Immobilization of the Amidohydrolase MxcM and Its Application for Biocatalytic Flow Synthesis of Pseudochelin A

The chemical synthesis of heterocycles typically requires elevated temperature and acid or base addition to form the desired product. Moreover, these reactions often involve hazardous reagents, which is why biocatalytic routes for heterocycle formation have gained increasing attention. In recent years, several enzymes belonging to the amidohydrolase superfamily have been identified to generate heterocycles via cyclocondensation reactions. Of particular interest is the amidohydrolase MxcM, which catalyzes the formation of an imidazoline moiety in the biosynthesis of the anti-inflammatory natural product pseudochelin A. In this study, we present a concept for the immobilization of this enzyme using a fused hexahistidine tag for fixation onto a solid, porous carrier. Notably, the immobilization improves the enzyme's tolerance to organic solvents. The immobilized MxcM exhibits a residual activity of 169% in the polar solvent acetonitrile compared to the free enzyme, and the storage stability in the presence of 20 vol% acetonitrile was ameliorated. In addition, an immobilized enzyme reactor (IMER) was designed that can be operated under flow conditions. The MxcM-IMER retains its biocatalytic activity and mechanic stability over the tested operation time. These results provide important insights for the integration of heterocycle-forming amidohydrolases in chemical processes.