6FZC

Crystal Structure of lipase from Geobacillus stearothermophilus T6 variant L184F/L360F

Summary for 6FZC

• Entry DOI: 10.2210/pdb6fzc/pdb
• Related: 4X6U 6FZ1 6FZ7 6FZ8 6FZ9 6FZA
• Descriptor: Lipase, ZINC ION, CALCIUM ION, ... (4 entities in total)
• Functional Keywords: lipase, methanol, organic solvent, hydrolase
• Biological source: Geobacillus stearothermophilus
• Total number of polymer chains: 1
• Total formula weight: 44106.52

Authors

Gihaz, S.,Kanteev, M.,Pazy, Y.,Fishman, A. (deposition date: 2018-03-14, release date: 2018-10-17, Last modification date: 2024-01-17)

Primary citation

Gihaz, S.,Kanteev, M.,Pazy, Y.,Fishman, A.
Filling the Void: Introducing Aromatic Interactions into Solvent Tunnels To Enhance Lipase Stability in Methanol.
Appl.Environ.Microbiol., 84:-, 2018

PubMed Abstract: 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 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 ( ) of 7°C in methanol and a 3-fold increase in biodiesel synthesis yield from waste chicken oil. A kinetic analysis with -nitrophenyl laurate revealed that all mutants displayed lower hydrolysis rates (), 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. Enzymatic synthesis in organic solvents holds increasing industrial opportunities in many fields; however, one major obstacle is the limited stability of biocatalysts in such a denaturing environment. Aromatic interactions play a major role in protein folding and stability, and we were inspired by this to redesign enzyme voids. The rational protein engineering of solvent tunnels of lipase from is presented here, offering a promising approach to introduce new aromatic interactions within the enzyme core. We discovered that longer tunnels leading from the surface to the enzyme active site were more beneficial targets for mutagenesis for improving lipase stability in methanol during biodiesel biosynthesis. A structural analysis of the variants confirmed the generation of new interactions involving aromatic residues. This work provides insights into stability-driven enzyme design by targeting the solvent channel void.

PubMed: 30217852
DOI: 10.1128/AEM.02143-18

Experimental method

X-RAY DIFFRACTION (2.7 Å)