## 3 Results and Discussion
### Product Profile and Activity of IsPETase and Trx-IsPETase  
The products of both enzymes against PET were evaluated by HPLC. DMSO was used as a co-solvent for the depolymerization products. TPA, MHET, and BHET have low solubility in water. All controls containing the polymers, buffer, and DMSO did not show any detectable degrading effect of the DMSO under the same experimental conditions as the enzymatic reactions. Furthermore, dissolved products are required for analysis by HPLC. The chromatograms showed the same product profiles (Figure 3A). With terephthalatic acid (TPA) eluted at -0.61 min, other peaks at -0.76 and -0.96 min were identified by HPLC-MS (Figure 3B) as mono-(2-hydroxyethyl) terephthalate (MHET) and bis-(2-hydroxyethyl) terephthalate (BHET), respectively. According to the relative area of the peaks, MHET was the main product, followed by TPA, while BHET was present in a significantly smaller fraction. Trx-IsPETase showed slightly higher activity than IsPETase (Figure 3C). All resulting peaks from HPLC analysis mentioned in this section were regarded as terephthalatic acid equivalents (TPAeq), assuming the same molar extinction coefficient \(\varepsilon\) = 17,000 M\({}^{-1}\)cm\({}^{-1}\) conferred by the aromatic ring. Thus, the calculated TPAeq produced could be determined. The total amount of degraded polymer could be calculated using the TPAeq and the repeating molecular weight (192 g/mol). The results of the depolymerization are shown in Table 1. The degree of depolymerization after 72 h by IsPETase was 23%, and for Trx-IsPETase, it was 32%. The enzyme/PET ratio was slightly higher for Trx-IsPETase (0.005) than IsPETase (0.004). However, the specific activity, defined here as the mg of product per mg of enzyme and time (mg degradation products/mg enzyme h\({}^{-1}\) or h\({}^{-1}\)), gave a higher value for Trx-IsPETase (0.7 h\({}^{-1}\)) than IsPETase (0.9 h\({}^{-1}\)) (Table 2). Although the differences were not remarkable, the student's t-test gave a statistically significant higher product formation for Trx-IsPETase than IsPETase (Figure 3C). \begin{table}
\begin{tabular}{l c c c c c} \hline \hline
**Host** & & **Plasmid** & **Production (mg/L)** & **Reference** \\ \hline \multicolumn{5}{c}{_Cytosolic systems_} \\ _E. coli_ & & & & \\ Rosetta-gamiTM2 & pET28b(+):IsPETase & 79 & This work \\ _E. coli_ & & & & \\ Rosetta-gamiTM2 & pET32b(+):Trx-IsPETase & 93 & This work \\ _E. coli_ & & & & \\ _Extracellular systems_ & & & \\ _E. coli_ & & & \\ (DE3)-T1R & pET22b-SPMalE:IsPETase & 6.2 & [43] \\ _E. coli_ & & & \\ (DE3)-T1R & pET22b-SPLamB:IsPE-Tase & 3 & [43] \\ _E. coli_ & & & \\ _E. coli_ & & & \\ _Bacillus subtilis_ & & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Production of recombinant _Is_PETase in different systems. \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline
**Enzyme** & **Enzyme/PET Ratio** & **Degradation Products** & \multicolumn{2}{c}{**Relative Production (\%)**} & \multicolumn{2}{c}{**Depolymerization PET**} & \multicolumn{2}{c}{**Specific Activity**} \\ \cline{3-8}  & **(mg/mg)** & **(mg/L)** & **TPA** & **MHET** & **BHET** & **(\%)** & **(h\({}^{-1}\))** \\ \hline _Is_PETase & 0.004 & 2304 & 24.2 & 73.7 & 2.1 & 23 & 0.7 \\ Trx-IsPETase & 0.005 & 3230 & 31.5 & 66.7 & 1.8 & 32 & 0.9 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: PET depolymerization activity of _Is_PETase and Trx-IsPETase. Degradation products consist of the sum of the TPA, MHET, and BHET quantified. The dynamic stability of Trx-_Is_PETase was determined by monitoring the formation of the product for 240 h. The degradation products increased in concentration during the first 72 h until reaching saturation (Figure 4). The saturation was attributed to the inactivation of the enzyme and not to the completion of the reaction, since the substrate conversion reached \(\sim\)35%.