【摘要】 以腺嘌呤核苷三磷酸(ATP)等为原料，来源于乳酸克鲁维酵母(Kluyveromyces lactis)的ATP硫酸化酶(ATPS)和来源于产黄青霉菌(Penicillium chrysogenum)的APS激酶(APSK)构建的多酶催化体系可合成3′-磷酸腺苷-5′-磷酸硫酸(PAPS)。同时，在酶法合成PAPS的反应体系中引入铜绿假单胞菌(Pseudomonas aeruginosa)来源的无机焦磷酸水解酶(PPase),消耗反应过程中的副产物PPi,使得反应向生成PAPS的方向进行，大幅提高多酶法合成PAPS的产率。本研究构建了重组质粒pET-28a-sumo-ATPS、pET-28a-APSK和pET-28a-PPase,并在大肠杆菌中表达了重组酶，研究酶学性质和优化多酶催化反应合成PAPS的反应条件，以提高PAPS的产量水平，最终PAPS的累积量为14.47 g/L。该工艺可大幅降低PAPS的制备成本。更多还原

【Abstract】 The application of two enzymes, ATP sulfatase(ATPS) from Kluyveromyces lactis and APS kinase(APSK) from Penicillium chrysogenum,could readily convert adenine nucleoside triphosphate(ATP) to 3′-phosphoadenosine-5′-phosphosulfate(PAPS).More importantly, the addition of inorganic pyrophosphorylase(PPase) from Pseudomonas aeruginosa could hydrolyze the corresponding by-product PPi generated in this enzymatic process, which assisted the reaction to proceed in the direction of PAPS biosynthesis.In this study, three recombinant plasmids pET-28a-sumo-ATPS,pET-28a-APSK,and pET-28a-PPase were constructed to express the corresponding proteins in E.coli.Through the investigation of enzymatic properties and the optimization of reaction conditions, the cumulative yield of PAPS was greatly improved to 14.47 g/L.This one-pot enzymatic process can potentially offer an alternative approach for the production of PAPS at relatively low cost.更多还原