Real-Time Observation of Enzymatic Polyhydroxyalkanoate Polymerization Using High-Speed Scanning Atomic Force Microscopy

[Image: see text] The initial stage of in vitro polyhydroxyalkanoate (PHA) polymerization by PHA synthase from Ralstonia eutropha (PhaC(Re)) on a mica substrate in water was observed using high-speed scanning atomic force microscopy (HS-AFM). Before PHA polymerization, the adsorption–desorption cycle of the PhaC(Re) molecule on mica was observed in real time. For approximately 30 s after the addition of the PHA monomer, no significant change was observed on the mica substrate, but PhaC(Re) could be transformed into an active enzyme in water upon contact with the monomer during this period. Subsequently, linearly elongating rod-shaped objects were observed on the mica substrate, plausibly as a result of the polymerization reaction. The height of these elongating objects was considerably larger than the expected height for a single PHA chain. This observation suggests that PHA chains generated during the reported experiments might form some kind of a semiregular structure.