Polyhydroxyalkanoate film formation and synthase activity during in vitro and in situ polymerization on hydrophobic surfaces.
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.
Sato
Shun
S
Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan.
Ono
Yusuke
Y
Mochiyama
Yukiko
Y
Sivaniah
Easan
E
Kikkawa
Yoshihiro
Y
Sudesh
Kumar
K
Hiraishi
Tomohiro
T
Doi
Yoshiharu
Y
Abe
Hideki
H
Tsuge
Takeharu
T
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
D054813:Polyhydroxyalkanoates; D011108:Polymers; D013438:Sulfhydryl Compounds; D011791:Quartz; D006046:Gold; D006108:Graphite; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase