Table 1. Selected IsPETase Engineering Studies Designed to Improve Thermal Stability.
| nomenclature and introduced mutations | improved stabilitya | design approach and interpretation | refs |
|---|---|---|---|
| S238F/W159H | Tm = 56.5 °C, ΔTm = +9.7 °C | Structural and sequence comparison with homologous PET hydrolases | (65,90) |
| ThermoPETase: S121E/D186H/R280A | Tm = 57.6 °C, ΔTm = +8.8 °C | Structure-based design; Water-mediated hydrogen bond between E121 and H186 | (54) |
| DuraPETase: A214H/I168R/W159H/S188Q/R280A/A180I/G165A/Q119Y/L17F/T140D | Tm = 77.0 °C, ΔTm = +31.0 °C | Structure-based design and machine learning approach; Multiple stabilizing interactions | (55) |
| W159H/F229Y | ΔTm = +10.4 °C | Sequence comparison with other homologous PET hydrolases | (91) |
| DuraPETase+N233K | Tm = 83.5 °C, ΔTm = +38.4 °C | Machine learning; Introduction of salt bridge between K233 and E204 | (88) |
| FAST-PETase: ThermoPETase+R224Q/N233K | Tm= 67.4 °C, ΔTm = +22.3 °C | Machine learning; Introduction of a hydrogen bond between Q224 and S192 | (88) |
| TS-PETase: ThermoPETase+N233C/S282C | Tm = 69.4 °C, ΔTm = +22.3 °C | Structural comparison with other homologous PET hydrolases | (83) |
| TM3: ThermoPETase+K95N/F201I/N233C/S282C | Tm = 70.8 °C, ΔTm = +25.8 °C | Random mutagenesis based on error prone PCR and structural comparison with LCCICCG mutant | (56) |
| D1: DuraPETase+N233C/S282C | Tm = 81.1 °C, ΔTm = +36.1 °C | Structural comparison with LCCICCG mutant | (56) |
| HotPETase: TS-PETase+P181V/S207R/S214Y/Q119K/S213E/R90T/Q182M/N212K/R224L/S58A/S61V/K95N/M154G/N241C/K252M/T270Q | Tm= 82.5 °C, ΔTm = +37.5 °C | Directed evolution | (89) |
a
ΔTm was estimated compared to Tm of the wild-type IsPETase either determined in the same publication or that of 45.1 °C determined by Brott et al.56Tm values were determined by circular dichroism, differential scanning calorimetry, or differential scanning fluorimetry.