WO2022068089A1 - 一种基于构建atp再生系统高效催化合成paps的方法 - Google Patents

一种基于构建atp再生系统高效催化合成paps的方法 Download PDF

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WO2022068089A1
WO2022068089A1 PCT/CN2020/137172 CN2020137172W WO2022068089A1 WO 2022068089 A1 WO2022068089 A1 WO 2022068089A1 CN 2020137172 W CN2020137172 W CN 2020137172W WO 2022068089 A1 WO2022068089 A1 WO 2022068089A1
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acid
atp
paps
polyphosphokinase
phosphoadenosine
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康振
堵国成
王阳
胥睿睿
陈坚
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Jiangnan University
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    • C12P19/30—Nucleotides
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    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
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Definitions

  • the invention relates to a method for efficiently catalyzing and synthesizing PAPS based on the construction of an ATP regeneration system, and belongs to the technical field of biological engineering.
  • 3'-phosphoadenosine-5'-phosphosulfate (3'-phosphoadenosine-5'-phosphosulfate, PAPS) is the most important active sulfonic acid donor for sulfonic acid reaction in vitro, and can be used for chondroitin sulfate, heparin and sulfated skin Supplementation of the sulfonated group of the element.
  • the purchase channels of PAPS are narrow, the price is high and the purity is not high.
  • the main reason is that the conversion rate of in vitro enzymatic synthesis of PAPS is low, the by-products are difficult to remove, and the purification of PAPS is difficult.
  • the inventors selected a polyphosphokinase with high enzymatic activity and efficient utilization of ADP and pyrophosphate from the few polyphosphokinases capable of simultaneously converting ADP and pyrophosphate. Based on the screened polyphosphokinase, it can be coupled with the system for enzymatically catalyzing the generation of PAPS, and the by-product pyrophosphate and ADP synthesis substrate ATP can be simultaneously recovered, and continue to participate in the synthesis of PAPS to realize the synthesis of PAPS without by-products. , in the catalytic system has a very high purity of PAPS, widely used. Thus, the conversion rate of the entire reaction process is improved, the production cost is significantly reduced, and the efficiency is improved.
  • the invention provides a method for synthesizing PAPS, adding polyphosphokinase in the reaction of enzymatically converting and synthesizing PAPS; the enzymatic converting and synthesizing PAPS uses ATP as a substrate and an enzyme capable of converting ATP into PAPS as a catalyst .
  • the polyphosphokinase includes, but is not limited to, derived from Corynebacterium glutamicum, Mycobacterium tuberculosis, Bacillus aeruginosa or Streptomyces.
  • the Gene ID of the polyphosphokinase derived from Mycobacterium tuberculosis is 888760.
  • the polyphosphokinase is derived from Corynebacterium glutamicum, and the Gene ID is 1020661.
  • the enzyme capable of converting ATP into PAPS is APS kinase and ATP sulfurylase; or a bifunctional PAPS synthesis enzyme obtained by linking APS kinase and ATP sulfurylase through a linker.
  • the Gen Bank No. of the APS kinase is M74586.1.
  • the Gene ID of the ATP sulfurylase is 853466.
  • sequence of the linker is shown in SEQ ID NO.1.
  • the reaction system also contains sulfate as a sulfate radical donor, and sulfate includes but is not limited to magnesium sulfate, sodium sulfate, and potassium sulfate.
  • the sulfate may preferably be magnesium sulfate.
  • the substrates ATP and magnesium sulfate are added to the reaction system, and then the PAPS synthetic bifunctional enzyme is added, or APS kinase and ATP sulfurylase are added simultaneously, and the catalyzed reaction starts 0-24 hours after the addition of polyphosphokinase reaction.
  • the buffer in the catalytic system for catalyzing the synthesis of PAPS is 50-100 mM Tris-HCl buffer pH 7.0-8.5, the PAPS synthesis bifunctional enzyme is 1-5 mg/mL, and the polyphosphokinase is 1-5 mg/mL. mL, substrate ATP 5 ⁇ 25g/L, magnesium sulfate 3 ⁇ 15g/L.
  • the reaction is deemed complete when no ATP is detected in the reaction system.
  • the present invention provides a method for improving the conversion efficiency of PAPS, by adding a polyphosphokinase and a phosphate donor to the conversion substrate using ATP as the substrate.
  • the phosphoric acid donor includes tripolyphosphoric acid, tetraphosphoric acid and/or hexaphosphoric acid.
  • the phosphoric acid donor includes sodium tripolyphosphate, sodium tetrapolyphosphate and/or sodium hexapolyphosphate.
  • the polyphosphokinase includes, but is not limited to, derived from Corynebacterium glutamicum, Mycobacterium tuberculosis, Bacillus aeruginosa or Streptomyces.
  • the Gene ID of the polyphosphokinase derived from Mycobacterium tuberculosis is 888760.
  • the polyphosphokinase is derived from Corynebacterium glutamicum, and the Gene ID is 1020661.
  • the reaction system also contains sulfate as a sulfate radical donor, and sulfate includes but is not limited to magnesium sulfate, sodium sulfate, and potassium sulfate.
  • the sulfate may preferably be magnesium sulfate.
  • the substrates ATP and magnesium sulfate are added to the reaction system, and then the PAPS synthetic bifunctional enzyme is added, or APS kinase and ATP sulfurylase are added simultaneously, and the catalyzed reaction starts 0-24 hours after the addition of polyphosphokinase reaction.
  • the buffer in the catalytic system for catalyzing the synthesis of PAPS is 50-100 mM Tris-HCl buffer pH 7.0-8.5, the PAPS synthesis bifunctional enzyme is 1-5 mg/mL, and the polyphosphokinase is 1-5 mg/mL. mL, substrate ATP 5 ⁇ 25g/L, magnesium sulfate 3 ⁇ 15g/L.
  • the reaction is deemed complete when no ATP is detected in the reaction system.
  • the invention provides a method for improving the recovery efficiency and rate of ADP.
  • polyphosphokinase is added, and a phosphate donor is added at the same time; the ADP is a by-product of synthesizing PAPS with ATP as a substrate.
  • the polyphosphokinase includes, but is not limited to, derived from Corynebacterium glutamicum, Mycobacterium tuberculosis, Bacillus aeruginosa or Streptomyces.
  • the Gene ID of the polyphosphokinase derived from Mycobacterium tuberculosis is 888760.
  • the polyphosphokinase is derived from Corynebacterium glutamicum, and the Gene ID is 1020661.
  • the phosphoric acid donor includes but is not limited to pyrophosphoric acid, tripolyphosphoric acid, tetraphosphoric acid and/or hexaphosphoric acid.
  • the phosphoric acid donor includes sodium tripolyphosphate, sodium tetrapolyphosphate and/or sodium hexapolyphosphate.
  • the reaction system also contains sulfate as a sulfate radical donor, and sulfate includes but is not limited to magnesium sulfate, sodium sulfate, and potassium sulfate.
  • the sulfate may preferably be magnesium sulfate.
  • the substrates ATP and magnesium sulfate are added to the reaction system, and then the PAPS synthetic bifunctional enzyme is added, or APS kinase and ATP sulfurylase are added simultaneously, and the catalyzed reaction starts 0-24 hours after the addition of polyphosphokinase reaction.
  • the buffer in the catalytic system for catalyzing the synthesis of PAPS is 50-100 mM Tris-HCl buffer pH 7.0-8.5, the PAPS synthesis bifunctional enzyme is 1-5 mg/mL, and the polyphosphokinase is 1-5 mg/mL. mL, substrate ATP 5 ⁇ 25g/L, magnesium sulfate 3 ⁇ 15g/L.
  • the reaction is deemed complete when no ATP is detected in the reaction system.
  • the present invention also protects the application of the method for synthesizing PAPS, or the method for improving the conversion efficiency of PAPS, or the method for improving the recovery efficiency and rate of ADP in the preparation of PAPS.
  • the present invention also protects the application of the method for synthesizing PAPS, or the method for improving the conversion efficiency of PAPS, or the method for improving the recovery efficiency and rate of ADP, in the preparation of products using PAPS as raw materials.
  • the present invention also protects the application of the method for synthesizing PAPS, or the method for improving the conversion efficiency of PAPS, or the method for improving the recovery efficiency and rate of ADP, in the preparation of products requiring the supply of sulfonic acid groups.
  • the present invention utilizes the PAPS synthesis bifunctional enzyme to prepare PAPS. Compared with the traditional bienzyme synthesis method, the catalyst preparation steps are simple, the catalytic efficiency is high, and the stability is good.
  • the present invention introduces a by-product recovery system coupled with polyphosphokinase in the PAPS synthesis reaction, which can simultaneously recover by-product pyrophosphate and ADP, and realize the equimolar conversion of the substrates ATP and PAPS, so that the system after the synthesis is completed.
  • a by-product recovery system coupled with polyphosphokinase in the PAPS synthesis reaction, which can simultaneously recover by-product pyrophosphate and ADP, and realize the equimolar conversion of the substrates ATP and PAPS, so that the system after the synthesis is completed.
  • Contains higher purity PAPS which can be used for the supply of sulfonic acid groups in most sulfonic acid transfer reactions without additional purification.
  • Figure 1 is a schematic diagram of the simultaneous regeneration of two by-products in the PAPS synthesis system.
  • Figure 2 is the SDS-PAGE chart of polyphosphokinases from different sources; lane 1 is the polyphosphokinase derived from mycobacteria binding; lane 2 is the polyphosphokinase derived from Corynebacterium glutamicum.
  • Fig. 3 is an enzymatic activity measurement of polyphosphokinase derived from Corynebacterium glutamicum and Mycobacterium.
  • Figure 4 shows the synthesis conversion of PAPS under different catalytic systems.
  • LB medium g/L: peptone 10, yeast powder 5, sodium chloride 10.
  • TB medium g/L: peptone 12, yeast powder 24, K 2 HPO 4 9.4, KH 2 PO 4 2.2, glycerol 5.
  • ATP detection method purchased from Beijing Soleibao Technology Co., Ltd., please refer to the instruction manual for the usage method.
  • PAPS detection method Agilent 1600HPLC system, Polyamine II column (4.6 ⁇ 250mm, 12nm), mobile phase: 50mM KH 2 PO 4 and 0.1% triethylamine solution, flow rate: 0.6mL ⁇ min -1 , injection volume: 5 ⁇ L , detection time 35min, detector: UV 254nm.
  • polyphosphokinase activity the amount of enzyme required to synthesize 1 ⁇ M ATP per hour at 37°C.
  • Specific enzymatic activity is defined as the number of enzymatic activity units per unit weight (mg) of protein at 37°C.
  • ATP conversion the ratio of moles of product PAPS to moles of substrate ATP.
  • step (2) Find the Gene ID: 888760 of the polyphosphokinase derived from Mycobacterium tuberculosis, obtain the nucleotide sequence through codon optimization, and use a method similar to step (1) to amplify and connect the target gene to the vector pET32a (+), and transfer the verified correct recombinant plasmid into E.coli BL21 (DE3) to obtain transformants, culture and sequence the transformants for verification, and record the verified correct transformants as strain E.coli MycPPK.
  • Expression conditions Pick a single colony and cultivate it in LB medium overnight, inoculate the seeds in TB medium at 2mL/100mL, and continue to culture until the OD600 is 0.6-0.8 to induce expression.
  • strain E.coli GluPPK The induction conditions of strain E.coli GluPPK are: 0.3-0.5mM IPTG to induce expression (30°C, 220rpm), and the expression time is 10-12h; the final concentration of 50 ⁇ g/L kanamycin sulfate is added to the medium to ensure the plasmid expression. stability.
  • strain E.coli MycPPK The induction conditions of strain E.coli MycPPK are: 0.1-0.2mM IPTG to induce expression (20°C, 220rpm), and the expression time is 20-24h; ampicillin with a final concentration of 100 ⁇ g/L is added to the medium.
  • Example 2 Purification of two polyphosphokinases and comparison of their enzymatic activities
  • Example 2 After the recombinant strain obtained in Example 1 was cultured in LB medium for 6-20 hours, the collected cells were sonicated and then centrifuged at high speed to remove cell debris. The supernatant was filtered through a 0.22 ⁇ m aqueous membrane, and a Ni-NTA affinity layer was used. analysis to purify the target protein. After equilibrating the column with liquid A, load the crude enzyme solution, and then use liquid A to equilibrate the chromatography column, then use liquid B of different concentrations to wash the chromatography column and collect the washing liquid, and use SDS-PAGE to verify the purified components ( Figure 2).
  • the purest fraction was desalted with a PD-10 desalting column, and a low-salt buffer (10 mM Tris-HCl, 0.1 M NaCl; pH 6.0) was used for desalting, and the purified and desalted protein was collected.
  • a low-salt buffer (10 mM Tris-HCl, 0.1 M NaCl; pH 6.0) was used for desalting, and the purified and desalted protein was collected.
  • Solution A 20mM Tris-HCl buffer pH 7.5, 500mM NaCl
  • Solution B 20mM Tris-HCl buffer pH 7.5, 500mM NaCl, 500mM imidazole.
  • polyphosphokinase After obtaining the pure enzymes of polyphosphokinase from different sources, firstly add the same amount of pure enzyme 0.5-1.0mg/L and ADP 5g/L to the catalytic system 50-100mM Tris-HCl buffer pH 7.5, and then add the same amount respectively Different phosphate donors sodium pyrophosphate, sodium tripolyphosphate and sodium metahexaphosphate 2-5g/L.
  • the specific enzyme activity of polyphosphokinase was determined by detecting the production of ATP. The reaction temperature was 30-35 °C and the reaction time was 12-24 h. The results are shown in Figure 3.
  • the polyphosphokinase derived from Corynebacterium glutamicum can be When using sodium pyrophosphate, sodium tripolyphosphate and sodium metahexaphosphate as substrates, the specific enzyme activity is higher.
  • ATP sulfurylase (Gene ID: 853466) derived from Saccharomyces cerevisiae and APS kinase (GenBank No. M74586.1) derived from Escherichia coli were fused into a fragment (before and after the two enzymes) according to gene manipulation methods. The sequence does not affect its expression), add different fusion linker sequences (linkers) to the linker part, and fuse them into a fragment, so that both enzymes maintain a certain spatial position, and the catalysis is more orderly.
  • linkers linker sequences
  • the linker sequence was: (GGGGS) 6
  • the fusion fragment was connected to the plasmid pET28a(+), and transferred into E. coli BL21 (DE3)
  • the transformants were obtained, and the transformants were cultured and sequenced for verification, and the correct transformants were recorded as strain E.coli CaePAPS.
  • the specific expression method and the purification method of the enzyme are the same as those in Examples 1 and 2.
  • Enzyme activity assay Add 0.5-1.0mg/mL of pure enzyme of bifunctional enzyme, 1-5g/L of substrate ATP and 0.5-2.5g/L of magnesium sulfate to 50-100mM Tris-HCl buffer pH 7.5 in the catalytic system, The enzymatic activity of the PAPS-synthesizing bifunctional enzyme was determined by detecting the production of PAPS in the reaction system. The reaction temperature was 30-35° C. and the reaction time was 24-48 h. The results showed that the enzyme activity was 300 ⁇ 20U, which could be used for subsequent transformation.
  • additional phosphoric acid donors can be added to the catalytic system to prepare synthetic PAPS by the same method as above, but in During the reaction, phosphoric acid donors (sodium pyrophosphate, sodium tripolyphosphate and sodium metahexaphosphate) with a final concentration of 2 g/L were added to the reaction system to carry out the reaction, and after the reaction, the PAPS was purified and the yield was measured.

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Abstract

提供了一种基于构建ATP再生系统催化合成PAPS的方法，包括通过微生物重组表达人工构建PAPS合成双功能酶，耦联谷氨酸棒杆菌和结核分枝杆菌来源的聚磷酸激酶的ATP再生系统，可同时回收两种副产物焦磷酸和ADP，实现底物与产物的等量转化，催化体系中生成的PAPS具有较高的纯度，能实现大部分磺酸转移反应中磺酸基团的供给。

Description

一种基于构建ATP再生系统高效催化合成PAPS的方法 技术领域
本发明涉及一种基于构建ATP再生系统高效催化合成PAPS的方法，属于生物工程技术领域。
背景技术
3’-磷酸腺苷-5’-磷酰硫酸(3’-phosphoadenosine-5’-phosphosulfate，PAPS)是体外磺酸反应最重要的活性磺酸供体，可用于硫酸软骨素、肝素和硫酸皮肤素的磺酸化基团供给。
目前PAPS购买渠道狭窄，价格昂贵且纯度不高，主要原因为体外酶法合成PAPS转化率低，副产物难以去除，PAPS纯化困难。
在PAPS酶法合成中，主要产生两种副产物焦磷酸和ADP，使得底物的理论转化率仅为50％。副产物的重新利用是PAPS高效生产的重要研究内容，在目前的研究中多从聚磷酸激酶的角度入手，以期将副产物转化再利用，但是大多数的聚磷酸激酶只能转化ADP，而并不能转化焦磷酸，仍然存在着副产物不能得到充分利用的问题。
发明内容
[技术问题]
在酶法合成PAPS的过程中，由于副产物焦磷酸和ADP的产生，大量物质流和能量流被浪费，导致PAPS合成价格高昂且难于得到纯度较高的PAPS。已有报道的聚磷酸激酶大部分都只能转化ADP，而不能转化焦磷酸，但是在酶法制备PAPS中，副产物焦磷酸的量也比较大，如果只能转化ADP的话，那么剩余的焦磷酸还是不能被充分利用，造成了资源的浪费。
[技术方案]
发明人从少数能够同时转化ADP和焦磷酸的聚磷酸激酶中选出了一种酶活力高且能高效利用ADP和焦磷酸的聚磷酸激酶。基于筛选出的聚磷酸激酶，将其与酶法催化生成PAPS的系统进行耦联，可同时回收副产物焦磷酸和ADP合成底物ATP，继续参加PAPS的合成，实现无副产物的PAPS的合成，在催化体系中有极高的PAPS纯度，应用广泛。从而提高了整个反应过程的转化率、显著降低了生产成本、提升了效率。
本发明提供了一种合成PAPS的方法，在酶法转化合成PAPS的反应中加入聚磷酸激酶；所述酶法转化合成PAPS是以ATP为底物，以能将ATP转化为PAPS的酶为催化剂。
在本发明的一种实施方式中，所述聚磷酸激酶包括但不限于来源于谷氨酸棒杆菌、结核分枝杆菌、铜绿芽孢杆菌或链霉菌。
在本发明的一种实施方式中，来源于结核分枝杆菌的聚磷酸激酶的Gene ID为888760。
在本发明的一种实施方式中，所述聚磷酸激酶来源于谷氨酸棒杆菌，Gene ID为1020661。
在本发明的一种实施方式中，所述能将ATP转化为PAPS的酶为APS激酶和ATP硫酸化酶；或是通过linker将APS激酶和ATP硫酸化酶连接得到的PAPS合成双功能酶。
在本发明的一种实施方式中，所述APS激酶的Gen Bank号为M74586.1。
在本发明的一种实施方式中，所述ATP硫酸化酶的Gene ID为853466。
在本发明的一种实施方式中，所述linker的序列如SEQ ID NO.1所示。
在本发明的一种实施方式中，反应体系中还含有硫酸盐作为硫酸根供体，硫酸盐包括但不限于硫酸镁、硫酸钠、硫酸钾。
在本发明的一种实施方式中，硫酸盐可优选为硫酸镁。
在本发明的一种实施方式中，在反应体系中加入底物ATP和硫酸镁，再加入PAPS合成双功能酶，或同时加入APS激酶和ATP硫酸化酶，催化反应开始后的0-24h添加聚磷酸激酶进行反应。
在本发明的一种实施方式中，催化合成PAPS的催化体系中缓冲液为50-100mM Tris-HCl buffer pH 7.0～8.5，PAPS合成双功能酶1～5mg/mL，聚磷酸激酶1～5mg/mL，底物ATP 5～25g/L，硫酸镁3～15g/L。
在本发明的一种实施方式中，反应体系中没有ATP检出时即视为反应结束。
本发明提供了一种提高PAPS转化效率的方法，在以ATP为底物的转化底物中加入聚磷酸激酶和磷酸供体。
在本发明的一种实施方式中，所述磷酸供体包括三聚磷酸、四聚磷酸和/或六聚磷酸。
在本发明的一种实施方式中，所述磷酸供体包括三聚磷酸钠、四聚磷酸钠和/或六聚磷酸钠。
在本发明的一种实施方式中，所述聚磷酸激酶包括但不限于来源于谷氨酸棒杆菌、结核分枝杆菌、铜绿芽孢杆菌或链霉菌。
在本发明的一种实施方式中，来源于结核分枝杆菌的聚磷酸激酶的Gene ID为888760。
在本发明的一种实施方式中，所述聚磷酸激酶来源于谷氨酸棒杆菌，Gene ID为1020661。
在本发明的一种实施方式中，反应体系中还含有硫酸盐作为硫酸根供体，硫酸盐包括但不限于硫酸镁、硫酸钠、硫酸钾。
在本发明的一种实施方式中，硫酸盐可优选为硫酸镁。
在本发明的一种实施方式中，在反应体系中加入底物ATP和硫酸镁，再加入PAPS合成双功能酶，或同时加入APS激酶和ATP硫酸化酶，催化反应开始后的0-24h添加聚磷酸激酶进行反应。
在本发明的一种实施方式中，催化合成PAPS的催化体系中缓冲液为50-100mM Tris-HCl buffer pH 7.0～8.5，PAPS合成双功能酶1～5mg/mL，聚磷酸激酶1～5mg/mL，底物ATP 5～25g/L，硫酸镁3～15g/L。
在本发明的一种实施方式中，反应体系中没有ATP检出时即视为反应结束。
本发明提供了一种提高ADP回收效率和速率的方法，在合成PAPS的反应中加入聚磷酸激酶，同时再加入磷酸供体；所述ADP是以ATP为底物合成PAPS的副产物。
在本发明的一种实施方式中，所述聚磷酸激酶包括但不限于来源于谷氨酸棒杆菌、结核分枝杆菌、铜绿芽孢杆菌或链霉菌。
在本发明的一种实施方式中，来源于结核分枝杆菌的聚磷酸激酶的Gene ID为888760。
在本发明的一种实施方式中，所述聚磷酸激酶来源于谷氨酸棒杆菌，Gene ID为1020661。
在本发明的一种实施方式中，所述磷酸供体包括但不限于焦磷酸、三聚磷酸、四聚磷酸和/或六聚磷酸。
在本发明的一种实施方式中，所述磷酸供体包括三聚磷酸钠、四聚磷酸钠和/或六聚磷酸钠。
在本发明的一种实施方式中，反应体系中还含有硫酸盐作为硫酸根供体，硫酸盐包括但不限于硫酸镁、硫酸钠、硫酸钾。
在本发明的一种实施方式中，硫酸盐可优选为硫酸镁。
在本发明的一种实施方式中，在反应体系中加入底物ATP和硫酸镁，再加入PAPS合成双功能酶，或同时加入APS激酶和ATP硫酸化酶，催化反应开始后的0-24h添加聚磷酸激酶进行反应。
在本发明的一种实施方式中，催化合成PAPS的催化体系中缓冲液为50-100mM Tris-HCl buffer pH 7.0～8.5，PAPS合成双功能酶1～5mg/mL，聚磷酸激酶1～5mg/mL，底物ATP 5～25g/L，硫酸镁3～15g/L。
在本发明的一种实施方式中，反应体系中没有ATP检出时即视为反应结束。
本发明还保护所述合成PAPS的方法，或提高PAPS转化效率的方法，或提高ADP回收效率和速率的方法在制备PAPS中的应用。
本发明还保护所述合成PAPS的方法，或提高PAPS转化效率的方法，或提高ADP回收效率和速率的方法在制备以PAPS为原料的产品中的应用。
本发明还保护所述合成PAPS的方法，或提高PAPS转化效率的方法，或提高ADP回收效率和速率的方法在制备需要有磺酸基团供给的产品中的应用。
[有益效果]
1、本发明利用PAPS合成双功能酶制备PAPS，与传统双酶合成法相比，催化剂制备步骤简单，催化效率高，稳定性好。
2、本发明在PAPS合成反应中引入耦联聚磷酸激酶的副产物回收系统，能同时将副产物焦磷酸和ADP回收，实现底物ATP和PAPS的等摩尔转化，使得合成完成后的体系中含有较高纯度的PAPS，无需额外纯化也可用于大部分磺酸转移反应中磺酸基团的供给。
附图说明
图1是PAPS合成体系中两种副产物同时再生示意图。
图2是不同来源聚磷酸激酶SDS-PAGE图；泳道1为结合分枝杆菌来源的聚磷酸激酶；泳道2为谷氨酸棒杆菌来源的聚磷酸激酶。
图3是谷氨酸棒杆菌及分支杆菌来源的聚磷酸激酶的酶活测定。
图4是不同催化体系下PAPS合成转化率。
具体实施方式
材料：
实验中所涉及的菌株、质粒均为本实验室保藏。
各种分析纯试剂购自国药集团。
基因操作工具、试剂购买于上海生物生工。
LB培养基(g/L)：蛋白胨10，酵母粉5，氯化钠10。
TB培养基(g/L)：蛋白胨12，酵母粉24，K 2HPO 4 9.4，KH 2PO 4 2.2，甘油5。
ATP检测方法：购买于北京索莱宝科技有限公司，使用方法详见说明书。
PAPS检测方法：采用Agilent 1600HPLC系统，Polyamine II柱(4.6×250mm,12nm)，流动相：50mM KH 2PO 4和0.1％三乙胺溶液，流速：0.6mL·min -1，进样量：5μL，检测时间35min，检测器：UV 254nm。
PAPS合成双功能酶酶活定义：37℃条件下，每小时合成1μM PAPS所需要的酶量。
聚磷酸激酶酶活定义：37℃条件下，每小时合成1μM ATP所需要的酶量。
比酶活定义：37℃条件下，单位重量(mg)蛋白质所具有的酶活力单位数。
ATP转化率：产品PAPS的摩尔数与底物ATP的摩尔比值。
实施例1：聚磷酸激酶的获得
(1)在NCBI网站获得来源谷氨酸棒杆菌的聚磷酸激酶的Gene ID:1020661，通过密码子优化获得核苷酸序列，利用聚合酶链式反应扩增目的基因，并接至pRSFDuet-1载体上，得到重组质粒，将验证正确的重组质粒转入E.coli BL21(DE3)得到转化子，将转化子培养并测序验证，将验证正确的转化子处记为菌株E.coli GluPPK。
(2)查得结核分枝杆菌来源的聚磷酸激酶的Gene ID:888760，通过密码子优化获得核苷酸序列，利用与步骤(1)相似的方法，将目的基因扩增并连接到载体pET32a(+)上，并将验证正确的重组质粒转入E.coli BL21(DE3)得到转化子，将转化子培养并测序验证，将验证正确的转化子记为菌株E.coli MycPPK。
表达条件：挑取单菌落于LB培养基中过夜培养，将种子按照2mL/100mL接种于TB培养基中，继续培养至OD600为0.6-0.8时进行诱导表达。
菌株E.coli GluPPK诱导条件为：0.3-0.5mM的IPTG诱导表达(30℃，220rpm)，表达时间为10-12h；培养基中加入终浓度为50μg/L硫酸卡那霉素以保证质粒的稳定性。
菌株E.coli MycPPK诱导条件为：0.1-0.2mM的IPTG诱导表达(20℃，220rpm)，表达时间为20-24h；培养基中加入终浓度为100μg/L的氨苄青霉素。
实施例2：两种聚磷酸激酶的纯化及酶活比较
将实施例1中得到的重组菌株在LB培养基中培养6-20h之后，将收集的菌体超声破碎后高速离心去除细胞碎片，上清经由0.22μm水系膜过滤，利用Ni-NTA亲和层析对目的蛋白进行纯化。A液平衡柱子后上样粗酶液，再利用A液平衡层析柱，之后利用不同浓度的B液冲洗层析柱并收集冲洗液，利用SDS-PAGE验证纯化的组分(图2)，并将最纯的组分用PD-10脱盐柱进行脱盐，脱盐时使用低盐缓冲液(10mM Tris-HCl，0.1M NaCl；pH 6.0)，收集得到纯化脱盐的蛋白。
A液：20mM Tris-HCl buffer pH 7.5，500mM NaCl
B液：20mM Tris-HCl buffer pH 7.5，500mM NaCl，500mM咪唑。
在获得不同来源的聚磷酸激酶的纯酶后，先向催化体系50-100mM Tris-HCl buffer pH 7.5 中同时添加等量纯酶0.5-1.0mg/L和ADP 5g/L，再分别添加等量不同的磷酸供体焦磷酸钠、三聚磷酸钠和偏六磷酸钠2-5g/L。通过检测ATP的产生来测定聚磷酸激酶的比酶活，反应温度为30-35℃，反应时间为12-24h，结果如图3所示，来源于谷氨酸棒杆菌的聚磷酸激酶能在以焦磷酸钠、三聚磷酸钠和偏六磷酸钠为底物时，比酶活更高。
实施例3：合成双功能酶的表达、纯化及酶活测定
双功能酶的构建：按照基因操作手段将酿酒酵母来源的ATP硫酸化酶(Gene ID为853466)和大肠杆菌来源的APS激酶(GenBank号为M74586.1)融合为一个片段(两种酶的前后顺序不影响其表达)，分别在接头部分添加不同融合接头序列(linker)，融合为一个片段，使两种酶均保持一定的空间位置，催化更加有序，具体为，将前面一个基因的终止密码子去除，与linker直接连接，再与另一个基因的起始密码子连接，所述linker序列为：(GGGGS) 6，将融合片段连接至质粒pET28a(+)，并转入E.coli BL21(DE3)得到转化子，将转化子培养并测序验证，将验证正确的转化子处记为菌株E.coli CaePAPS。具体的表达方式及酶的纯化方法同实施例1和2。
菌株E.coli CaePAPS诱导条件：0.1-0.2mM的IPTG诱导表达(25℃，220rpm)，表达时间为10-15h。
酶活测定：向催化体系中50-100mM Tris-HCl buffer pH 7.5中添加双功能酶的纯酶0.5-1.0mg/mL、底物ATP 1-5g/L和硫酸镁0.5-2.5g/L，通过检测反应体系中的PAPS的产生测定PAPS合成双功能酶的酶活，反应温度为30-35℃，反应时间为24-48h。结果表明酶活为300±20U，可用于后续的转化。
实施例4：耦联ATP再生系统高效合成PAPS
利用实施例3获得的合成双功能酶和实施例2获得的谷氨酸棒杆菌来源的聚磷酸激酶，合成PAPS。
准备1.5mL的反应体系(50-100mM Tris-HCl buffer pH 7.5)，其中，PAPS合成双功能酶1mg/mL，聚磷酸激酶1mg/mL，底物ATP 5g/L，硫酸镁3g/L。其中PAPS合成双功能酶加入时间为反应开始后0h，聚磷酸激酶加入的时间为反应开始后0～24h，反应35～50h完成反应(反应体系中没有ATP检出时即视为反应结束)，反应结束后测定PAPS的产量。
为了达到更高的产量和更快的转化速率，催化体系中可额外添加磷酸供体(焦磷酸钠、三聚磷酸钠和偏六磷酸钠)，利用和上述相同的方法制备合成PAPS，只是在反应时，在反应 体系中加入终浓度为2g/L的磷酸供体(焦磷酸钠、三聚磷酸钠和偏六磷酸钠)，进行反应，反应结束后对PAPS纯化并测定产量。
产量结果如图4所示，耦联了聚磷酸激酶之后，底物ATP转化效率得到显著提升，可从耦联前的42％提升至83％～95％。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (12)

  1. 一种合成3’-磷酸腺苷-5’-磷酰硫酸的方法，其特征在于，以能将ATP转化为3’-磷酸腺苷-5’-磷酰硫酸的酶为催化剂，并在反应中加入聚磷酸激酶；所述催化剂为APS激酶和ATP硫酸化酶，APS激酶的Gen Bank号为M74586.1，ATP硫酸化酶的Gene ID为853466，APS激酶和ATP硫酸化酶通过氨基酸序列如SEQ ID NO.1所示的序列连接；所述聚磷酸激酶的Gene ID为1020661。
  2. 一种合成3’-磷酸腺苷-5’-磷酰硫酸的方法，其特征在于，以能将ATP转化为3’-磷酸腺苷-5’-磷酰硫酸的酶为催化剂，并在反应中加入聚磷酸激酶。
  3. 根据权利要求2所述的方法，其特征在于，所述聚磷酸激酶包括但不限于来源于谷氨酸棒杆菌、结核分枝杆菌、铜绿芽孢杆菌或链霉菌的酶。
  4. 根据权利要求2所述的方法，其特征在于，所述能将ATP转化为3’-磷酸腺苷-5’-磷酰硫酸的酶为APS激酶和ATP硫酸化酶。
  5. 根据权利要求1所述的方法，其特征在于，所述能将ATP转化为3’-磷酸腺苷-5’-磷酰硫酸的酶是通过linker将APS激酶和ATP硫酸化酶连接得到的合成双功能酶。
  6. 根据权利要求5所述的方法，其特征在于，所述linker的序列如SEQ ID NO.1所示。
  7. 根据权利要求2所述的方法，其特征在于，反应体系中还含有硫酸盐。
  8. 一种提高3’-磷酸腺苷-5’-磷酰硫酸转化效率的方法，其特征在于，在以ATP为底物的转化底物中加入聚磷酸激酶和磷酸供体。
  9. 根据权利要求8所述的方法，其特征在于，所述磷酸供体包括三聚磷酸、四聚磷酸和/或六聚磷酸。
  10. 一种提高ADP回收效率和速率的方法，其特征在于，在合成3’-磷酸腺苷-5’-磷酰硫酸的反应中加入聚磷酸激酶，同时再加入磷酸供体；所述ADP是以ATP为底物合成3’-磷酸腺苷-5’-磷酰硫酸的副产物。
  11. 根据权利要求10所述的方法，其特征在于，所述磷酸供体包括但不限于焦磷酸、三聚磷酸、四聚磷酸和/或六聚磷酸。
  12. 权利要求1～11任一所述方法在制备3’-磷酸腺苷-5’-磷酰硫酸，或在制备以3’-磷酸腺苷-5’-磷酰硫酸为原料的产品，或在制备需要有磺酸基团供给的产品中的应用。
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