3'-phosphoadenosine-5'-phosphosulfate (PAPS) is the only "active" sulfo group donor found currently, which plays an important role in enzymatic synthesis of biological engineered heparin. However, chemical synthesis of PAPS is very expensive. Therefore, large-scale production of PAPS at low cost is the key factor for the industrialization of enzymatic synthesis of heparin. In this study, adenosine triphosphate (ATP) sulfurylase and adenosine 5'-phosphosulfate (APS) kinase were cloned and expressed, and an enzymatic synthesis pathway of PAPS was constructed. ATP was first converted to APS by ATP sulfurylase, with the formation of pyrophosphoric acid (PPi), and APS was then converted to PAPS by APS kinase. Effects of Mg2+concentration, buffer system, pH, temperature and inorganic pyrophosphohydrolase (PPase) on PAPS synthesis were studied. The results show that the presence of PPase greatly increases PAPS conversion. Investigation on PAPS accumulation as a function of time under different ATP concentrations and optimal conditions reveals that substrate inhibition happens during reaction, and PAPS accumulation reaches to 18.87 mmol?L-1at 50 mmol?L-1ATP after 25 h. Moreover, the results indicate that catalytic efficiency can be greatly improved when ATP is added under fed-batch conditions, and PAPS accumulation reaches to 20.36 mmol·L-1 (10.32 g·L-1) with conversion of 81.4% after 11.5 h.%3'-磷酸腺苷-5'-磷酰硫酸(PAPS)是目前发现的唯一的"活性"硫酸基团供体,在生物肝素酶法硫磺化反应中起到重要作用,但化学合成的成本高.如何大量低成本制备PAPS是实现酶法合成肝素工业化的关键因素.本文克隆表达了酶法合成PAPS的两个酶:三磷酸腺苷(ATP)硫化酶和5'-腺苷磷酰硫酸(APS)激酶.构建了一条体外合成PAPS的途径.首先利用ATP硫化酶将ATP转化生成APS,同时产生副产物无机焦磷酸(PPi),APS再在APS激酶作用下转化生成PAPS.研究了Mg2+浓度、缓冲体系和pH、温度以及无机焦磷酸水解酶(PPase)的添加对该酶反应的影响.通过添加无机焦磷酸水解酶(PPase),极大提高了PAPS的转化率.在最优条件下考察不同ATP浓度下PAPS随时间的积累量变化,发现反应过程中存在底物抑制作用;ATP浓度为50 mmol?L-1时,反应25 h,PAPS的积累量为18.87 mmol?L-1.反应过程中分批补料ATP,能极大提高催化效率.反应11.5 h,PAPS积累量可达20.36 mmol?L-1(10.32 g·L-1),转化率为81.4%.
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