Work

Functional characterization and domain shuffling of poly(3-hydroxybutyrate) depolymerases from Cupriavidus malaysiensis and Pseudomonas guguanensis for sustainable bioplastic degradation

Year: 2025

Type: article

Abstract: • Two novel PHB depolymerases identified from C. malaysiensis and P. guguanensis. • Enzymes exhibit distinct kinetics, catalytic and thermal stability profiles. • Cma PHBd shows broad PHA specificity and high solid-PHB degradation efficiency. • Domain shuffling enhances Pgu PHBd degradation efficiency toward solid films. 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 ( Cma PHBd) and from Pseudomonas guguanensis ( Pgu PHBd). 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 Cma PHBd and Pgu PHBd. The results demonstrated that Cma PHBd was most active in acidic conditions (pH 5.0–6.0), while Pgu PHBd 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 Cma PHBd, while Pgu PHBd 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, Cma PHBd exhibited superior degradation efficiency towards solid PHB films. Furthermore, Cma PHBd displayed promiscuous activity against other solid polyhydroxyalkanoate (PHA) films. A domain-shuffling strategy was employed, to enhance the activity Pgu PHBd towards solid plastic films, by fusing the SBD of Cma PHBd to the CD of Pgu PHBd. 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 Cma PHBd and Pgu PHBd, highlighting their potential application in bioplastic degradation, reusability and biocatalysis.

Source: Bioresource Technology

DOI: 10.1016/j.biortech.2025.133571

Authors: Charoutioun S. Bodourian, Nikolaos D. Georgakis, Christos Margioulas, Apostolis Koutinas, Nikolaos E. Labrou

Institution: Agricultural University of Athens

Language: English

Topic: biodegradable polymer synthesis and properties

Subfield: Biomaterials

Field: Materials Science

Domain: Physical Sciences

SDG: Responsible consumption and production

Open Access status: hybrid

Estimated APC paid: $4,880