多酶一锅法合成硫酸基供体3′-磷酸腺苷-5′-磷酸硫酸(PAPS)  

One-pot biosynthesis of sulfate group donor 3′-phosphoadenosine-5′-phosphosulfate(PAPS) through catalysis with multiple enzymes

作　　者：岳继恒 储梦萍 储建林[1,3] 何冰芳 YUE Jiheng;CHU Mengping;CHU Jianlin;HE Bingfang(College of Biotechnology and Pharmaceutical Engineering,Nanjing Tech University,Nanjing 211800,China;Yixing People′s Hospital,Yixing 214200,China;School of Pharmaceutical Sciences,Nanjing Tech University,Nanjing 211800,China)

机构地区：[1]南京工业大学生物与制药工程学院,江苏南京211800 [2]宜兴市人民医院,江苏宜兴214200 [3]南京工业大学药学院,江苏南京211800

出　　处：《生物加工过程》2023年第6期632-641,共10页Chinese Journal of Bioprocess Engineering

基　　金：国家重点研发计划(2018YFA0902000);江苏省自然科学基金面上项目(BK20181379)。

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。(PAPS),the most common sulfate donor for the in vivo sulfation reactions,is a high-energy phosphate compound and relatively unstable at room temperature.The commercial PAPS is usually quite expensive due to the high cost in its chemical synthesis.Recently,enzymatic transformations provided an attractive approach to produce PAPS.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.

关 键 词：3′-磷酸腺苷-5′-磷酸硫酸 ATP硫酸化酶 APS激酶 无机焦磷酸水解酶 

分 类 号：Q81[生物学—生物工程]