## 2 Materials and Methods  
### Production and Purification of Enzymes  
_Is_PETase and Trx-_Is_PETase were produced in _Escherichia coli_ Rosetta-gamiTM2 (DE3) according to standard protocols [15; 16]. The gene encoding the _Is_PETase from _I. sakaiensis_ (GenBank accession code GAP38373.1) without a signal peptide (first 29 amino acids from the N-terminal) was chemically synthesized (GenScript USA Inc., Piscataway, NJ, USA) with codons optimized for _E. coli_ and cloned into the expression vectors pET28b(+) and pET32b(+). The latter added a thioredoxin fusion domain (Trx) in the N-terminal of the _Is_PETase. The obtained plasmids, pET28b(+):_Is_PETase and pET32b(+):Trx-_Is_PETase, were introduced into _E. coli_ Rosetta-gamiTM2 (DE3) (Novagen, U.S) by heat shock transformation. The recombinant strains were grown at 37 \({}^{\circ}\)C in an LB medium supplemented with 35 \(\mu\)g/mL kanamycin or 100 \(\mu\)g/mL ampicillin as selection factors for the cells harboring pET28b(+):_Is_PETase or pET32b(+):_Trx-_Is_PETase plasmids, respectively. The recombinant strains were cultivated in 300 mL of the LB medium at 37 \({}^{\circ}\)C until OD\({}_{600\text{nm}}\) = 0.6-1.0 was reached. Then, the production of _Is_PETase or Trx-_Is_PETase was induced by adding 1 mM isopropyl \(\beta\)-d-1-thiogalactopyranoside (IPTG) and overnight incubation at 20 \({}^{\circ}\)C. _Is_PETase and Trx-_Is_PETase were purified from cell extracts by Immobilized Metal Affinity Chromatography (IMAC). A HisTrapTM Fast Flow column installed in an AKTA start protein purification system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) was equilibrated with a binding buffer (100 mM Tris-HCl, 500 mM NaCl, pH 7.4). Then, the cell extract was loaded. After a washing step, the bound proteins were eluted with an elution gradient using a buffer (100 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 7.4). The protein purity was analyzed by sodium dodecyl sulphate-polyacrylamideallel gel electrophoresis (SDS-PAGE), while the concentration was determined spectrophotometrically at 280 nm.  
_Is_PETase and Trx-_Is_PETase were produced in _Escherichia coli_ Rosetta-gamiTM2 (DE3) according to standard protocols [15; 16]. The gene encoding the _Is_PETase from _I. sakaiensis_ (GenBank accession code GAP38373.1) without a signal peptide (first 29 amino acids from the N-terminal) was chemically synthesized (GenScript USA Inc., Piscataway, NJ, USA) with codons optimized for _E. coli_ and cloned into the expression vectors pET28b(+) and pET32b(+). The latter added a thioredoxin fusion domain (Trx) in the N-terminal of the _Is_PETase. The obtained plasmids, pET28b(+):_Is_PETase and pET32b(+):_Trx-_Is_PETase, were introduced into _E. coli_ Rosetta-gamiTM2 (DE3) (Novagen, U.S) by heat shock transformation. The recombinant strains were grown at 37 \({}^{\circ}\)C in an LB medium supplemented with 35 \(\mu\)g/mL kanamycin or 100 \(\mu\)g/mL ampicillin as selection factors for the cells harboring pET28b(+):_Is_PETase or pET32b(+):_Trx-_Is_PETase plasmids, respectively. The recombinant strains were cultivated in 300 mL of the LB medium at 37 \({}^{\circ}\)C until OD\({}_{600\text{nm}}\) = 0.6-1.0 was reached. Then, the production of _Is_PETase or Trx-_Is_PETase was induced by adding 1 mM isopropyl \(\beta\)-d-1-thiogalactopyranoside (IPTG) and overnight incubation at 20 \({}^{\circ}\)C. _Is_PETase and Trx-_Is_PETase were purified from cell extracts by Immobilized Metal Affinity Chromatography (IMAC). A HisTrapTM Fast Flow column installed in an AKTA start protein purification system (GE Healthcare Bio-Sciences AB Uppsala, Sweden) was equilibrated with a binding buffer (100 mM Tris-HCl, 500 mM NaCl, pH 7.4). Then, the cell extract was loaded. After a washing step, the bound proteins were eluted with an elution gradient using a buffer (100 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 7.4). The protein purity was analyzed by sodium dodecyl sulphate-polyacrylamideamide gel electrophoresis (SDS-PAGE), while the concentration was determined spectrophotometrically at 280 nm.