Pazy Y

References (2)

Title : Bridges to Stability: Engineering Disulfide Bonds Towards Enhanced Lipase Biodiesel Synthesis - Gihaz_2020_ChemCatChem_12_181
Author(s) : Gihaz S , Bash Y , Rush I , Shahar A , Pazy Y , Fishman A
Ref : ChemCatChem , 12 :181 , 2020
Abstract : Computational design of disulfide bonds was performed for lipase from Geobacillus stearothermophilus T6 (LipT6) for enhanced methanol stability and improved biodiesel production. Thirteen double mutants comprising new cysteine pairs were screened and evaluated for their stability in 70% methanol. Superior stability was found with variant E251C/ G332C (M13) having a 5.5-fold higher hydrolysis activity and enhanced unfolding temperature (Tm) of +7.9 C in methanol compared with wild-type. Moreover, M13 converted nearly 80% waste chicken oil to biodiesel, representing a 2.4-fold improvement relative to the WT. Structural studies using X-ray crystallography confirmed the existence of the engineered disulfide bonds shedding light on the link between the bond location and backbone architecture with its stabilization impact. Rational integration of disulfide bonds is suggested to be a feasible method to promote elevated stability in organic solvents for various industrial applications such as biodiesel synthesis
ESTHER : Gihaz_2020_ChemCatChem_12_181
PubMedSearch : Gihaz_2020_ChemCatChem_12_181
Gene_locus related to this paper: geose-q93a71

Title : Filling the Void: Introducing Aromatic Interactions into Solvent Tunnels To Enhance Lipase Stability in Methanol - Gihaz_2018_Appl.Environ.Microbiol_84_
Author(s) : Gihaz S , Kanteev M , Pazy Y , Fishman A
Ref : Applied Environmental Microbiology , 84 : , 2018
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 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 (Tm ) of 7 degrees 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 pi-pi or CH/pi 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.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 redesign enzyme voids. The 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. 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.
ESTHER : Gihaz_2018_Appl.Environ.Microbiol_84_
PubMedSearch : Gihaz_2018_Appl.Environ.Microbiol_84_
PubMedID: 30217852
Gene_locus related to this paper: geose-q93a71