title: Polyhydroxyalkanoate film formation and synthase activity during in vitro and in situ polymerization on hydrophobic surfaces.
abstract: In vitro and in situ enzymatic polymerization of polyhydroxyalkanoate (PHA) on two hydrophobic surfaces, a highly oriented pyrolytic graphite (HOPG) and an alkanethiol self-assembled monolayer (SAM), was studied by atomic force microscopy (AFM) and quartz crystal microbalance (QCM), using purified Ralstonia eutropha PHA synthase (PhaC(Re)) as a biocatalyst. (R)-Specific enoyl-CoA hydratase was used to prepare R-enantiomer monomers [(R)-3-hydroxyacyl-CoA] with an acyl chain length of 4-6 carbon atoms. PHA homopolymers with different side-chain lengths, poly[(R)-3-hydroxybutyrate] [P(3HB)] and poly[(R)-3-hydroxyvalerate] [P(3HV)] were successfully synthesized from such R-enantiomer monomers on HOPG substrates. After the reaction, the surface morphologies were analyzed by AFM, revealing a nanometer thick PHA film. The same biochemical polymerization process was observed on an alkanethiol (C18) SAM surface fabricated on a gold electrode using QCM. This analysis showed that a complex sequence of PhaC(Re) adsorption and PHA polymerization has occurred on the hydrophobic surface. On the basis of these observations, the possible mechanisms of the PhaC(Re)-catalyzed polymerization reaction on the surface of hydrophobic substrates are proposed.
authors.affiliation: Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan.
authors.forename: Shun
authors.initials: S
authors.lastname: Sato
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Ono
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Mochiyama
authors.forename: Easan
authors.initials: E
authors.lastname: Sivaniah
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kikkawa
authors.forename: Kumar
authors.initials: K
authors.lastname: Sudesh
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Hiraishi
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Doi
authors.forename: Hideki
authors.initials: H
authors.lastname: Abe
authors.forename: Takeharu
authors.initials: T
authors.lastname: Tsuge
mesh_terms: D000217:Acyltransferases / Q000737:chemistry; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003460:Crystallization; D020118:Cupriavidus necator; D004566:Electrodes; D006046:Gold / Q000737:chemistry; D006108:Graphite / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D018625:Microscopy, Atomic Force; D054813:Polyhydroxyalkanoates / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry; D011791:Quartz; D013438:Sulfhydryl Compounds; D013499:Surface Properties
chemical_list: D054813:Polyhydroxyalkanoates; D011108:Polymers; D013438:Sulfhydryl Compounds; D011791:Quartz; D006046:Gold; D006108:Graphite; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase