Comparative Biochemistry of Four Polyester (PET) Hydrolases*

Area of Science:

• Biotechnology
• Polymer Science
• Biochemistry

Background:

• Bioprocessing offers potential for a circular plastic economy.
• Enzymatic degradation of synthetic polymers, especially poly(ethylene terephthalate) (PET), is under active research.
• A standardized kinetic framework for comparing plastic-degrading enzymes on insoluble substrates is lacking.

Purpose of the Study:

• To establish a kinetic framework for comparing PET hydrolases.
• To analyze the kinetics of four different PET hydrolases.
• To compare the efficiency of enzymes on intact PET versus PET fragments.

Main Methods:

• Development and application of a kinetic framework for enzyme activity on insoluble substrates.
• Measurement of kinetic parameters (kcat, KM, apparent specificity constant) for four PET hydrolases.
• Quantification of enzyme attack sites on PET surfaces.

Main Results:

• A kinetic framework was established, yielding kcat, KM, and specificity constants.
• A variant of Ideonella sakaiensis PET hydrolase demonstrated superior efficiency at ambient conditions, driven by a high kcat.
• Hydrolysis rates were significantly higher for soluble and insoluble PET fragments compared to intact PET.
• Enzymes exhibited high substrate affinity, reaching half-saturation at low attack site concentrations (~50 nM).

Conclusions:

• The proposed kinetic framework enables comparative analysis of PET hydrolases.
• Enzyme efficiency is influenced by factors beyond catalysis, including polymer chain interactions.
• Nonspecific adsorption likely plays a role in enhancing enzyme-substrate proximity and degradation rates.