高校化学工程学报2017，Vol.31Issue(1)：119-125,7.DOI:10.3969/j.issn.1003-9015.2017.01.017
多酶催化合成3'-磷酸腺苷-5'-磷酰硫酸反应体系的构建及优化
Construction and Optimization of a Multi-Enzyme Catalytic System for PAPS Biosynthesis
摘要
Abstract
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.关键词
3'-磷酸腺苷-5'-磷酰硫酸/ATP硫化酶/APS激酶/分批补料Key words
3'-phosphoadenosine-5'-phosphosulfate/ATP sulfurylase/APS kinase/fed-batch分类
生物科学引用本文复制引用
李晓燕,蔡谨,杭宝建,黄磊,徐志南..多酶催化合成3'-磷酸腺苷-5'-磷酰硫酸反应体系的构建及优化[J].高校化学工程学报,2017,31(1):119-125,7.基金项目
国家自然科学基金资助(21276226). （21276226）
