Chimeric yeast VLPs expressing bacterial esterase (EstA) function as recyclable bioreactor and show efficient substrate conversion.
(A) Electron micrograph of recombinant Gag/EstA particles prepared from yeast were purified by sucrose gradient centrifugation, negatively stained with uranyl acetate/methyl cellulose and subsequently used for electron microscopy (magnification 340,000). (B) Linear correlation between the 4-nitrophenol concentration of up to 1 mM and its absorption at 405 nm. (C) Kinetics of Gag/EstA-driven hydrolysis of 4-nitrophenylacetate (280 µM) to 4-nitrophenol and acetate at 25°C in PBS50 buffer (pH 7.0). (D) Coomassie-Blue staining and western analysis of Gag/EstA particles before and after cata-lysis and recycling by ultracentrifugation. BSA (1 and 3 µg) was used as loading control [M, full range rainbow marker, Amersham]. (E) Specific activity of chimeric Gag/EstA particles before and after recycling.