Biocatalytic application and structural elucidation of robust bacterial protein nanocages†

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.