The invention relates to 3apos; -adenosine monophosphate-5apos; phosphoryl sulfate synthetase mutant and application thereof

Abstract

The invention discloses a 3 '-phosphoadenosine-5'-phosphoryl sulfate synthetase mutant and an application of the 3 '-phosphoadenosine-5'-phosphoryl sulfate synthetase mutant. Compared with a wild enzyme amino acid sequence as shown in SEQ ID NO.1, the amino acid sequence of the 3 '-adenosine-5'-phosphoryl sulfate synthetase mutant has one or two mutations in the 101 site, the 207 site and the 560 site; preferably, phenylalanine at the 101 site is mutated into tryptophan; cysteine at the 207th site is mutated into glycine; phenylalanine at the 560th site is mutated into tryptophan. The invention also provides a synthesis method and application of the 3 '-adenosine-5'-phosphoryl sulfuric acid. The APS intermediate product inhibition effect of hPAPSS1 is relieved through a point mutation technology, the substrate affinity of hPAPSS1 to ATP is improved, and a mutant enzyme with a high PAPS conversion rate is obtained; and the bifunctional enzyme hPAPSS1 is synthesized by heterologous expression of PAPS in escherichia coli, so that the acquisition of the enzyme is simplified, and the synthesis efficiency of PAPS is improved.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly to a 3′-phosphoadenosine-5′-phosphosulfate synthase mutant and its application. Background Art

[0002] 3′-phosphoadenosine-5′-phosphosulfate (PAPS), as an important sulfate group donor, is almost used in the sulfation reactions of all valuable sulfate compounds. Many compounds, such as polysaccharides, peptides, flavonoids, neurotransmitters, secondary metabolites, and lipids, etc., rely on sulfation modification to have biological activities. With the rapid development of the biomanufacture of sulfate-containing compounds such as heparan sulfate, chondroitin sulfate, and sulfated peptides, the demand for large-scale production of PAPS at low cost is increasing day by day, thus promoting the research on in vitro enzymatic synthesis of PAPS.

[0003] PAPS can be synthesized by catalyzing the sulfation and phosphorylation of ATP through adenosine 5′-triphosphate sulfurylase (ATPS) and adenosine 5′-phosphosulfate kinase (APSK) in two-step catalytic reactions. However, the separate expression of the two proteins causes the two consecutive reactions to be further spatially separated, which is not conducive to the conversion of intermediate metabolites into products and makes the enzyme acquisition more complex. The currently reported 3′-phosphoadenosine 5′-phosphosulfate synthase (hPAPSS1) has the activity of catalyzing the formation of PAPS from sulfate and ATP substrates, making the synthesis of PAPS more efficient.

[0004] However, the method of synthesizing PAPS using hPAPSS1 still has the defects of the inhibition of the intermediate product adenosine 5′-phosphosulfate (APS) and poor substrate binding efficiency, resulting in low conversion efficiency of PAPS and restricting its industrial application process. So far, the modification of hPAPSS1 has focused on achieving the purpose of improving enzyme activity by relieving the rigidity of its flexible loop, but the inhibition of the intermediate product APS has not been relieved, and the conversion rate of PAPS is still at a relatively low level. Summary of the Invention

[0005] In order to solve at least one of the above problems, the present invention provides a 3′-phosphoadenosine-5′-phosphosulfate synthase mutant and its application.

[0006] In order to achieve the above object, the present invention adopts the following technical means:

[0007] The first aspect of the present invention provides a 3′-phosphoadenosine-5′-phosphosulfate synthase mutant, and the amino acid sequence of the 3′-phosphoadenosine-5′-phosphosulfate synthase mutant is compared with the amino acid sequence of the wild enzyme shown in SEQ ID NO.1:

[0008] (1) There is a site mutation at position 101, position 207 and position 560: phenylalanine at position 101 is mutated to tryptophan; cysteine at position 207 is mutated to one of glycine, lysine, alanine, glutamic acid or threonine; phenylalanine at position 560 is mutated to tryptophan; or,

[0009] (2) There are two site mutations at position 101, position 207 and position 560: the mutation sites are selected from any two of phenylalanine at position 101 being mutated to tryptophan; cysteine at position 207 being mutated to glycine; phenylalanine at position 560 being mutated to tryptophan.

[0010] In some embodiments of the present invention, the 3′-phosphoadenosine-5′-phosphosulfate synthase mutant is any one of the following mutants:

[0011] Mutant F101W, the amino acid sequence is as shown in SEQ ID NO: 21;

[0012] Mutant C207G, the amino acid sequence is as shown in SEQ ID NO: 25;

[0013] Mutant C207K, the amino acid sequence is as shown in SEQ ID NO: 27;

[0014] Mutant C207A, the amino acid sequence is as shown in SEQ ID NO: 29;

[0015] Mutant C207E, the amino acid sequence is as shown in SEQ ID NO: 31;

[0016] Mutant C207T, the amino acid sequence is as shown in SEQ ID NO: 33;

[0017] Mutant F560W, the amino acid sequence is as shown in SEQ ID NO: 35;

[0018] Mutant F101W / F560W, the amino acid sequence is as shown in SEQ ID NO: 39;

[0019] Mutant F101W / C207G, the amino acid sequence is as shown in SEQ ID NO: 41;

[0020] Mutant C207G / F560W, the amino acid sequence is as shown in SEQ ID NO: 43.

[0021] In some embodiments of the present invention, the nucleotide sequence of the 3'-phosphoadenosine-5'-phosphosulfate synthase mutant is as follows: The nucleotide sequence of the mutant F101W is shown in SEQ ID NO: 22;

[0022] The nucleotide sequence of the mutant C207G is shown in SEQ ID NO: 26;

[0023] The nucleotide sequence of the mutant C207K is shown in SEQ ID NO: 28;

[0024] The nucleotide sequence of the mutant C207A is shown in SEQ ID NO: 20;

[0025] The nucleotide sequence of the mutant C207E is shown in SEQ ID NO: 32;

[0026] The nucleotide sequence of the mutant C207T is shown in SEQ ID NO: 33;

[0027] The nucleotide sequence of the mutant F560W is shown in SEQ ID NO: 36;

[0028] The nucleotide sequence of the mutant F101W / F560W is shown in SEQ ID NO: 40;

[0029] The nucleotide sequence of the mutant F101W / C207G is shown in SEQ ID NO: 42;

[0030] The nucleotide sequence of the mutant C207G / F560W is shown in SEQ ID NO: 44.

[0031] The third aspect of the present invention provides an expression vector containing the nucleotide sequence described in the first aspect.

[0032] The fourth aspect of the present invention provides a recombinant strain containing the expression vector described in the third aspect.

[0033] In some embodiments of the present invention, the recombinant bacterium uses Escherichia coli as the host.

[0034] In some embodiments of the present invention, the Escherichia coli includes BL21(DE3).

[0035] In some embodiments of the present invention, the expression vector of the recombinant bacterium includes pET series vectors.

[0036] In some embodiments of the present invention, the expression vector of the recombinant bacterium includes pET32a.

[0037] The fifth aspect of the present invention provides a method for synthesizing 3′-phosphoadenosine-5′-phosphosulfate. The reaction system uses ATP and sodium sulfate as substrates, and adds the 3′-phosphoadenosine-5′-phosphosulfate synthase mutant described in the first aspect, potassium chloride, magnesium chloride, and Tris-HCl buffer solution for catalytic reaction.

[0038] In some embodiments of the present invention, the sodium sulfate can also be replaced by other sulfur donors, and the sulfur donors include but are not limited to sulfates, sulfites, thio phosphates, hydrogen sulfide; the sulfates include but are not limited to magnesium sulfate, potassium sulfate.

[0039] In some embodiments of the present invention, in terms of the final concentration of the reaction system, ATP is 2-20 mM, sodium sulfate is 1-100 mM, the 3′-phosphoadenosine-5′-phosphosulfate synthase mutant described in the first aspect is 0.8-1.0 mg / mL, potassium chloride is 100-500 mM, magnesium chloride is 1-10 mM, the Tris-HCl buffer solution is 10-50 mM, and the pH is 8.0-10.0.

[0040] In some embodiments of the present invention, the reaction system further includes 0.02-0.1 mg / mL of the stabilizer BSA.

[0041] In some embodiments of the present invention, the temperature of the catalytic reaction is 20-40 °C, and the catalytic time is 8-20 h.

[0042] The present invention also provides the use of the 3′-phosphoadenosine-5′-phosphosulfate synthase mutant described in the first aspect in the preparation of 3′-phosphoadenosine-5′-phosphosulfate or a product containing 3′-phosphoadenosine-5′-phosphosulfate.

[0043] The present invention also provides the use of the 3′-phosphoadenosine-5′-phosphosulfate synthase mutant described in the first aspect in the preparation of a sulfate-containing compound.

[0044] Advantages of the present invention

[0045] Compared with the prior art, the present invention has the following advantages: The present invention relieves the inhibition of the APS intermediate product of hPAPSS1 through site-directed mutagenesis technology, improves its substrate affinity for ATP, and obtains a mutant enzyme with a high PAPS conversion rate; and heterologously expresses the PAPS synthesis bifunctional enzyme hPAPSS1 in Escherichia coli, simplifies the acquisition of the enzyme, and improves the synthesis efficiency of PAPS.

[0046] By modifying the sites related to ATP substrate binding, APS intermediate binding and release of hPAPSS1, the constructed mutants have good catalytic properties, which is of great significance for promoting the green and efficient synthesis of PAPS. Compared with the original enzyme hPAPSS1, the specific enzyme activity of the mutant hPASS1-C183G / F536W is increased by 2.30 times compared with the wild type; the PAPS conversion rate is increased from 19.29% of the wild type hPAPSS1 to 44.37%; the present invention lays a theoretical and technical foundation for the in-situ synthesis of PAPS by hPAPSS1. Description of the Drawings

[0047] Figure 1 Shows the SDS-PAGE electrophoresis pattern of hPAPSS1;

[0048] Figure 2 Shows the schematic diagram of PAPS synthesis catalyzed by hPAPSS1;

[0049] Figure 3 Shows the HPLC chromatogram analysis of the PAPS product;

[0050] Figure 4 Shows the mass spectrometry analysis of PAPS;

[0051] Figure 5 Shows the crystal structure of hPAPSS1 and the diagram of active pocket residues;

[0052] Figure 6 Shows the comparison diagram of PAPS conversion rates between the wild type hPAPSS1 enzyme and the mutant enzyme. Detailed Embodiments

[0053] The following examples are used herein to demonstrate the preferred embodiments of the present invention. Those skilled in the art will understand that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention, and thus can be regarded as preferred embodiments for implementing the present invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein and still obtain the same or similar results without departing from the spirit or scope of the present invention.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs, and all materials cited herein and their citations will be incorporated by reference. Those skilled in the art will recognize or can learn by routine experimentation many equivalent techniques to the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0055] The technical solution of the present application will be further described in detail below in conjunction with specific embodiments.

[0056] Experimental consumables: Escherichia coli BL21(DE3) and pET32a(+) are laboratory-preserved materials; plasmid construction reagents are purchased from Hangzhou Youkang Biotechnology Co., Ltd., and sequencing verification is completed by Hangzhou Youkang Biotechnology Co., Ltd.; analytical pure reagents are purchased from the Sinopharm Group; low molecular weight standard proteins, DNA 5000 ladder, and DNA 10000 ladder are purchased from Shanghai Baosai Biotechnology Co., Ltd.; tryptone and yeast extract are purchased from Oxoid, UK; BCA kit is purchased from Tiangen Biochemical Technology Co., Ltd.

[0057] Reagent preparation:

[0058] LB liquid medium: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast powder.

[0059] TB liquid medium: 5 g / L glycerol, 11.8 g / L peptone, 23.6 g / L yeast powder, 9.4 g / L K2HPO4, 2.2 g / L KH2PO4.

[0060] Configuration of LB agar medium: Add 15 g of agarose powder to 1 L of LB liquid medium, autoclave and pour into a 9 cm sterile petri dish to obtain LB solid medium without antibiotics; adding antibiotics will result in the corresponding antibiotic-selective LB solid medium. After condensation, place it in a 4°C refrigerator for later use.

[0061] Reagents required for PAPS catalytic reaction:

[0062] Buffer: 50 mM Tris-HCl, pH 8.0;

[0063] 0.5 M sodium sulfate: Dissolve 0.71 g of sodium sulfate in 10 mL of buffer;

[0064] 0.5 M ATP: Dissolve 2.535 g of ATP in 10 mL of buffer;

[0065] 0.5 M magnesium chloride: Dissolve 0.476 g of magnesium chloride in 10 mL of buffer;

[0066] 20 mg / mL BSA: Dissolve 100 mg of BSA in 5 mL of buffer;

[0067] 0.5 M potassium chloride: Dissolve 0.373 g of potassium chloride in 10 mL of buffer.

[0068] Preparation and detection of PAPS reaction solution: Under the reaction conditions with 50 mM Tris-HCl as the buffer solution and pH 8.0, 10 mM ATP, 20 mM sodium sulfate, 10 mM magnesium chloride, and 100 mM potassium chloride were used as the initial substrates. Subsequently, the pure hPAPSS1 enzyme solution was added. The reaction temperature was 30 °C, and the catalytic time was 24 h. After heat treatment and centrifugation of the reaction solution, the proteins in the reaction solution were removed, and HPLC detection was carried out.

[0069] An Agilent 1260 HPLC system and an ultraviolet detector were used to separate and detect PAPS products. The HPLC detection conditions were as follows:

[0070] The chromatographic column was a YMC-Pack Polyamine II column (250×4.6 mm, S-5μm, 12 nm). The mobile phase was composed of 50 mM KH2PO4 containing 0.1% triethylamine and 10% acetonitrile by volume. The flow rate was 0.6 mL / min, and the injection volume was 5 μL. During the entire detection process, the temperature of the column oven was maintained at 30 °C, and the detection wavelength of the ultraviolet detector was set at 254 nm.

[0071] Detection and definition of specific enzyme activity: Under the conditions of enzyme activity determination, the amount of enzyme required to generate 1 mmol of PAPS per minute was defined as 1 U of enzyme activity. The enzyme activity corresponding to a unit mass of enzyme was the specific enzyme activity U / mg.

[0072] Definition of PAPS conversion rate: The ratio of the concentration of the product PAPS in the reaction system to the concentration of the initial substrate ATP.

[0073] The "mutant" involved in the following examples refers to a polypeptide with 3'-phosphoadenosine-5'-phosphosulfate synthase activity that contains changes (i.e., substitutions, insertions, and / or deletions) at one or more positions. Substitution refers to replacing the amino acid occupying a certain position with a different amino acid; insertion refers to adding an amino acid adjacent to and immediately following the amino acid occupying a certain position; deletion refers to removing the amino acid occupying a certain position. The mutant of the present invention has the amino acid sequence of the wild enzyme shown in SEQ ID NO.1, and at least one amino acid substitution occurs at position 101, position 207, and position 560: The amino acids substituting position 101 include tryptophan (W), tyrosine (Y); the amino acids substituting position 207 include glycine (G), lysine (K), alanine (A), glutamic acid (E), threonine (T); the amino acids substituting position 560 include tryptophan (W), tyrosine (Y).

[0074] In the description of the mutant of the present invention, for ease of reference, the accepted IUPAC single-letter or three-letter amino acid abbreviations are used. For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid.

[0075] Example 1: Expression and purification of hPAPSS1

[0076] The gene sequence (Gene ID AAF40236.1) of the human-derived hPAPSS1 enzyme (amino acid sequence shown in SEQ ID NO.1) was obtained from the NCBI website. The target gene was amplified by polymerase chain reaction and ligated between the BamHI restriction enzyme cleavage sites of pET32a(+), resulting in a recombinant plasmid.

[0077] The recombinant plasmid was transformed into E.coli DH5α. After verification, it was streaked on a plate containing 50 g / L ampicillin. Single colonies on the plate were picked for colony PCR, and agarose nucleic acid electrophoresis was used to detect the bands. Single colonies with the correct band size were selected for sequencing.

[0078] The plasmid was extracted from the successfully sequenced bacterial solution, and the plasmid was transformed into E.coli BL21(DE3). Colony PCR and sequencing were performed again for verification, and finally the recombinant plasmid pET-32a-hPAPSS1 was obtained.

[0079] The nucleotide sequence of the hPAPSS1 enzyme is shown in SEQ ID NO.2.

[0080] Single colonies were picked and inoculated into LB seed medium. After culturing to the seed liquid, the seed liquid was transferred to 200 mL of TB fermentation medium at an inoculation amount of 1%, and the culture was continued until the OD600 was between 0.8 and 1.0. IPTG with a final concentration of 0.1 mM was added and induced at 16 °C for 18 - 24 h. After completion, the bacteria were collected, ultrasonically disrupted, and used for purification and analysis.

[0081] After the collected bacteria were ultrasonically disrupted, they were centrifuged at high speed to remove cell debris. The supernatant was filtered through a 0.45 μm aqueous membrane, and the target protein was purified by Ni-NTA affinity chromatography. After equilibrating the column with solution A, the crude enzyme solution was loaded. Then, solution B was used to elute the impurity proteins. After that, solution C was used to wash the chromatography column and the washing solution was collected. SDS-PAGE was used to verify the purified components, and the component with the highest enzyme concentration was subjected to gradient dialysis for desalting. The gradient desalting buffer was 100 mM Tris-HCl, pH 8.0 and 50 mM Tris-HCl, pH 8.0. The purified and desalted protein was collected, as Figure 1 shown.

[0082] The solutions involved in the above purification are as follows:

[0083] Solution A: 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole;

[0084] Solution B: 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 30 mM imidazole;

[0085] Solution C: 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 500 mM imidazole.

[0086] Example 2: Synthesis of PAPS catalyzed by hPAPSS1

[0087] (1) Preparation of reaction system

[0088] The reaction schematic diagram of PAPS synthesis catalyzed by hPAPSS1 is as Figure 2 shown.

[0089] The purified hPAPSS1 obtained in Example 1 was dialyzed to remove salt ions, and then verified for enzyme purity by SDS-PAGE, and the concentration of pure enzyme was determined by BCA kit.

[0090] Using 50 mM Tris-HCl pH 8.0 as the buffer of the reaction system, the catalytic reaction system contained substrates ATP, sodium sulfate, potassium chloride and magnesium chloride. In addition, a certain concentration of BSA stabilizer was added. Subsequently, hPAPSS1 was added to catalyze the reaction, and the enzymatic reaction solution was obtained after reacting at 30 °C for 24 h. The reaction system is shown in Table 1.

[0091] Table 1 Reaction system for the synthesis of PAPS by hPAPSS1

[0092] Reagent Final Concentration / Enzyme Concentration hPAPSS1 0.8 mg / mL ATP 10 mM Sodium Sulfate 20 mM Potassium Chloride 100 mM Magnesium Chloride 10 mM BSA 0.02 mg / mL

[0093] The specific enzyme activity of hPAPSS1 was determined by detecting the production of PAPS.

[0094] (2) Detection and identification of PAPS product

[0095] The enzymatic reaction solution obtained in (1) above was centrifuged at 12000 rpm for 5 min to remove proteins. The supernatant was filtered through a 0.45 μm membrane and reserved. An Agilent 1260 HPLC system and an ultraviolet detector were used to separate and detect the PAPS product. The HPLC detection conditions were as follows:

[0096] The chromatographic column was a YMC-Pack Polyamine II column (250×4.6 mm, S-5 μm, 12 nm). The mobile phase was composed of 50 mM KH2PO4 containing 0.1% triethylamine and 10% acetonitrile by volume fraction. The flow rate was 0.6 mL / min, and the injection volume was 5 μL. During the whole detection process, the temperature of the column oven was maintained at 30 °C, and the detection wavelength of the ultraviolet detector was set at 254 nm. The chromatographic peaks of the reaction components are as Figure 3 shown.

[0097] Further identification of the target product by LCMS-IT-TOF mass spectrometry showed results as Figure 4 follows.

[0098] The results showed that the ion peak of PAPS was detected, indicating that hPAPSS1 achieved effective biosynthesis of PAPS. After detection, the specific enzyme activity of hPAPSS1 was 18.52 U / mg.

[0099] Example 3: Construction of mutant enzymes of hPAPSS1

[0100] (1) Analysis of key amino acid residues at the active site of hPAPSS1 to determine its mutation sites

[0101] The crystal structure of hPAPSS1 and the residues of the active pocket are as shown in Figure 5 the following. Among them, as shown in (A) on the left in Figure 5 , the APS kinase domain of hPAPS1 contains a Cys-Cys redox regulatory element. The formation of a disulfide bond between them reduces the flexibility of the C loop, hinders the catalytic binding of ATP, and thus reduces the enzyme activity of the APS kinase domain of hPAPSS1 for PAPS production. Therefore, by mutating the amino acid residue Cys207 into other amino acids with similar properties but without disulfide bonds, it is expected to improve the binding efficiency of hPAPSS1 to the substrate ATP and increase the enzyme activity of hPAPSS1.

[0102] Figure 5 As shown in (B) on the right in

[0103] , after the formation of a Π-Π interaction between the adenine base of the intermediate product APS and the residue Phe101, the active pocket becomes smaller, resulting in a decrease in APS kinase activity. At the substrate binding site of the ATP kinase, the aromatic amino acid Phe560 is located within a diameter of 5 m around APS in hPAPSS1. By forming a Π-Π interaction with the side chain of APS, it is not conducive to its release. Therefore, site-directed mutagenesis of residues Phe101 and Phe560 is expected to improve the affinity of the APS kinase domain for APS, promote the release of APS by the ATP kinase domain, and thus increase the enzyme activity of hPAPSS1. (2) Construction of a mutant library

[0104] In this invention, using the pET-32a-hPAPSS1 recombinant plasmid constructed in Example 1 as a template, primers were designed, and PCR was used to mutate the Phe101, Phe560, and Cys207 sites of the hPAPSS1 enzyme respectively to construct a mutant library. The mutant primer information used for constructing the mutant library is shown in Table 2 below.

[0105] Table 2 Mutant primer information​

[0106] Primer Name Sequence Sequence Number F101W-F 5'-ATCTTGGCtggAGTCCTGAAGACAGAGAAGAAAATGTT-3' SEQ ID NO.3 F101W-R 5'-AGGACTccaGCCAAGATTTTTATTGAGACCTTGA-3' SEQ ID NO.4 F101Y-F 5'-TCTTGGCtatAGTCCTGAAGACAGAGAAGAAAATGTT-3' SEQ ID NO.5 F101Y-R 5'-CAGGACTataGCCAAGATTTTTATTGAGACCTTGA-3' SEQ ID NO.6 C207K-F 5'-CAGACTCCaaaGATGTAAATGACTGTGTCCAGCAAG-3' SEQ ID NO.7 C207K-R 5'-TACATCtttGGAGTCTGTTTTCAGCACCAACT-3' SEQ ID NO.8 C207A-F 5'-AGACTCCgcaGATGTAAATGACTGTGTCCAGCAAG-3' SEQ ID NO.9 C207A-R 5'-TTACATCtgcGGAGTCTGTTTTCAGCACCAACT-3' SEQ ID NO.10 C207G-F 5'-AGACTCCggtGATGTAAATGACTGTGTCCAGCAAG-3' SEQ ID NO.11 C207G-R 5'-TTACATCaccGGAGTCTGTTTTCAGCACCAACT-3' SEQ ID NO.12 C207E-F 5'-CAGACTCCgaaGATGTAAATGACTGTGTCCAGCAAG-3' SEQ ID NO.13 C207E-R 5'-TACATCttcGGAGTCTGTTTTCAGCACCAACT-3' SEQ ID NO.14 C207T-F 5'-AGACTCCaccGATGTAAATGACTGTGTCCAGCAAG-3' SEQ ID NO.15 C207T-R 5'-TTACATCggtGGAGTCTGTTTTCAGCACCAACT-3' SEQ ID NO.16 F560W-F 5'-TAGTTCCCtggCGAGTTGCAGCTTACAACAAGAA-3' SEQ ID NO.17 F560W-R 5'-AACTCGccaGGGAACTATTTCTAAAGTGATTAAACCA-3' SEQ ID NO.18 F560Y-F 5'-AGTTCCCtatCGAGTTGCAGCTTACAACAAGAA-3' SEQ ID NO.19 F560Y-R 5'-CAACTCGataGGGAACTATTTCTAAAGTGATTAAACCA-3' SEQ ID NO.20

[0107] Purify the PCR product using a PCR product purification kit to obtain a high-quality DNA purification product. Add QuickCut Dpn I enzyme to the purified PCR reaction solution and digest at 37°C for 5 min; then transform it into the competent E. coli DH5α system for cloning host; spread the transformation solution on an LB plate containing ampicillin and culture it in a constant temperature and humidity incubator at 37°C for 12 h.

[0108] It should be noted that the construction method of the double-point mutant recombinant plasmid is the same as that of the single-point mutant and is achieved through two rounds of mutant amplification.

[0109] Taking hPAPSS1-C207G / F560W as an example: First, use the recombinant plasmid of hPAPSS1 wild enzyme as a template, and use the primer pair at the C207 site described above to perform PCR amplification to obtain a linear mutant plasmid. After digestion with QuickCut Dpn I enzyme, further transform it into the E. coli DH5α system for cloning host. Spread the transformation solution on an LB plate containing ampicillin and culture it in a constant temperature and humidity incubator at 37°C for 12 h. Then, after amplification verification, obtain the recombinant plasmid of the positive mutant enzyme hPAPSS1-C207G. Then, use the recombinant plasmid of the mutant enzyme hPAPSS1-C207G as a template, and use the primer pair at the F560W site described above to perform PCR amplification to obtain a linear mutant plasmid. After digestion with QuickCut Dpn I enzyme, further transform it into the E. coli DH5α system for cloning host, and obtain the recombinant plasmid of the mutant enzyme hPAPSS1-C207G / F560W.

[0110] (3) Screening and purification of mutants

[0111] Pick single colonies on the plate in step (2), sequence the positive mutants obtained after amplification verification, and then transfer the mutant plasmids into E. coli BL21(DE3) respectively. Spread the transformation solution on a plate and culture it overnight at 37°C; then pick single colonies and culture them overnight to obtain an activated bacterial solution. Prepare the mutant pure enzyme solution according to the protein expression and purification method described in Example 1 for subsequent experiments.

[0112] The amino acid sequence and nucleotide sequence information of the mutants are shown in Table 3.

[0113] Table 3 Amino acid sequence and nucleotide sequence information of mutants

[0114] Mutant Amino acid sequence Nucleotide sequence hPAPSS1-F101W SEQ ID NO.21 SEQ ID NO.22 hPAPSS1-F101Y SEQ ID NO.23 SEQ ID NO.24 hPAPSS1-C207G SEQ ID NO.25 SEQ ID NO.26 hPAPSS1-C207K SEQ ID NO.27 SEQ ID NO.28 hPAPSS1-C207A SEQ ID NO.29 SEQ ID NO.30 hPAPSS1-C207E SEQ ID NO.31 SEQ ID NO.32 hPAPSS1-C207T SEQ ID NO.33 SEQ ID NO.34 hPAPSS1-F560W SEQ ID NO.35 SEQ ID NO.36 hPAPSS1-F560Y SEQ ID NO.37 SEQ ID NO.38 hPAPSS1-F101W / F560W SEQ ID NO.39 SEQ ID NO.40 hPAPSS1-C207G / F101W SEQ ID NO.41 SEQ ID NO.42 hPAPSS1-C207G / F560W SEQ ID NO.43 SEQ ID NO.44

[0115] Example 4: Determination of the conversion rate of mutant enzyme to synthesize PAPS

[0116] According to the PAPS synthesis method described in Example 2, reaction solutions of the original enzyme hPAPSS1 and the mutant enzyme were prepared separately, and the conversion rate of PAPS was determined by HPLC. Specifically, the concentration of the PAPS product in the mutant enzyme reaction solution was calculated from the peak area of the PAPS product detected by HPLC, and the PAPS conversion rate was defined by calculating the ratio of the concentration of the product PAPS to the initial substrate ATP concentration. The enzyme catalytic activities are shown in Table 4, and the PAPS conversion rates are shown in Table 5 and Figure 6 as shown below.

[0117] Table 4 Determination of Enzyme Catalytic Activity

[0118]

[0119]

[0120] Table 5 PAPS Conversion Rates of Mutant Enzymes in the Synthesis of PAPS

[0121] Mutant PAPS conversion rate (%) hPAPSS1 19.29 hPAPSS1-F101W 25.27 hPAPSS1-F101Y 14.47 hPAPSS1-F560W 26.62 hPAPSS1-F560Y 13.50 hPAPSS1-C207G 32.99 hPAPSS1-C207K 22.14 hPAPSS1-C207A 24.20 hPAPSS1-C207E 22.46 hPAPSS1-C207T 25.25 hPAPSS1-F101W / F560W 21.22 hPAPSS1-C207G / F101W 25.46 hPAPSS1-C207G / F560W 44.37

[0122] The results showed that among the mutants with mutations at the hPAPSS1-F101, hPAPSS1-F560, and hPAPSS1-C207 sites, except for hPAPSS1-F101Y and hPAPSS1-F560Y which retained partial catalytic activity, the catalytic activities of most other mutants were significantly improved. Among them, when G mutation, K mutation, A mutation, E mutation, and A mutation were carried out at the hPAPSS1-C207 site, the catalytic activities of their mutants were all significantly improved.

[0123] Moreover, the catalytic activities of hPAPSS1-C207G and hPAPSS1-C207G / F560W were significantly improved, and compared with the wild type, the specific enzyme activities were increased by 1.71 times and 2.30 times, respectively.

[0124] Furthermore, the PAPS conversion rate of hPAPSS1-C207G was increased from 19.29% of the wild-type hPAPSS1 to 32.99%; the PAPS conversion rate of hPAPSS1-C207G / F560W was increased from 19.29% of the wild-type hPAPSS1 to 44.37%.

[0125] All documents mentioned in the present invention are cited herein as references, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by this application.

Claims

1. A 3′-phosphoadenosine-5′-phosphosulfate synthase mutant, characterized in that, The amino acid sequence of the 3'-phosphoadenosine-5'-phosphosulfate synthase mutant, compared with the wild enzyme amino acid sequence shown in SEQ ID NO.1: (1) There is a site mutation at position 101, 207, and 560: phenylalanine at position 101 is mutated to tryptophan; cysteine at position 207 is mutated to one of glycine, lysine, alanine, glutamic acid, or threonine; phenylalanine at position 560 is mutated to tryptophan; or, (2) There are two site mutations at position 101, 207, and 560: the mutation sites are selected from any two of phenylalanine at position 101 is mutated to tryptophan; cysteine at position 207 is mutated to glycine; phenylalanine at position 560 is mutated to tryptophan.

2. The 3'-phosphoadenosine-5'-phosphosulfate synthase mutant according to claim 1, characterized in that, The 3'-phosphoadenosine-5'-phosphosulfate synthase mutant is any one of the following mutants: Mutant F101W, the amino acid sequence is as shown in SEQ ID NO: 21; Mutant C207G, the amino acid sequence is as shown in SEQ ID NO: 25; Mutant C207K, the amino acid sequence is as shown in SEQ ID NO: 27; Mutant C207A, the amino acid sequence is as shown in SEQ ID NO: 29; Mutant C207E, the amino acid sequence is as shown in SEQ ID NO: 31; Mutant C207T, the amino acid sequence is as shown in SEQ ID NO: 33; Mutant F560W, the amino acid sequence is as shown in SEQ ID NO: 35; Mutant F101W / F560W, the amino acid sequence is as shown in SEQ ID NO: 39; Mutant F101W / C207G, the amino acid sequence is as shown in SEQ ID NO: 41; Mutant C207G / F560W, the amino acid sequence is as shown in SEQ ID NO:

43.

3. A 3'-phosphoadenosine-5'-phosphosulfate synthase mutant according to claim 2, characterized in that: The nucleotide sequence of the mutant F101W is as shown in SEQ ID NO: 22; The nucleotide sequence of the mutant C207G is as shown in SEQ ID NO: 26; The nucleotide sequence of the mutant C207K is as shown in SEQ ID NO: 28; The nucleotide sequence of the mutant C207A is as shown in SEQ ID NO: 20; The nucleotide sequence of the mutant C207E is as shown in SEQ ID NO: 32; The nucleotide sequence of the mutant C207T is as shown in SEQ ID NO: 33; The nucleotide sequence of the mutant F560W is as shown in SEQ ID NO: 36; The nucleotide sequence of the mutant F101W / F560W is as shown in SEQ ID NO: 40; The nucleotide sequence of the mutant F101W / C207G is as shown in SEQ ID NO: 42; The nucleotide sequence of the mutant C207G / F560W is as shown in SEQ ID NO:

44.

4. An expression vector containing the nucleotide sequence recited in claim 3.

5. A recombinant strain containing the expression vector recited in claim 4.

6. A method for synthesizing 3′-phosphoadenosine-5′-phosphosulfate, characterized in that: The reaction system uses ATP and sodium sulfate as substrates, and adds the mutant recited in claim 1, potassium chloride, magnesium chloride and Tris-HCl buffer solution to carry out a catalytic reaction.

7. A method for synthesizing 3'-phosphoadenosine-5'-phosphosulfate according to claim 6, characterized in that: In the reaction system, in terms of the final concentration, ATP is 2-20 mM, sodium sulfate is 1-100 mM, the mutant recited in claim 1 is 0.8-1.0 mg / mL, potassium chloride is 100-500 mM, magnesium chloride is 1-10 mM, the Tris-HCl buffer solution is 10-50 mM, and the pH is 8.0-10.

0.

8. A method for synthesizing 3′-phosphoadenosine-5′-phosphosulfate according to claim 7, characterized in that: The reaction system further includes 0.02-0.1 mg / mL of the stabilizer BSA.

9. A method for synthesizing 3′-phosphoadenosine-5′-phosphosulfate according to claim 5-7, characterized in that: The temperature of the catalytic reaction is 20-40 °C, and the catalytic time is 8-20 h.

10. Use of the 3′-phosphoadenosine-5′-phosphosulfate synthase mutant recited in claim 1 in the preparation of 3′-phosphoadenosine-5′-phosphosulfate or a product containing 3′-phosphoadenosine-5′-phosphosulfate.

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