Functional characterization and domain shuffling of poly(3-hydroxybutyrate) depolymerases from Cupriavidus...

Polyhydroxybutyrate (PHB) depolymerases (PHBds) enable bioplastic degradation into reusable monomers, making them essential for sustainable waste management and circular bioeconomy solutions. In this study, we computationally identified and characterized two new PHBds from Cupriavidus malaysiensis (CmaPHBd) and from Pseudomonas guguanensis (PguPHBd). Sequence analysis revealed that both enzymes are classified as extracellular type I depolymerases, containing a conserved N-terminal catalytic domain (CD) and a C-terminal substrate-binding domain (SBD). Recombinant expression in E. coli enabled purification and biochemical characterization of CmaPHBd and PguPHBd. The results demonstrated that CmaPHBd was most active in acidic conditions (pH 5.0-6.0), while PguPHBd exhibited optimum activity in alkaline conditions (pH 9.5). Differential scanning fluorimetry (DSF) analysis indicated a melting temperature (T_m) of 49.6 °C for CmaPHBd, while PguPHBd exhibited two unfolding transitions at 45.7 °C and 57.1 °C. Initial rate kinetics analysis indicated an allosteric regulation in both enzymes and significant substrate inhibition at high PHB concentrations. Among the two enzymes, CmaPHBd exhibited superior degradation efficiency towards solid PHB films. Furthermore, CmaPHBd displayed promiscuous activity against other solid polyhydroxyalkanoate (PHA) films. A domain-shuffling strategy was employed, to enhance the activity PguPHBd towards solid plastic films, by fusing the SBD of CmaPHBd to the CD of PguPHBd. The engineered variant, showed improved degradation efficiency on PHB and related biopolymers, confirming the critical role of SBD in overcoming the rate-limiting adsorption step towards solid plastic films. These findings provide valuable insights into the structural and functional properties of CmaPHBd and PguPHBd, highlighting their potential application in bioplastic degradation, reusability and biocatalysis.