IRTA, Carretera de Poble Nou, km 5.5, 43540 Sant Carles de la Ràpita, Spain.
J Biotechnol. 2012 Jan;157(1):89-95. doi: 10.1016/j.jbiotec.2011.11.020. Epub 2011 Dec 1.
This work presents the functional characterisation of a protein phosphatase 2A (PP2A) catalytic subunit obtained by genetic engineering and its conjugation to magnetic particles (MPs) via metal coordination chemistry for the subsequent development of assays for diarrheic lipophilic marine toxins. Colorimetric assays with free enzyme have allowed the determination of the best enzyme activity stabiliser, which is glycerol at 10%. They have also demonstrated that the recombinant enzyme can be as sensitive towards okadaic acid (OA) (LOD=2.3μg/L) and dinophysistoxin-1 (DTX-1) (LOD=15.2μg/L) as a commercial PP2A and, moreover, it has a higher operational stability, which makes possible to perform the protein phosphatase inhibition assay (PPIA) with a lower enzyme amount. Once conjugated to MPs, the PP2A catalytic subunit still retains its enzyme activity and it can also be inhibited by OA (LOD=30.1μg/L).
本工作通过遗传工程获得了蛋白磷酸酶 2A（PP2A）催化亚基，并通过金属配位化学将其偶联到磁性颗粒（MPs）上，用于开发针对腹泻性脂溶性海洋毒素的测定方法。游离酶的比色测定允许确定最佳的酶活性稳定剂，即 10%的甘油。它们还表明，重组酶对 okadaic acid（OA）（LOD=2.3μg/L）和 dinophysistoxin-1（DTX-1）（LOD=15.2μg/L）的敏感性与商业 PP2A 相当，而且，它具有更高的操作稳定性，使得可以用更少的酶量进行蛋白磷酸酶抑制测定（PPIA）。偶联到 MPs 后，PP2A 催化亚基仍保留其酶活性，并且也可以被 OA 抑制（LOD=30.1μg/L）。
