Abstract：
3\'-Phosphoadenosine-5\'-phosphosulfate (PAPS) is the bioactive form of sulfate and is involved in all biological sulfation reactions. The enzymatic transformation method for PAPS is promising, but the low efficiency and high cost of enzyme purification and storage restrict its practical applications. Here, we reported PAPS biosynthesis with a protein crystalline inclusion (PCI)-based enzyme immobilization system. First, the in vivo crystalline inclusion protein CipA was identified as an efficient auto-assembly tag for immobilizing the bifunctional PAPS synthase (ASAK). After characterizing the pyrophosphokinase activity of a polyphosphate exonuclease PaPPX from Pseudomonas aeruginosa, and optimizing the linker fragment, auto-assembled enzymes ASAK-PT-CipA and PaPPX-PT-CipA were constructed. Then, the auto-assembled enzymes ASAK-PT-CipA and PaPPX-PT-CipA with high stability were co-expressed and immobilized for constructing a transformation system. The highest transformation rate of PAPS from ATP and sulfate reached 90%, and the immobilized enzyme can be reused 10 times. The present work provided a convenient, efficient, and easy to be enlarged auto-immobilization system for PAPS biosynthesis from ATP and sulfate. The immobilization system also represented a new approach to reduce the production cost of PAPS by facilitating the purification, storage, and reuse of related enzymes, and it would boost the studies on biotechnological production of glycosaminoglycans and sulfur-containing natural compounds.
摘要：
3'-磷酸腺苷-5'-磷酸硫酸盐（PAPS）是硫酸盐的生物活性形式，参与所有生物硫酸化反应。PAPS的酶转化方法是有前途的，但酶的纯化和储存效率低、成本高限制了其实际应用。这里,我们报道了一种基于蛋白质晶体包合物（PCI）的酶固定系统的PAPS生物合成。首先,体内结晶包涵蛋白CipA被鉴定为用于固定双功能PAPS合酶（ASAK）的有效自动组装标签。在表征了铜绿假单胞菌的多磷酸盐外切核酸酶PaPPX的焦磷酸激酶活性后，并优化接头片段，构建了自组装酶ASAK-PT-CipA和PaPPX-PT-CipA。然后，将具有高稳定性的自组装酶ASAK-PT-CipA和PaPPX-PT-CipA共表达并固定化以构建转化系统。ATP和硫酸盐对PAPS的最高转化率达到90%，固定化酶可重复使用10次。目前的工作提供了一个方便，高效,易于扩大ATP和硫酸盐生物合成PAPS的自动固定化系统。固定化系统还代表了一种通过促进纯化来降低PAPS生产成本的新方法。storage,以及相关酶的重复使用，这将促进糖胺聚糖和含硫天然化合物生物技术生产的研究。
