Filling the void: Introducing aromatic interactions into solvent tunnels to enhance lipase stability in methanol

Shalev Gihaz, Margarita Kanteev, Yael Pazy, Ayelet Fishman

Research output: Contribution to journalArticlepeer-review


An enhanced stability of enzymes in organic solvents is desirable under industrial conditions. The potential of lipases as biocatalysts is mainly limited by their denaturation in polar alcohols. In this study, we focused on selected solvent tunnels in lipase from Geobacillus stearothermophilus T6 to improve its stability in methanol during biodiesel synthesis. Using rational mutagenesis, bulky aromatic residues were incorporated to occupy solvent channels and induce aromatic interactions leading to a better inner core packing. The chemical and structural characteristics of each solvent tunnel were systematically analyzed. Selected residues were replaced with Phe, Tyr, or Trp. Overall, 16 mutants were generated and screened in 60% methanol, from which 3 variants showed an enhanced stability up to 81-fold compared with that of the wild type. All stabilizing mutations were found in the longest tunnel detected in the "closed-lid" X-ray structure. The combination of Phe substitutions in an A187F/L360F double mutant resulted in an increase in unfolding temperature (T m ) of 7°C in methanol and a 3-fold increase in biodiesel synthesis yield from waste chicken oil. A kinetic analysis with p-nitrophenyl laurate revealed that all mutants displayed lower hydrolysis rates (k cat ), though their stability properties mostly determined the transesterification capability. Seven crystal structures of different variants were solved, disclosing new π-π or CH/π intramolecular interactions and emphasizing the significance of aromatic interactions for improved solvent stability. This rational approach could be implemented for the stabilization of other enzymes in organic solvents.

Original languageEnglish
Article numbere02143-18
JournalApplied and Environmental Microbiology
Issue number23
StatePublished - 1 Dec 2018


  • Biodiesel
  • Lipase
  • Organic solvents
  • Protein engineering
  • Solvent tunnel
  • Stability

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Food Science
  • Ecology
  • Applied Microbiology and Biotechnology


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