Polyhydroxyalkanoates (PHAs) are biodegradable polyesters that accumulate
in the cytoplasm of certain bacteria. One promising biotechnological application
utilizes these biopolymers as supports for protein immobilization. Here, the
PHA-binding domain of the Pseudomonas putida KT2440 PhaF phasin (BioF polypeptide)
was investigated as an affinity tag for the in vitro functionalization of poly-3-
hydroxybutyrate (PHB) particles with recombinant proteins, namely, full-length PhaF and two fusion proteins tagged to BioF (BioF–C-LytA and BioF–_-galactosidase, containing the choline-binding module C-LytA and the _-galactosidase enzyme, respectively).
The protein-biopolyester interaction was strong and stable in a wide range
of pHs and temperatures, and the bound protein was highly protected from selfdegradation, while the binding strength could be modulated by coating with amphiphilic compounds. Finally, BioF–_-galactosidase displayed very stable enzymatic activity after several continuous activity-plus-washing cycles when immobilized in a minibioreactor. Our results demonstrate the potentialities of PHA and the BioF tag for the construction of novel bioactive materials.