Structural insights into methanol-stable variants of lipase T6 from Geobacillus stearothermophilus

Abstract

Enzymatic production of biodiesel by transesterification of triglycerides and alcohol, catalyzed by lipases, offers an environmentally friendly and efficient alternative to the chemically catalyzed process while using low-grade feedstocks. Methanol is utilized frequently as the alcohol in the reaction due to its reactivity and low cost. However, one of the major drawbacks of the enzymatic system is the presence of high methanol concentrations which leads to methanol-induced unfolding and inactivation of the biocatalyst. Therefore, a methanol-stable lipase is of great interest for the biodiesel industry. In this study, protein engineering was applied to substitute charged surface residues with hydrophobic ones to enhance the stability in methanol of a lipase from Geobacillus stearothermophilus T6. We identified a methanol-stable variant, R374W, and combined it with a variant found previously, H86Y/A269T. The triple mutant, H86Y/A269T/R374W, had a half-life value at 70 % methanol of 324 min which reflects an 87-fold enhanced stability compared to the wild type together with elevated thermostability in buffer and in 50 % methanol. This variant also exhibited an improved biodiesel yield from waste chicken oil compared to commercial Lipolase 100L® and Novozyme® CALB. Crystal structures of the wild type and the methanol-stable variants provided insights regarding structure-stability correlations. The most prominent features were the extensive formation of new hydrogen bonds between surface residues directly or mediated by structural water molecules and the stabilization of Zn and Ca binding sites. Mutation sites were also characterized by lower B-factor values calculated from the X-ray structures indicating improved rigidity.

Bridges to Stability: Engineering Disulfide Bonds Towards Enhanced Lipase Biodiesel Synthesis

“…The crystal structure of M8 (A187C/F291C) displays a disulfide bond connecting the enzyme helix‐lid (α9, residues F177‐A192) with position 291 in the neighboring α‐helix (Figure A). Complete and continuous electron density affirms the S−S bond and the blockage of the previously described active site tunnel . In Figure B, moderate changes in secondary structure are observed, in the Cys291 α‐helix, now in the form of a helical loop.…”

“…The second characterization assay preformed was unfolding temperature (T m ) measurements in buffer and in 70 % methanol (resembling methanol concentration during biodiesel production). Previous studies on LipT6 showed good correlations between methanol stability and thermostability, though the opposite correlation was found to be more complex . Mutants stable in methanol were also more thermostable, but improved thermal stability did not necessarily project stability in methanol.…”

“…This enzyme belongs to the I.5 lipase subfamily and the crystal structure was solved revealing a fairly compressed packing with a helix lid (α9, residues F177 to A192) covering the enzymes’ active site pocket . Surface hydrogen bonds and core aromatic interactions were identified as stabilizing forces in previous studies . LipT6 WT does not possess disulfide bonds but has two unpaired cysteine residues (Cys65 and Cys296) found as thiols in different regions of the enzyme structure.…”

“…Methods such as random mutagenesis, structure guided consensus and rational solvent tunnels design were found effective in establishing methanol‐stable variants with improved biodiesel yield, compared with wild‐type (WT) LipT6 . This enzyme belongs to the I.5 lipase subfamily and the crystal structure was solved revealing a fairly compressed packing with a helix lid (α9, residues F177 to A192) covering the enzymes’ active site pocket . Surface hydrogen bonds and core aromatic interactions were identified as stabilizing forces in previous studies .…”

Pseudomonas fluorescensStrain R124 Encodes Three Different MIO Enzymes

“…To characterize the enzymes, unfolding events of the proteins were measured with a nanoDSF device by monitoring the intrinsic tryptophan fluorescence at emission wavelengths of λ =330 and 350 nm. Because the melting temperature ( T m ) of a lipase determined by differential scanning calorimetry (DSC) was identical to that obtained by nanoDSF, this technique proved to be a straightforward, simple, and highly correlative method to traditional techniques.…”