Sustainable engineering has become an integral part of the overall effort toward establishing a circular bioeconomy, including emerging topics such as plastic degradation and synthesis. In the example of polyesters, which are primarily produced by melt polycondensation from petroleum-based feedstocks, their production is energy-intensive processes with high carbon dioxide (CO
2) emission. Enzymatic polymerization provides a new platform that enables selectivity and mild reaction conditions. Since the ’90s, exploratory work on polyester enzymatic synthesis has been conducted with hydrolases for polycondensation, transesterification, and ring-opening mechanisms. In the past decades, the aim of enzymatic polyester synthesis often targets high molecular weight, particularly in aliphatic polyester synthesis. Recently, research interests have shifted to bio-based substrates and to obtain novel molecular architecture, although the model enzyme,
Candida Antarctica lipase (CALB), has never changed. The lack of understanding of the kinetics and thermodynamics of the reaction, and limited methods that can be used to correlate protein sequences to the polymerization performance, posed challenges to engineering enzymes toward effective and selective polymerization, instead, engineering targets are enzyme thermostability, hydrolysis activity, and solvent tolerance.
In this work, we used serine superfamily hydrolases as the model enzymes for ring-opening polymerization (ROP) and developed a workflow to compare the enzyme performance. In addition to the physical-chemical properties of obtained polymer and oligomers, such as molecular weight and crystallinity, we conducted time-resolved measurement using nuclear magnetic resonance (NMR) spectroscopy and small-angle x-ray scattering (SAXS) to study the reaction kinetics. We also used open-port sampling interface mass spectrometry (OPSI-MS) to monomer the depletion and to realize the limits of different characterization techniques. The developed workflow can evaluate a panel of enzymes and lysates, thus providing a versatile platform for rapid enzyme engineering. By combining enzymatic method and polymer characterization methods, the study will lay down the groundwork for sustainable polyester synthesis and supplement enzyme engineering efforts towards engineering effective enzymes suitable for large-scale application and accessing diverse macromolecular architectures.
