We selected a variant of a leaf-branch compost cutinase (LCC WCCG ) for its ability to depolymerize PET polyesters (PES), and a variant of a cutinase from Thermobifida cellulosilytica (Tc_Cut2_R29N_A30V; herein referred as Tc_Cut2NV) for its catalytic activity toward PET and polylactic acid (PLA). p-Nitrophenyl butyrate (PNPB, 7) was used as model substrate to determine the specific activity of both enzymes at 40 °C. Although this work aimed at targeting the hydrolysis of insoluble polymers, 7 is the commonly used substrate for initial evaluation of the catalytic performance of this type of hydrolytic enzymes. LCC WCCG and Tc_Cut2NV showed 11828 U mg -1 and 9904 U mg -1 specific activity, respectively. As the depolymerization reaction rates for the polyesters increased at higher temperatures, we hypothesized that the reactions could be run at a temperature closer to the glass transition temperature (Tg) of the polymers, therefore increasing polymer chain accessibility (Tg, between 58 and 87 °C, SI Table , Fig. ). We therefore attempted to increase the thermostability of Tc_Cut2NV (>60 °C). Tc_Cut2NV (PDB: 5LUK) has a high structural similarity with the high thermostable variant LCC WCCG (PDB: 4EB0 for wild-type LCC). Both Tc_Cut2NV and wild-type LCC display a typical α/β-hydrolase fold in which nine antiparallel β-strands are enveloped by ten α-helices, five from each side. The enzymes contain an active site serine residue (S131 for Tc_Cut2NV and S138 for LCC) along with the proximal aspartic acid and histidine residues that create a catalytic triad, which is essential for hydrolysis. Figure depicts the X-ray crystal structure of Tc_Cut2NV where the catalytic triad is highlighted (magenta), whereas Figure displays a structural alignment between Tc_Cut2NV (grey) and wild-type LCC (magenta). Due to their high structural similarity, we speculated that introducing similar mutations of the variant LCC WCCG to the scaffold of Tc_Cut2NV might also increase the thermal stability of the latter. Therefore, via 3D-structural alignment, the corresponding residues were identified in the Tc_Cut2NV scaffold and the mutations (Q93G, D205C, F210W and E254C) were introduced to create the Tc_Cut2NV WCCG gene, which was recombinantly expressed in E. coli and the enzyme was purified by Ni 2+ affinity chromatography (SI, Fig. ) as previously done for Tc_Cut2NV and LCC WCCG . Thermostability measurements revealed that the mutations in the Tc_Cut2NV WCCG scaffold enhanced the stability of the enzyme, which displayed an impressive melting temperature (Tm) of 91 °C (~27 °C higher than the parental Tc_Cut2NV) (SI, Fig. ). The new variant also showed a 16% increase in catalytic activity for the hydrolysis of the model substrate 7 compared with the Tc_Cut2NV (11460 U mg -1 and 9904 U mg -1 for Tc_Cut2NV WCCG and Tc_Cut2NV, respectively). These results motivated us to conduct long-term thermostability measurements at 40 °C and, for up to 6 days, at 71 °C using purified Tc_Cut2NV WCCG . The enzyme showed a similar specific activity for substrate 7 at 71 °C and at 40 °C (ca. 5000 U mg -1 ) at the beginning, but the activity at 71 °C decreased 1.6-fold and 6-fold after 3 and 6 days, respectively (Table ). [a] Activity was measured by monitoring the increase of the absorbance of 7a at λ of 405 nm for 1 min using the extinction coefficient (ε405nm = 9.36 mL/(μmol*cm)). Reactions were performed with 7 (4 mM) at 71 °C in 50 mM KPi pH 7.6 with the pure enzymes. Measurements were carried out using a spectrophotometer equipped with temperature control. In all measurements, the enzymes were incubated at the desired temperature prior to the assay.