Mutation Background
In our project, we selected specific mutations in the TfCut2 enzyme to explore their potential for enhancing plastic degradation...
- Thermostability
- Residues around active sites
- Computational models
Thermal Stability
Thermostability is critical for degrading PET and PBAT...
- S121P/D174S/D204P:
- Melting temp: 80.7°C
- Reduces hydrogen bonding
- D204C/E253C:
- Disulfide bond enhances stability
Active Site and Binding Mutations
- G62A: Reduces MHET binding
- L90A: Enhances cotton/PET binding
Visualization with PyMOL
We used AlphaFold and PyMOL to dock ligands and visualize key interactions...
Understanding Byproducts
- Substrate interaction analysis
- Bond cleavage mapping
Multiple Sequence Alignment (MSA)
- Conserved catalytic residues identified
- Guided rational mutagenesis
Alanine Scanning Mutagenesis (ASM)
High-impact vs low-impact residues ("head" vs "hair") were tested...
Site Saturation Mutagenesis (SSM)
Each chosen site was mutated into all 20 amino acids; filtered by binding and structure.
Mutants from DBTL Cycle 1
- G62A/P193H/S197D: forms catalytic triad, reduces MHET inhibition
- H77R/D204C/E253C: disulfide bridge-stabilized
Citations:
- Li, Q., et al. (2022)...
- Wei, R., et al. (2016)...