Vault Nanoparticles Packaged with Enzymes as an Efficient Pollutant Biodegradation Technology

Abstract

Vault nanoparticles packaged with enzymes were synthesized as agents for efficiently degrading environmental contaminants. Enzymatic biodegradation is an attractive technology for in situ cleanup of contaminated environments because enzyme-catalyzed reactions are not constrained by nutrient requirements for microbial growth and often have higher biodegradation rates. However, the limited stability of extracellular enzymes remains a major challenge for practical applications. Encapsulation is a recognized method to enhance enzymatic stability, but it can increase substrate diffusion resistance, lower catalytic rates, and increase the apparent half-saturation constants. Here, we report an effective approach for boosting enzymatic stability by single-step packaging into vault nanoparticles. With hollow core structures, assembled vault nanoparticles can simultaneously contain multiple enzymes. Manganese peroxidase (MnP), which is widely used in biodegradation of organic contaminants, was chosen as a model enzyme in the present study. MnP was incorporated into vaults via fusion to a packaging domain called INT, which strongly interacts with vaults' interior surface. MnP fused to INT and vaults packaged with the MnP-INT fusion protein maintained peroxidase activity. Furthermore, MnP-INT packaged in vaults displayed stability significantly higher than that of free MnP-INT, with slightly increased Km value. Additionally, vault-packaged MnP-INT exhibited 3 times higher phenol biodegradation in 24 h than did unpackaged MnP-INT. These results indicate that the packaging of MnP enzymes in vault nanoparticles extends their stability without compromising catalytic activity. This research will serve as the foundation for the development of efficient and sustainable vault-based bioremediation approaches for removing multiple contaminants from drinking water and groundwater.

Synthesis and assembly of human vault particles in yeast

“…INT‐fused MnP (rMnP) was produced and secreted by insect Sf9 cells as previously described (M. Wang et al, ). The Sf9 culture infected with rMnP baculoviruses was collected at 72 hr, and spun at 3000 g for 5 min at 4°C to remove cells and cell debris.…”

“…Supernatant was decanted, and the rMnP‐yMVP pellet was resuspended in buffer A. rMnP and rMnP‐yMVP were stored at −20°C and 4°C before use, respectively. Fungus produced native MnP (nMnP) was purified from the fungus Phanerochaete chrysosporium as described previously (M. Wang et al, ).…”

“…A previous study using insect vaults packaged with rMnP also showed similar phenolic compound removal improvement (M. Wang et al, 2015), which further suggests yeast vaults are comparable to insect vaults and P. pastoris is a viable alternative to insect cells for producing recombinant vaults. Interestingly, although nMnP showed better thermal stability than rMnP, and maintained 78% of its initial activity in 6 hr at 25°C, it did not show statistically higher BPA removal than rMnP, and did not induce significant BPA removal after 6.5 hr.…”

“…Taking advantage of these properties, recombinant vaults have been engineered to enhance their functionality with various added domains to impart new activities like: cell targeting (Kickhoefer et al, ), cytoplasmic targeting (Han, Kickhoefer, Nemerow, & Rome, ), fluorescence (Kickhoefer et al, ), and amphiphilicity (Buehler, Toso, Kickhoefer, Zhou, & Rome, ), which are being explored in therapeutic applications (Kar et al, ). We recently reported that the encapsulation of enzymes in recombinant vault particles can also improve their longevity and catalytic activities (M. Wang, Abad, Kickhoefer, Rome, & Mahendra, ). Manganese peroxidase (MnP), which is a widely used lignin‐degrading enzyme in treating environmental contaminants, such as phenolics (Hirano, Honda, Watanabe, & Kuwahara, ), aromatic hydrocarbons (Eibes, Cajthaml, Moreira, Feijoo, & Lema, ), and azo dyes (Spadaro & Renganathan, ), was packaged into vaults using the INT strategy.…”

Eukaryotically expressed encapsulins as orthogonal compartments for multiscale molecular imaging

“…With regards to protein complexes as building blocks for addressable nanocompartments, viruses and virus-like particles have been expressed in bacterial hosts to encase fluorescent proteins [8][9][10][11][12] , enzymes [13][14][15][16] and even multi-enzymatic processes 17,18 . In mammalian systems, vault proteins (vaults) have been explored, which are ribonucleoprotein complexes enclosed by~60 nm large envelope structures 19 into which foreign cargo proteins such as fluorescent proteins or enzymes can be packaged [20][21][22] . However, vaults have openings on both ends and are endogenously expressed by many eukaryotic cells such that overexpressing them may interfere with their natural functions 23 .…”