Degradation kinetics of medium chain length Polyhydroxyalkanoate degrading enzyme: a quartz crystal microbalance study

Abstract

This study investigates the enzymatic degradation processes of different classes of polyhydroxyalkanoates (PHAs), a group of biopolymers naturally synthesized by various microorganisms. Medium chain length PHAs (mcl-PHAs) are distinguished biopolymers due to their biodegradability and diverse material properties. Using quartz crystal microbalance measurements as a valuable tool for accurate real-time monitoring of the enzymatic degradation process, the research provides detailed kinetic data, describing the interaction between enzymes and substrates during the enzymatic degradation process. Thin films of poly-3-hydroxybutyrate (PHB) and polyhydroxyoctanoate copolymer (PHO), containing molar fractions of about 84% 3-hydroxyoctanoate and 16% 3-hydroxyhexanoate, were exposed to scl-depolymerases from Pseudomonas lemoignei LMG 2207 and recombinant mcl-depolymerase produced in Escherichia coli DH5α harboring the plasmid pMAD8, respectively. Analyses based on a heterogeneous kinetic model for the polymer degradation indicated a six-fold stronger adsorption equilibrium constant of mcl-depolymerase to PHO. Conversely, the degradation rate constant was approximately twice as high for scl-depolymerases acting on PHB. Finally, the study highlights the differences in enzyme-substrate interactions and degradation mechanisms between the investigated scl- and mcl-PHAs.

Keywords: biodegradable polymers; degradation kinetics; depolymerase enzymes; enzymatic degradation; polyhydroxyalkanoates; quartz crystal microbalance.

FIGURE 1

Degradation of PHB thin films exposed to depolymerases from Pseudomonas lemoignei LMG 2207 at various protein concentrations. The heterogeneous degradation model was fitted to the observed data through non-linear regression using the Levenberg-Marquardt algorithm. The degradation rate measurement for an enzyme concentration of C _prot = 82.8 µg mL⁻¹ was carried out in triplicate and the standard deviation was used to calculate the error bars assuming a constant error.

FIGURE 2

Degradation of PHO thin films exposed to recombinant mcl-depolymerases from Escherichia coli DH5α harboring the plasmid pMAD8, at various protein concentrations. The heterogeneous degradation model was fitted to the observed data through non-linear regression using the Levenberg-Marquardt algorithm. The degradation rate measurement for an enzyme concentration of C _prot = 2.4 µg mL^-1 was carried out in triplicate and the standard deviation was used to calculate the error bars assuming a constant error.