Method for efficient catalytic synthesis of PAPS based on constructing ATP regeneration system

Abstract

The present disclosure discloses a method for efficient catalytic synthesis of PAPS based on constructing an ATP regeneration system, and belongs to the technical field of bioengineering. Efficient production of PAPS is realized through microbial recombination expression and artificial construction of PAPS bifunctional synthetase. On the basis, an ATP regeneration system coupling with polyphosphate kinase from Corynebacterium glutamicum and Mycobacterium tuberculosis can be used for recovering two byproducts: pyrophosphoric acid and ADP at the same time, the equivalent conversion of a substrate and a product is realized, the PAPS generated in a catalysis system has high purity, and the sulfonic acid group donation in most sulfonic acid transfer reactions can be realized.

Claims

1. A method for synthesizing 3′-phosphoadenosine-5′-phosphosulfate (PAPS), comprising: using enzymes capable of converting ATP into PAPS as catalysts, and adding polyphosphate kinase into a reaction, wherein the catalysts are adenosine 5′-phosphosulfate(APS kinase) and ATP sulfurylase, the amino acid sequence of the APS kinase is as set forth in SEQ ID NO.2, the coding DNA sequence of the ATP sulfurylase is as set forth in SEQ ID NO.3, and the APS kinase and the ATP sulfurylase are linked through a sequence with the amino acid sequence as set forth in SEQ ID NO.1; and the coding DNA sequence of the polyphosphate kinase is as set forth in SEQ ID NO.4.

2. A method for synthesizing PAPS, comprising: using enzymes capable of converting ATP into 3′-phosphoadenosine-5′-phosphosulfate (PAPS) as catalysts, and adding polyphosphate kinase into a reaction.

3. The method according to claim 2, wherein the polyphosphate kinase comprises enzymes from Corynebacterium glutamicum, Mycobacterium tuberculosis, Bacillus aeruginosa or Streptomyces.

4. The method according to claim 2, wherein the enzymes capable of converting ATP into PAPS are APS kinase and ATP sulfurylase.

5. The method according to claim 1, wherein the enzymes capable of converting ATP into PAPS are bifunctional synthetase obtained by linking the APS kinase and the ATP sulfurylase by a linker.

6. The method according to claim 5, wherein the sequence of the linker is as set forth in SEQ ID NO.1.

7. The method according to claim 2, wherein sulfate is comprised in a reaction system.

8. A method for improving the conversion efficiency of 3′-phosphoadenosine-5′-phosphosulfate (PAPS), comprising: adding polyphosphate kinase and a phosphoric acid donor into a conversion substrate using ATP as a substrate.

9. The method according to claim 8, wherein the phosphoric acid donor comprises triphosphoric acid, tetraphosphoric acid and/or hexaphosphoric acid.

Description

BRIEF DESCRIPTION OF FIGURES

[0048] FIG. 1 is a schematic diagram of simultaneous regeneration of two byproducts in a PAPS synthesis system.

[0049] FIG. 2 is an SDS-PAGE diagram of polyphosphate kinase with different sources. Lane 1 is polyphosphate kinase from Mycobacterium tuberculosis. Lane 2 is polyphosphate kinase from Corynebacterium glutamicum.

[0050] FIG. 3 is enzyme activity determination of polyphosphate kinase from Corynebacterium glutamicum and Mycobacterium tuberculosis.

[0051] FIG. 4 is the PAPS synthesis conversion rate in different catalysis systems.

DETAILED DESCRIPTION

Materials:

[0052] Bacterial strains and plasmids used in experiments are all preserved in this laboratory.

[0053] Various analytical pure reagents are purchased from China National Pharmaceutical Group.

[0054] Gene manipulation tools and reagents are purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0055] LB culture medium (g/L): 10 of peptone, 5 of yeast powder, and 10 of sodium chloride.

[0056] TB culture medium (g/L): 12 of peptone, 24 of yeast powder, 9.4 of K.sub.2HPO.sub.4, 2.2 of KH.sub.2PO.sub.4, and 5 of glycerol.

[0057] ATP detection method: purchased from Beijing Solarbio Science & Technology Co.,Ltd. See instructions for use.

[0058] PAPS detection method: the following are adopted: an Agilent 1600 HPLC system, a Polyamine II column (4.6×250 mm, 12 nm), a mobile phase: 50 mM of KH.sub.2PO.sub.4 and 0.1% triethylamine solution, a flow velocity: 0.6 mL.Math.min.sup.−1, an injection volume: 5 μL, detection time: 35 min, and a detector: UV 254 nm.

[0059] PAPS bifunctional synthetase: with ATP sulfurylase and APS kinase activities at the same time.

[0060] Definition of enzyme activity of PAPS bifunctional synthetase: the enzyme amount required by synthesis of 1 μM of PAPS per hour under a condition of 37° C.

[0061] Definition of enzyme activity of polyphosphate kinase: the enzyme amount required by synthesis of 1 μM of ATP per hour under a condition of 37° C.

[0062] Definition of specific enzyme activity: enzyme activity unit number of unit weight (mg) of protein at 37° C.

[0063] ATP conversion rate: mole ratio of mole number of product PAPS to substrate ATP.

Example 1: Obtaining of Polyphosphate Kinase

[0064] (1) The Gene ID of the polyphosphate kinase from Corynebacterium glutamicum obtained from NCBI website was 1020661. Through codon optimization, a nucleotide sequence was obtained. A target gene was amplified through a polymerase chain reaction, and was linked to a pRSFDuet-1 vector to obtain recombinant plasmids. The correctly verified recombinant plasmids were transfected into E. coli BL21 (DE3) to obtain a transformant. The transformant was cultured and subjected to sequencing verification, and the correctly verified transformant position was recorded as bacterial strain E. coli GluPPK.

[0065] (2) Through inquiry, the Gene ID of the polyphosphate kinase from Mycobacterium tuberculosis was 888760. Through codon optimization, a nucleotide sequence was obtained. A method similar to step (1) was used to amplify the target gene and link the target gene onto a vector pET32a (+), and the correctly verified recombinant plasmids were transfected into E. coli BL21 (DE3) to obtain a transformant. The transformant was cultured and subjected to sequencing verification, and the correctly verified transformant position was recorded as bacterial strain E. coli MycPPK.

[0066] Induction conditions of bacterial strain E. coli GluPPK: 0.3-0.5 mM of IPTG induced expression (30° C., 220 rpm), and expression time of 10-12 h. Kanamycin sulfate with a final concentration of 50 μg/L was added into a culture medium to ensure the plasmid stability.

[0067] Induction conditions of bacterial strain E. coli MycPPK: 0.1-0.2 mM of IPTG induced expression (20° C., 220 rpm), and expression time of 20-24 h. Ampicillin with a final concentration of 100 μg/L was added into a culture medium.

Example 2: Purification and Enzyme Activity Comparison of Two Kinds of Polyphosphate Kinase

[0068] Recombination bacterial strains obtained in Example 1 were cultured for 6-20 h in an LB culture medium. After ultrasonic disruption, collected thalli were subjected to high-speed centrifugation to remove cell debris. The supernatant was filtered by a 0.22 μm water system membrane, and target protein was purified through Ni-NTA affinity chromatography. After column balance of solution A, a crude enzyme was loaded. Then, a chromatographic column was balanced through solution A. Next, the chromatographic column was flushed by B solution of different concentrations, and flushing solution was collected. Purified ingredients (FIG. 2) were verified by SDS-PAGE. The purest ingredients were desalinized through a PD-10 desalinizing column. During desalinization, low-salt buffer (10 mM Tris-HCl, 0.1 M NaCl; pH 6.0) was used, and purified desalinized protein was obtained through collection.

[0069] Solution A: 20 mM of Tris-HCl buffer with a pH value of 7.5, and 500 mM of NaCl.

[0070] Solution B: 20 mM of Tris-HCl buffer with a pH value of 7.5, 500 mM of NaCl, and 500 mM of imidazole.

[0071] After pure enzymes of polyphosphate kinase from different sources were obtained, the same amount of 0.5-1.0 mg/L of pure enzyme and 5 g/L of ADP were added into 50-100 mM of Tris-HCl buffer with a pH value of 7.5 of a catalysis system at the same time. Then, different amount of 2-5 g/L of phosphoric acid donors of sodium pyrophosphate, sodium tripolyphosphate and sodium hexametaphosphate were respectively added. The specific enzyme activity of the polyphosphate kinase was determined by detecting the production of ATP. A reaction temperature was 30-35° C., a reaction time was 12-24 h, and a result was as shown in FIG. 3. The polyphosphate kinase from Corynebacterium glutamicum might have higher specific enzyme activity when the sodium pyrophosphate, sodium tripolyphosphate and the sodium hexametaphosphate were used as substrates.

Example 3: Expression, Purification and Enzyme Activity Determination of Bifunctional Synthetase

[0072] Construction of bifunctional synthetase: ATP sulfurylase (Gene ID: 853466) from Saccharomyces cerevisiae and APS kinase (GenBank number: M74586.1) from Escherichia coli were fused into one segment (the expression was not influenced by the sequential order of two enzymes) according to a gene manipulation measure. Different fusion linker sequences (linker) were respectively added to linker portions to obtain one segment through fusion, so that the two enzymes maintained a certain spatial position, and the catalysis was more ordered. Specifically, a termination codon of a former gene was removed, the former gene was directly linked to the linker, and was then linked with an initiation codon of another gene. The sequence of the linker was (GGGGS).sub.6. The fusion segment was linked to plasmids pET28a (+), and was transfected into E. coli BL21(DE3) to obtain a transformant. The transformant was cultured and subjected to sequencing verification, and the correctly verified transformant position was recorded as bacterial strain E. coli CaePAPS. The specific expression manner and enzyme purification methods are the same as those in Examples 1 and 2.

[0073] Induction conditions of bacterial strain E. coli CaePAPS: 0.1-0.2 mM of IPTG induced expression (25° C., 220 rpm), and expression time of 10-15 h.

[0074] Enzyme activity determination: 0.5-1.0 mg/mL of pure enzyme of bifunctional synthetase, 1-5 g/L of a substrate ATP and 0.5-2.5 g/L of magnesium sulfate were added into 50-100 mM of Tris-HCl buffer with a pH value of 7.5 of a catalysis system. The enzyme activity of the PAPS bifunctional synthetase was determined by detecting the production of PAPS in a reaction system. A reaction temperature was 30-35° C., and a reaction time was 24-48 h. The result showed that the enzyme activity is 300±20 U, and the enzyme may be used for subsequent conversion.

Example 4: Efficient Synthesis of PAPS by Coupling ATP Regeneration System

[0075] The PAPS was synthesized by using the bifunctional synthetase obtained in Example 3 and the polyphosphate kinase from Corynebacterium glutamicum obtained in Example 2.

[0076] A 1.5 mL reaction system (50-100 mM Tris-HCl buffer with pH value of 7.5) including 1 mg/mL of PAPS bifunctional synthetase, 1 mg/mL of polyphosphate kinase, 5 g/L of a substrate ATP and 3 g/L of magnesium sulfate was prepared. The adding time of the PAPS bifunctional synthetase was 0 h after the starting of the reaction. The adding time of the polyphosphate kinase was 0-24 h after the starting of the reaction. The reaction ended after 35-50 h of the reaction (no ATP detection in the reaction system was regarded as the end of the reaction). The yield of PAPS was determined after the end of the reaction.

[0077] In order to reach higher yield and higher conversion rate, phosphoric acid donors (sodium pyrophosphate, sodium tripolyphosphate and sodium hexametaphosphate) might be additionally added into the catalysis system. The PAPS was prepared and synthesized by using the same method. The only difference was that during reaction, the phosphoric acid donors (sodium pyrophosphate, sodium tripolyphosphate and sodium hexametaphosphate) with a final concentration of 2 g/L were added into the reaction system for reaction. After the end of the reaction, the PAPS was purified, and the yield was determined.

[0078] The result of the yield was as shown in FIG. 4. After the coupling with the polyphosphate kinase, the conversion efficiency of the substrate ATP is obviously improved, and may be raised from 42% before coupling to 83%-95%.

[0079] Although the exemplary Examples of the disclosure have been provided above, they are not intended to limit the disclosure. Those skilled in the art will appreciate that various changes and modifications may be made without departing from the spirit and scope of the disclosure. Therefore, the protection scope of the disclosure shall be defined by the claims.