Free enzymes are promising catalytic agents because their activities can be achieved in the absence of growth substrates and conditions required for live microorganisms. Immobilizing enzymes on solid supports have been shown to improve their stability and longevity. To date, numerous enzyme immobilization supports and techniques have been developed, however, several associated issues, such as reduced enzymatic activity, low immobilization efficiency, and involvement of toxic substances, still limit the application of enzymes, particularly in environmental remediation.
In this research, a novel enzyme immobilization approach was developed and evaluated for removing environmental contaminants. The vault particles, members of protein nanocages, are entirely biocompatible and biodegradable, and have large empty cores for anchoring enzyme molecules, thus serving as eco-friendly enzyme carriers in environmental applications. Manganese peroxidase (MnP), which has been demonstrated to be efficient in biodegrading various environmental contaminants, was employed as a model enzyme in the present study.
MnP was encapsulated into vault nanoparticles through the previously developed INT strategy, and the resultant vault-encapsulated MnP showed improved thermal stability and wider pH adaptability than free enzymes. The encapsulated MnP exhibited significantly better biodegradation of phenol than that catalyzed by native MnP. The performance of vault-encapsulated MnP was subsequently evaluated for removing and detoxifying three bisphenolic compounds, BPA, BPF, BPAP, which are trace contaminants of concern in water supplies. Compared with free MnP enzymes, encapsulated MnP showed longer durability in reactions, high removal rates and efficiency, and different product profiles. Further reproductive toxicity studies in Caenorhabditis elegans demonstrated that products resulted from catalysis by vault-encapsulated MnP induced less germline apoptosis and led to lower fertility damage.
Synthesis and proper assembly of vault particles in yeast Pichia pastoris cells were demonstrated for the first time in this study. The yeast production system showed comparable vault yield to the currently used insect cell system, but at more than ten times lower cost, which makes production of large quantities of vault particles for industrial, commercial, and environmental applications possible.
A vault-templating route to porous silica nanoparticles was developed. Combining it with vault encapsulation, a new enzyme immobilization approach with high immobilization efficiency and enzyme activity yield, and low leakage was demonstrated. MnP immobilized in vault/silica was more stable than free MnP as well as vault-encapsulated MnP, and accomplished high activity and excellent reusability.
Taken together, this research demonstrates the feasibility of using vault particles as enzyme carriers for water treatment and environmental applications. This work serves as a foundation for using customized vaults packaged with biodegradative enzymes to target specific contaminant groups in various industrial and environmental applications.