6fzd | pdb_00006fzd
Crystal Structure of lipase from Geobacillus stearothermophilus T6 variant L184F/A187F/L360F
Structural highlights
Publication Abstract from PubMedEnhanced stability in organic solvents is a desirable feature for enzymes implemented under industrial conditions. Lipases potential as biocatalysts is mainly limited by 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 in order to occupy solvent channels and induce aromatic interactions leading to a better inner core packing. Each solvent tunnel was systematically analyzed with respect to its chemical and structural characteristics. Selected residues were replaced with Phe, Tyr or Trp. Overall, 16 mutants were generated and screened in 60% methanol, from which 3 variants showed elevated stability up to 81-fold compared with wild-type. All stabilizing mutations were found in the longest tunnel detected in the "closed-lid" x-ray structure. Combining the Phe substitutions created double mutant A187F/L360F with an increase in Tm of +7 degrees C in methanol and a 3-fold increase in biodiesel synthesis yield from waste chicken oil. Kinetic analysis with p-nitrophenyl laurate revealed that all mutants displayed lower hydrolysis rates (kcat ), though mostly their stability properties determined the transesterification capability. Seven crystal structures of different variants were solved disclosing new pi-pi or CH/pi intramolecular interactions emphasizing the significance of aromatic interactions to improved solvent stability. This rational approach could be implemented in other enzymes for stabilization in organic solvents.IMPORTANCE 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 re-design enzyme voids. Rational protein engineering of solvent tunnels of lipase from Geobacillus stearothermophilus 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. 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 solvent channels void. Filling the void: Introducing aromatic interactions into solvent tunnels towards lipase stability in methanol.,Gihaz S, Kanteev M, Pazy Y, Fishman A Appl Environ Microbiol. 2018 Sep 14. pii: AEM.02143-18. doi:, 10.1128/AEM.02143-18. PMID:30217852[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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