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Open Access Publications from the University of California

Peroxidase Enzymes Packaged in Vaults as an Innovative Bioremediation Technology

  • Author(s): Wang, Meng
  • Advisor(s): Mahendra, Shaily
  • et al.

Bioremediation is a promising technology for low-cost, effective, in-situ clean up of contaminated soil and water environments, and has been successfully applied for treating a variety of metallic and organic pollutants. Current bioremediation research and practice mostly focuses on processes driven by microbial whole cells, which are sensitive to biogeochemical conditions, such as oxygen, pH, temperature, soil permeability, organic content, dissolved ions, and co-contaminants. In addition, microbe based bioremediation generally takes longer to accomplish, because it depends on microbial viability and activity to degrade the contaminants. Using free enzymes instead of microbes is a potential alternative approach, as in-vitro enzyme-catalyzed reactions are not constrained by nutrient requirements for microbial growth, and often have higher biodegradation rates. Moreover, enzymatic bioremediation is considered to be safer because of the elimination of potentially pathogenic microbes. Peroxidases, which compromise a class of non-specific heme enzymes, are attractive candidates due to their ability to oxidize a broad range of contaminants, including phenolic compounds, organic dyes, etc. However, a limitation of applying purified enzymes is their limited stability under various environments. Immobilization of peroxidases on solid surfaces or entrapment in matrices has been previously attempted. Even though immobilization gives higher stability, it limits enzyme-substrate interactions, which decreases the accessibility and efficiency of the enzymes.

This thesis describes the synthesis of highly stable and active peroxidase nanocapsules using vault nanoparticles, and demonstrates the effectiveness of vaults packaged peroxidases using phenol as a model contaminant. Two peroxidases from the wood degrading fungus Phanerochaete chrysosporium, including lignin peroxidase (LiP) and manganese peroxidase (MnP), were tested in this study. Degradation of phenol by these enzymes has been previously confirmed in whole cells as well as purified enzyme assays. Both LiP and MnP were fused to an INT domain, which strongly interacts with vault nanoparticles and drives the packaging of the enzyme, and heterologously expressed in Sf9 insect cells intracellularly as nsLiP-INT and nsMnP-INT via baculovirus expression systems. Cell lysates containing nsLiP-INT or nsMnP-INT showed 35-45% higher peroxidase activity than the negative control, suggesting INT-fused nsLiP and nsMnP were actively expressed. The nsMnP-INT was successfully packaged into vaults, but the activity of packaged nsMnP-INT was below the assay detection limit. INT-fused MnP was also expressed extracellularly in Sf9 cells as sMnP-INT using its natural signal sequence, and packaged into vaults. Both free sMnP-INT and vaults packaged sMnP-INT showed Mn2+ dependent activity identical to that of MnP produced by P. chrysosporium. Thermal stability of packaged sMnP-INT was compared with that of free sMnP-INT and MnP from P. chrysosporium, and it was found that vaults packaged sMnP-INT was more stable at 20⁰C, 30⁰C, and 40⁰C. Furthermore, P. chrysosporium MnP, free recombinant sMnP-INT, as well as vaults packaged sMnP-INT were able to catalyze the degradation of phenol, indicating that sMnP-INT maintained its catalytic ability while packaged in vaults. This study demonstrated that packaging of peroxidases in vault nanoparticles extends their stability and catalytic ability. These results will contribute to the development of innovative and sustainable vault-based bioremediation approaches for multiple contaminants in wastewater and groundwater.

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