Residue-Specific Incorporation of the Non-Canonical Amino Acid Norleucine Improves Lipase Activity on Synthetic Polyesters

Karolina Haernvall, Patrik Fladischer, Heidemarie Schoeffmann, Sabine Zitzenbacher, Tea Pavkov-Keller, Karl Gruber, Michael Schick, Motonori Yamamoto, Andreas Kuenkel, Doris Ribitsch*, Georg M. Guebitz, Birgit Wiltschi

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Environmentally friendly functionalization and recycling processes for synthetic polymers have recently gained momentum, and enzymes play a central role in these procedures. However, natural enzymes must be engineered to accept synthetic polymers as substrates. To enhance the activity on synthetic polyesters, the canonical amino acid methionine in Thermoanaerobacter thermohydrosulfuricus lipase (TTL) was exchanged by the residue-specific incorporation method for the more hydrophobic non-canonical norleucine (Nle). Strutural modelling of TTL revealed that residues Met-114 and Met-142 are in close vicinity of the active site and their replacement by the norleucine could modulate the catalytic activity of the enzyme. Indeed, hydrolysis of the polyethylene terephthalate model substrate by the Nle variant resulted in significantly higher amounts of release products than the Met variant. A similar trend was observed for an ionic phthalic polyester containing a short alkyl diol (C5). Interestingly, a 50% increased activity was found for TTL [Nle] towards ionic phthalic polyesters containing different ether diols compared to the parent enzyme TTL [Met]. These findings clearly demonstrate the high potential of non-canonical amino acids for enzyme engineering.

Original languageEnglish
Article number769830
JournalFrontiers in Bioengineering and Biotechnology
Publication statusPublished - 26 Jan 2022


  • enzyme hydrolysis
  • genetic code engineering
  • lipase
  • norleucine
  • polyester modification
  • Thermoanaerobacter thermohydrosulfuricus
  • TTL

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Histology
  • Biomedical Engineering


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