One-phase synthesis of single enzyme nanoparticles (SENs) of Trametes versicolor laccase by in situ acrylamide polymerisation

Single enzyme nanoparticles (SENs) are a state of art technology that allows immobilisation of individual enzyme molecules in a nanometre scale cross-linked polymer matrix that is covalently bonded to the enzyme. Although the method has great potential in the enzyme immobilisation, it has attracted relatively low attention from researchers over the last decade. Therefore, it can be very fruitful to apply this method to several enzymes in order to increase the understanding about the mechanisms and parameters important for the technique. In this study, Trametes versicolor laccase enzyme (TvL) was immobilised as SENs without phase transfer. The enzyme was immobilised via acryloyl chloride modification that is followed by an in situ polyacrylamide polymerisation in the water phase. The resulting single TvL nanoparticles were found to be less than 50 nm in diameter and round in shape with an activity recovery of 66.33 +/- 2.57%. Both V-max and K-m values of the TvL SENs were lower than the free counterpart. However, approximately the same K-cat/K-m values suggested similar catalytic efficiencies for the free and the immobilised enzymes. The significantly lower (similar to 2 fold) K-m value of the immobilised enzyme suggested an affinity increase towards 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) upon immobilisation. Whilst the immobilised and the free enzymes showed similar thermal stabilities at 60 degrees C for 240 min, the immobilised form showed higher stability in solution at ambient temperature after 15 days which may suggest an increase in microbial stability.