Browsing by Author "Yildirim, Deniz"

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Engineering of Geobacillus Kaustophilus Lipase for Enhanced Catalytic Efficiency and Methanol Tolerance in Biodiesel Production from Sunflower Oil
(Elsevier, 2025) Tulek, Ahmet; Poyraz, Yagmur; Sukur, Gozde; Pacal, Nurettin; Ozdemir, F. Inci; Yildirim, Deniz; Essiz, Sebnem
Lipase-mediated biodiesel production offers a sustainable and environmentally friendly alternative to conventional chemical methods. However, enzyme limitations such as low activity, poor thermal stability, and limited solvent tolerance remain challenges. In this study, a lipase from Geobacillus kaustophilus (Gklip) was engineered for improved biodiesel production using molecular docking, molecular dynamics (MD) simulations, and molecular mechanics/generalized born surface area (MM/GBSA) free energy calculations. Five mutants (Y29S, Q114T, F289D, Q184M, and Q114F) were generated via site-directed mutagenesis and expressed in Escherichia coli. Biochemical characterization revealed that all mutants retained the wild-type's optimal temperature (50 degrees C) and pH (8.0), while showing varying pH ranges, with the broadest observed in Q184M. Thermal stability increased significantly in Q184M (32.86-fold) and Q114F (5.93-fold). Catalytic efficiencies improved by 2.07-, 2.05-, and 2.63-fold in Q184M, F289D, and Y29S, respectively, compared to the wild-type (0.57). In the presence of 60 % methanol, the wild-type retained only 30.4 % activity, while Q184M maintained 67.5 %, highlighting superior solvent tolerance. Biodiesel conversion assays using sunflower oil showed no product formation by the wild-type, whereas Q184M, Q114F, and F289D achieved yields of 58.7 %, 56.3 %, and 49.2 %, respectively. These findings identify Q184M and Q114F as promising enzyme candidates for enzymatic biodiesel production.
Citation - WoS: 35
Citation - Scopus: 35
Sustainable production of formic acid from CO2 by a novel immobilized mutant formate dehydrogenase
(Elsevier, 2023) Tulek, Ahmet; Gunay, Elif; Servili, Burak; Essiz, Sebnem; Binay, Baris; Yildirim, Deniz; Core Program; Molecular Biology and Genetics; 05. Faculty of Engineering and Natural Sciences; 07. Core Program; 01. Kadir Has University
Formate dehydrogenase (NAD+-dependent FDH) is an enzyme that catalyzes the reversible oxidation of formate to CO2 while reducing NAD+ to NADH. The enzyme has been used in industrial and chemical applications for NADH regeneration for a long time. However, discovering the unique ability of FDHs, which is to reduce CO2 and produce formic acid, leads studies focusing on discovering or redesigning FDHs. Despite using various protein engineering techniques, these studies mostly target the same catalytic site amino acids of FDHs. Here, for the first time, the effect of an Asp188 mutation on a potential allosteric site in NAD+-dependent CtFDH around its subunit-subunit interface was studied by molecular modelling and simulation in the presence of bicarbonate and formate. Biochemical and kinetic characterization of this Asp188Arg mutant and wild type CtFDH enzymes were performed in detail. Both enzymes were also immobilized on newly synthesized MWCNT-Ni-O-Si/Ald and MWCNT-Ni-O-Si/Glu supports designed to overcome well-known CtFDH stability problems including thermostability and reuse resistance. Integrating mutation and immobilization provided about a 25-fold increase in catalytic efficiency for carbonate activity. The one-way ANOVA analysis also ensured significant effect of the mutation and immobilization on kinetic constants. After characterizing the immobilization of highly purified wild type and mutant enzyme with instrumental analysis techniques, the thermal stability of MWCNT-Ni-Si@wtCtFDH and MWCNT-Ni-Si@mt-CtFDH was found to increase about 11-and 18-fold, respectively, compared to their free counterparts at 50 degrees C. The mutant CtFDH and its immobilized counterpart produced around 2-fold more formic acid than those of wild type CtFDH and its immobilized counterpart under the same conditions. MWCNT-Ni-Si@wt-CtFDH and MWCNT-Ni-Si@mt-CtFDH remained around 82 % and 86 % of their initial activities respectively after lots of recycling. Integration of subunit interface amino acid position of NAD+ dependent FDHs engineering and immobilization provides a new insight can be scientifically and rationally employed for this current application FDHs as a solution to produce formic acids from renewable sources.