Enzyme purification and sustained enzyme activity for pharmaceutical biocatalysis by fusion with phase-separating intrinsically disordered protein.

ABSTRACT

In recent decades, biocatalysis has emerged as an important alternative to chemical catalysis in pharmaceutical manufacturing. Biocatalysis is attractive because enzymatic cascades can synthesize complex molecules with incredible selectivity, yield, and in an environmentally benign manner. Enzymes for pharmaceutical biocatalysis are typically used in their unpurified state, since it is time-consuming and cost-prohibitive to purify enzymes using conventional chromatographic processes at scale. However, impurities present in crude enzyme preparations can consume substrate, generate unwanted byproducts, as well as make the isolation of desired products more cumbersome. Hence, a facile, nonchromatographic purification method would greatly benefit pharmaceutical biocatalysis. To address this issue, here we have captured enzymes into membraneless compartments by fusing enzymes with an intrinsically disordered protein region, the RGG domain from LAF-1. The RGG domain can undergo liquid-liquid phase separation, forming liquid condensates triggered by changes in temperature or salt concentration. By centrifuging these liquid condensates, we have successfully purified enzyme-RGG fusions, resulting in significantly enhanced purity compared to cell lysate. Furthermore, we performed enzymatic reactions utilizing purified fusion proteins to assay enzyme activity. Results from the enzyme assays indicate that enzyme-RGG fusions purified by the centrifugation method retain enzymatic activity, with greatly reduced background activity compared to crude enzyme preparations. Our work focused on three different enzymes-a kinase, a phosphorylase, and an ATP-dependent ligase. The kinase and phosphorylase are components of the biocatalytic cascade for manufacturing molnupiravir, and we demonstrated facile co-purification of these two enzymes by co-phase separation. To conclude, enzyme capture by RGG tagging promises to overcome difficulties in bioseparations and biocatalysis for pharmaceutical synthesis.