This report investigates for the first time stability, scalability, and reusability characteristics of a protein nano-bioreactor useful for green synthesis of fine chemicals in aqueous medium extracting maximum enzyme efficiency. Enzyme catalysts conjugated with magnetic nanomaterials allow easy product isolation after a reaction involving simple application of a magnetic field. In this study, we examined a biocatalytic system made of peroxidase-like myoglobin (Mb), as a model protein, to covalently conjugate with poly(acrylic acid) functionalized magnetic nanoparticles (MNPs, 100 nm hydrodynamic diameter) to examine the catalytic stability, scalability, and reusability features of this bioconjugate. Application of the conjugate was effective for electrochemical reduction of organic and inorganic peroxides, and for both peroxide-mediated and electrocatalytic oxidation of the protein substrate 2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) with greater turnover rates and product yields than Mb prepared in solution or MNP alone. Mb-attached MNPs displayed extensive catalytic stability even after 4 months of storage compared to Mb present in solution. Five- and ten-fold scale up of MNPs in the bioconjugates resulted in two- and four-fold increases in protein-catalyzed oxidation products, respectively. Nearly 40% of the initial product was present even after four reuses, which is advantageous for synthesizing sufficient products with a minimal investment of precious enzymes. Thus, the results obtained in this study are highly significant in guiding cost-effective development and efficient multiple uses of enzyme catalysts for biocatalytic, electrocatalytic, and biosensing applications via magnetic nanomaterials conjugation.

To develop bioelectrodes with high catalytic efficiency in a minimal reactor volume, elegant tailoring of remarkable enzyme catalysts with nanomaterials is promising. In particular, the role of magnetic nanoparticles (MNPs) in enzyme electrocatalysis and electrochemical biosensors has received notable attention recently. Biomolecules bound to magnetic nanomaterials allow easy isolation from the bulk solution involving simple application of a magnetic field. In this study, we designed a biocatalytic system made of peroxidase-like myoglobin, as a model protein, to covalently conjugate with carboxylic acid functionalized MNPs to examine the catalytic stability, scalability, and reusability features of this bioconjugate. Application of the conjugate was effective for electrochemical reduction of organic and inorganic peroxides, and for both peroxide-mediated and electrocatalytic oxidation of the protein substrate 2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) with greater turnover rates and product yields than myoglobin prepared in solution or MNP alone. Enzymes are often difficult to purify and obtaining high yields for practical applications can be very challenging. Moreover, the electrocatalytic stability and achieving direct electron transfer of purified enzyme films on electrodes is another bottleneck. These challenges are even more drastic in the case of enzymes bound to phospholipid membranes. Despite these limitations, we show that drug metabolizing human liver microsomes, a rather complex enzyme matrix, can be successfully attached to MNPs and immobilized on graphite electrodes with enhanced electrocatalytic activity than microsomal film alone in converting drug into its metabolite. In summary, this presentation will focus on cost-effective development and efficient multiple uses of enzyme catalysts and microsomes for biocatalytic, electrocatalytic, and biosensing applications via magnetic nanomaterials conjugation.