Insight in the role of bovine serum albumin for promoting the in situ surface growth of polyhydroxybutyrate (PHB) on patterned surfaces via enzymatic surface-initiated polymerization

Polyhydroxyalkanoates (PHAs) are a family of aliphatic polyesters produced by a variety of microorganisms as a reserve of carbon and energy. Enzymes involved in the synthesis of PHAs can be utilized to produce polymers in vitro, both in bulk and on solid surfaces. Here, site-specific attachment of the key catalytic enzyme, PHA synthase, on lithographically patterned surfaces and subsequent addition of (R)-3-hydroxybutyryl-CoA substrate allowed us to fabricate spatially ordered polyhydroxybutyrate (PHB) polymeric structures via an in situ enzymatic surface-initiated polymerization (ESIP). By varying the reaction conditions, we enhanced the growth of PHB on solid surfaces and analyzed the resulting structures by fluorescence microscopy, atomic force microscopy (AFM), attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, and gel permeation chromatography (GPC). We found that stabilization of smaller PHB granule structures by an addition of bovine serum albumin (BSA) was the most important factor for a successful synthesis of a PHB layer up to 1 μm in thickness, consisting mainly of larger cluster assemblies of PHB granules that cover the entire patterned area. Immunofluorescence detection and surface contact angle analysis revealed that BSA was physically bound to the PHB polymer all through the cluster, and reduced the overall hydrophobicity of the polymer surface. Based on information obtained from AFM, kinetic measurements and various polymer characterization methods, a plausible model for roles of BSA in the enhancement of PHB formation on surfaces is discussed. Furthermore, by using biotinylated BSA conjugates, we were able to incorporate biotin groups into the PHB polymer matrix, thus generating a bioactive surface that can be used for displaying other functional biomolecules through streptavidin-biotin interaction on the PHB structures. Because of its versatility, our fabrication strategy is expected to be a useful surface modification tool for numerous biomedical and biotechnological applications.