Browsing by Author "Pacal, Nurettin"

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    Biocontrol Potential of Vibrio Maritimus Chitinase: Heterologous Expression and Insecticidal Activity Against Acanthoscelides Obtectus
    (Elsevier, 2025) Dikbas, Neslihan; Tulek, Ahmet; Ucar, Sevda; Alim, Seyma; Servili, Burak; Pacal, Nurettin; Ercisli, Sezai; Core Program; 07. Core Program; 01. Kadir Has University
    In this study, the chitinase gene from the marine bacterium Vibrio maritimus was heterologously expressed in Escherichia coli, purified via affinity chromatography and tested for its insecticidal activity against the storage pest Acanthoscelides obtectus. The recombinant VmChiA protein exhibited a molecular mass of similar to 60 kDa, with optimum activity observed at pH 6.0 and 40 degrees C. Enzyme kinetic analysis revealed a K-m value of 0.042 mM, V-max of 17.48 mu mol min(-1), k(cat) of 1.75 min(-1) and catalytic efficiency of 41.61 mM(-1) min(-1), respectively. Furthermore, a dose of 40 U mL(-1) of recombinant VmChiA showed similar efficacy to malathion insecticide against A. obtectus, with 100 % mortality in both treatments. LC50 and LC90 values of VmChiA were 13.95 U mL(-1) and 27.66 U mL(-1), respectively. Furthermore, the three-dimensional structure of the catalytic site of VmChiA was modeled. Molecular dynamics simulation technique was used to explore and analyze the dynamics and interactions. A salt bridge (GLU274-ARG296) in the alpha + beta domain was observed as a critical feature facilitating substrate (GlcNAc)(2) binding and enzymatic activity. These findings demonstrate that recombinant VmChiA possesses potent insecticidal properties, highlighting its potential as a bio-based, eco-friendly alternative for managing significant agricultural pests.
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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.