Related Experiment Video
Updated: Jul 14, 2026
08:37
Measuring Enzymatic Stability by Isothermal Titration Calorimetry
Published on: March 26, 2019
Isosteric Engineering of Enzymes: Overcoming Activity-Stability Trade-Offs by Site-Selective CH → N Substitutions
Elwy H Abdelkader1, Haocheng Qianzhu2, Gottfried Otting1
1ARC Centre of Excellence for Innovations in Peptide and Protein Science, Research School of Chemistry, Australian National University, Canberra, ACT, Australia.
Angewandte Chemie (International Ed. in English)
|July 13, 2026
Summary
Enzymes engineered with azatryptophans, a type of non-canonical amino acid, overcome performance limits. This breakthrough enhances polyethylene terephthalate hydrolase (PETase) activity and stability, offering cost-effective industrial applications.
Area of Science:
- Biochemistry
- Protein Engineering
- Synthetic Biology
Background:
- Industrial enzymes are engineered for peak performance, but canonical amino acids limit further optimization.
- Non-canonical amino acids (ncAA) offer expanded chemical space but are often costly and disrupt enzyme structure.
- Polyethylene terephthalate hydrolases (PETases) are crucial for plastic degradation but face an evolutionary ceiling.
Purpose of the Study:
- To overcome the evolutionary ceiling in enzyme engineering using genetically encoded non-canonical amino acids.
- To introduce azatryptophans as cost-effective, isosteric replacements for tryptophan in industrial enzymes.
- To develop a robust assay for benchmarking PETase activity.
Main Methods:
- Genetic encoding systems for 4-, 5-, and 6-azatryptophan were established.
- Site-selective incorporation of azatryptophans into polyethylene terephthalate hydrolases (PETases).
- Development and application of a fluorescence-based kinetic assay (PETra) for PETase activity measurement.
Main Results:
- Azatryptophan incorporation enhanced PETase catalytic activity while maintaining thermal stability.
- 6-azatryptophan (6AW) served as a sensitive reporter for side-chain solvent exposure, crucial for PETase function.
- Enzymes bearing azatryptophans were produced cost-effectively via enzymatic biosynthesis.
- The PETra assay demonstrated reproducibility and strong correlation with solid PET hydrolysis.
Conclusions:
- Azatryptophans represent a viable strategy to break the evolutionary ceiling for industrial enzyme optimization.
- Genetically encoded azatryptophans offer a cost-effective and structurally compatible method for enhancing enzyme performance.
- The PETra assay provides a reliable tool for evaluating PETase activity and engineering efforts.
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