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 LCCICCG, 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, LCCICCG 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. Conversion of monomers, as determined by nuclear magnetic resonance (NMR) analysis, was ~90% for all enzymes while the average molecular weights (Mn) of the polyesters, analyzed by gel permeation chromatography, were between 3600 Da, for LCC and its variant LCCICCG, and 3800 Da for Thb.