Next, we tested whether the introduced mutations improved the tolerance of the enzyme against organic solvents. We chose hexafluoro-2-propanol (HFIP) and tetrahydrofuran (THF) because these solvents can solubilize polyesters, thereby making possible to increase the accessibility of their chains. Unfortunately, the catalytic activity of Tc_Cut2NV WCCG decreased 30-fold or 40-fold at the tested concentration of added HFIP (10-90% v v -1 ) (SI, Table . S1) or THF (50%). This outcome indicated that none of these solvents could be used in the enzymatic depolymerization reactions. Therefore, we decided to conduct all reactions in aqueous buffers without any organic cosolvent.