Biocatalytic application and structural elucidation of robust bacterial protein nanocages

Abstract

Encapsulins, bacterial protein nanocompartments, have emerged as promising platforms for enhancing biocatalyst stability. This study presents the identification and structural characterization of two encapsulins: ArthroEnc from Arthrobacter sp. SLBN-53 and DendroEnc from Dendrosporobacter quercicolus. Both bacterial encapsulins were successfully overexpressed in Escherichia coli and purified. Cryo-electron microscopy revealed that ArthroEnc assembles into a 20 nm T = 1 icosahedral capsid composed of 60 subunits, with its structure determined at 2.9 Å resolution, whereas DendroEnc forms a 40 nm T = 4 icosahedral complex with 240 subunits, resolved at 3.4 Å resolution. Both structures exhibit the characteristic HK97 phage-like fold. To explore their functional potential, the encapsulins were used to pack two distinct enzymes: CyanoPOX, a heme-containing peroxidase, and PTDH-mFMO, a fusion enzyme combining phosphite dehydrogenase with a flavin-containing monooxygenase. DendroEnc packed with CyanoPOX exhibited enhanced proteolytic stability, effectively shielding its cargo from chymotrypsin degradation. Activity assays with ABTS and guaiacol confirmed that encapsulated CyanoPOX retained enzymatic function within DendroEnc. Finally, mutating the pore of DendroEnc resulted in a fivefold reduction in activity, demonstrating the potential for tuning substrate diffusion. This study advances our understanding of encapsulin diversity and highlights their potential as versatile platforms for enzyme stabilization, biocatalytic and other biotechnological applications.