Nanoengineering of solid surfaces using an in-vitro synthesized biological polymer.

摘要

Polyhydroxyalkanotes (PHAs) are aliphatic polyesters produced by a wide range of microorganisms as intracellular carbon and energy storage compounds. PHA has received significant interest from industry and academia because it is a biocompatible and biodegradable thermoplastic with potential applications in consumer and medical products. One of the key enzymes in PHA biosynthesis is PHA synthase, which catalyzes the polymerization of 3-(R)-hydroxyacyl-CoA to poly (3-hydroxyalkanoate) [PHA].; In this work, different types of solid substrates, such as agarose, silicon and gold were modified by the in situ synthesis of PHA on the surface. In order to carry out these surface modifications, the PHA synthase from Ralstonia eutropha H16 was immobilized onto solid substrates through a transition-metal complex, Ni2+-nitrilotriacetic acid (Ni-NTA). Immobilized PHA synthase catalyzed the surface-initiated polymerization of 3-(R)-hydroxybutyryl-CoA, resulting in the formation of a polymer film on the surface. The immobilization of intact enzymes onto patterned silicon substrates was also conducted to demonstrate specific binding of the enzyme through a Ni-NTA linker. The subsequent polymerization within the patterned areas was characterized by fluorescent microscopy, atomic force microscopy, InfraRed spectroscopy and wide angle X-ray diffraction. In addition, the polymer growth on the gold surface was directly monitored by surface plasmon resonance (SPR). SPR experiments revealed the polymerization kinetics on the surface and the effect of enzyme concentration on the final film thickness. Significantly, the process of surface modifications could be reversed from “graft from” to “graft onto” by exploiting a unique characteristic of the catalytic mechanism of this enzyme. In PHA synthase, the polymer chain remains covalently attached to the synthase once synthesis has terminated, resulting in the formation of a highly stable polymer-protein complex structure. The introduction of a histidine tag to the protein thus allowed the polymer-protein complexes to be reversibly grafted onto Ni-NTA-coated surfaces. This work overall demonstrates how variable functional peptide units can be introduced into a polymeric material through genetic engineering and used for mediating specific biomolecular interactions with a given ligand or receptor.