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Using Enzymes as Promiscuous Biocatalysts for C-C and C-X Bond Forming Reactions, Enhancing the Catalytic Performance by Encapsulation and Investigating the Reaction Mechanism by Computational Simulation

Piraman, Zeinab | 2025

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  1. Type of Document: Ph.D. Dissertation
  2. Language: Farsi
  3. Document No: 58263 (03)
  4. University: Sharif University of Technology
  5. Department: Chemistry
  6. Advisor(s): Kalhor, Hamid Reza
  7. Abstract:
  8. This research focuses on the promiscuous activity of bacterial tautomerase and subsequently hen egg white lysozyme in facilitating carbon-carbon and carbon-heteroatom bond formation. Tautomerases refer to a class of enzymes that catalyze tautomerization reactions particularly keto-enol reactions. Initially, YDCE (a 4-oxalocrotonate tautomerase homologue) was cloned from E.coli genome and expressed in E.coli BL21. YDCE with 75 amino acids, after expression and purification using a cation-exchange chromatography column, was utilized to catalyze the synthesis of chroman derivatives from available precursors. Lysozyme, which belongs to the class of hydrolase, consists of 129 amino acids, 4 disulfide bonds, and possesses the appropriate folding to hydrolyze the glycosidic bonds in the cell wall of Gram-positive bacteria. In the first project, lysozyme was used to catalyze the Mannich reaction. Efforts were made to enhance the enzyme's activity and recovery by immobilizing it on a zeolite-imidazole network for the biocatalysis of the Mannich reaction. Furthermore, molecular dynamics simulations highlighted the critical role of tryptophan-63 over Asp52 in the catalytic mechanism. In the second project, given the significance of triazole scaffolds in pharmaceutical compounds, the synthesis of triazole derivatives using lysozyme was investigated. Immobilization of lysozyme on metal-free carbon-nitride supports enabled efficient, metal-free biocatalytic transformations and improved enzyme recovery. To elucidate the specific roles of active site residues, molecular docking, molecular dynamics, and computational mutations were conducted. The results emphasized the importance of Arg-112, Trp-62, and Trp-63 residues in the catalytic mechanism. Ultimately, a plausible mechanism was proposed for the lysozyme-catalyzed promiscuous reaction. Regioselective disubstituted-1,2,3-triazole derivatives were synthesized using sodium azid and flavine reductase. X-ray crystallography of the derivatives obtained with NaN3 confirmed regioselective substitution at the N2 position, leading to the thermodynamically favored 2,4-disubstituted products. In contrast, flavin reductase catalysis resulted in the formation of 1,4-disubstituted derivatives, representing the kinetically controlled products
  9. Keywords:
  10. Hen Egg White Lysozyme Enzyme ; Promiscuous Reactions ; Biocatalyst ; Molecular Docking ; Molecular Dynamics ; 1,2,3-Triazole Containing ; Mannich Reaction ; Cascade Reactions

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