Thermostable Biocatalytic Films of Enzymes and Polylysine on Electrodes and Nanoparticles in Microemulsions

Abstract

Microemulsions of oil, water and surfactant were evaluated as media for biocatalysis at high temperatures employing films of polylysine (PLL) and the enzymes horseradish peroxidase (HRP), soybean peroxidase (SBP) and the protein myoglobin (Mb). PLL was covalently linked to oxidized pyrolytic graphite electrodes or carboxylated 500 nm diameter silica nanoparticles, then cross-linked by amidization to HRP, SBP and Mb. The resulting film systems were stable at 90 °C for >12 h in microemulsions. Characterization of the microemulsions by conductivity, viscosity and probe diffusion coefficients suggested that these media have bicontinuous microstructures from 25 to 90 °C. UV circular dichroism and visible spectroscopy confirmed that the enzymes retained near-native conformation in the films at temperatures as high as 90 °C. Oxidation of o-methoxyphenol to 3,3′-dimethoxy-4,4′-biphenoquinone by enzyme-PLL films on silica nanoparticles gave yields 3−5-fold larger in microemulsions at 90 °C compared to the same reaction at 25 °C. The best yields were in CTAB microemulsions and were 3-fold larger than in buffers at 90 °C.