The quest for the new CaLB: Potential of three thermostable polyester hydrolases for esterification reactions

The potential of three thermophilic enzymes from the α,β-hydrolases superfamily, that have recently been described for polyester hydrolysis, was investigated for polyester synthesis, esterification and transesterification reactions. The hydrolases (LCC, leaf-branch compost cutinase; its variant LCC ICCG , Thb from Thermoanaerobacterales bacterium) were recombinantly expressed, purified and immobilized onto polypropylene beads. A design of experiments (DoE) assisted study was performed to investigate their synthetic potential to produce short flavor esters by analyzing their thermostability and selectivity towards alcohols and acids with different chain lengths. The factors considered in the DoE (i.e., temperature, alcohol chain length, acid chain length and reaction time) were optimized using MODDE® software to generate a predictive model defining the optimal synthetic conditions for the three enzymes. In each experiment, the monitored response was the acid conversion rate, quantified with GC-FID analysis. For synthesis, the temperature optima of LCC, LCC ICCG and Thb were 60 °C, 55 °C, and 80 °C, respectively, corresponding to the maximum percentage of monomers conversion for long-chain alcohols and acids as substrates. Polymerization of dimethyl adipate and 1,8-octanediol as building blocks was carried out to confirm the applicability of the obtained model for the synthesis of larger macromolecules via polycondensation reactions. The proposed approach highlights the innovative application of these novel thermophilic enzymes, traditionally associated with hydrolytic functions, as effective biocatalysts in synthetic processes, enabling the production of a well-known class of polyesters through an alternative and sustainable enzymatic route. Conversion of monomers, as determined by nuclear magnetic resonance (NMR) analysis, was ∼90 % for all enzymes while the average molecular weights (M n ) of the polyesters, analyzed by gel permeation chromatography, were between 3600 Da, for LCC and its variant LCC ICCG , and 3800 Da for Thb. A design of experiments (DoE) based approach was employed to investigate the thermostability and substrate selectivity of three enzymes described for polyester hydrolysis, namely LCC, LCC ICCG and Thb in short ester synthesis reactions involving alcohols and acids of varying chain length. The temperature optimum (T opt ) of the three enzymes was also tested for enzymatic polymerization of dimethyl adipate and 1,8-octanediol to confirm the applicability of the obtained model for the synthesis of larger macromolecules via polycondensation reactions. • Novel use of PET hydrolases (LCC, LCC ICCG and Thb) in synthetic applications. • DoE-assisted approach reveals Temperature optima (T opt ) and substrate selectivity. • LCC variants efficiently catalyze polycondensation of bio-based monomers. • Thb shows high polycondensation activity despite low performance on short molecules.