to 24 h.

9. A method for purifying 3',5'-adenosine diphosphate, characterized in that: The reaction solution obtained by the enzyme synthesis method according to claim 8 is separated by liquid chromatography, and the fractions are lyophilized to obtain 3',5'-adenosine diphosphate.

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