Poly-3-Hydroxybutyrate Functionalization with BioF-Tagged Recombinant Proteins

Abstract

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.