Ionic liquid activated Bacillus subtilis lipase A variants through cooperative surface substitutions

Biotechnol Bioeng. 2015 Oct;112(10):1997-2004. doi: 10.1002/bit.25617. Epub 2015 Jul 22.

Abstract

The interest in performing enzyme-catalyzed reactions in amphiphilic systems, e.g., imidazolium-based ionic liquids (ILs) or surfactants, has been increased over the past decades. Directed protein evolution has been successful in tailoring enzymes for desired properties. Herein, nine IL-resistant Bacillus subtilis lipase A variants, particularly an IL-activated variant M1 (M134N/N138S/L140S), were identified by directed evolution. For instance, variant M2 (M134R/L140S) showed almost doubled specific activity (16.9 vs. 9.4 U/mg) and resistance (233% vs. 111%) at 9 vol% 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([C4 mim][TfO]) compared with wild-type. The specific activities and resistance of purified individual single and double variants have been studied in five different IL-aqueous mixtures. The re-activation of lipase variant M1 (not wild-type) at high IL concentration was attributed to the cooperative effect of three surface substitutions (M134N, N138S, L140S) near the substrate-binding cleft. The presence of IL/substrate clusters under assay conditions was likely related to the re-activation effect. This study provides first example of IL-activated lipase variant generated by protein engineering, and helps to better understand the protein-IL interaction.

Keywords: clusters; cooperative effects; directed evolution; ionic liquids; lipase; protein engineering.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Bacillus subtilis / enzymology*
  • Binding Sites
  • Directed Molecular Evolution
  • Enzyme Activators / metabolism*
  • Ionic Liquids / metabolism*
  • Lipase / genetics
  • Lipase / metabolism*
  • Models, Molecular
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism
  • Protein Conformation
  • Protein Engineering

Substances

  • Enzyme Activators
  • Ionic Liquids
  • Mutant Proteins
  • Lipase