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Investigating One-pot Three Component Synthesis of ß-Amino Carbonyl Compounds Exploiting Hydrolases: Protein Splicing Enzyme and N-Acetylmuramide Glycanhydrolase

Fathali, Yasaman | 2019

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  1. Type of Document: M.Sc. Thesis
  2. Language: Farsi
  3. Document No: 51939 (03)
  4. University: Sharif University of Technology
  5. Department: Chemistry
  6. Advisor(s): Kalhor, Hamidreza
  7. Abstract:
  8. The application of biocatalysts in organic reactions refers to the use of enzymes, purified or crude, to increase the reaction rate. The usage of biocatalysts in organic reactions have many advantages, including biocompatibility, ease of separation, high yield, reusability without losing activity, high selectivity, and the use of water as the solvent. Furthermore, the carbon–carbon bond formation is one of the most important reactions in the synthesis of organic compounds, drugs, and biomolecules. Using enzymes in organic reactions can provide insightful information about the enzymes catalytic activities and paves the way for systematic investigation of their mechanisms. The enzymes belonging to hydrolase family such as lysozyme and protein splicing enzyme have been shown to carry carbon-carbon bond forming reactions in addition to their native reactions. However, the ability of the hen egg white lysozyme and the protein splicing enzyme (Intein) in forming such bonds has not been studied yet. Thus, in this project, the aforementioned enzymes were studied for their ability to catalyze the aldol and Mannich reactions. The protein splicing enzyme (Mycobacterium tuberculosis-H37Rv) was cloned and the corresponding recombinant protein was expressed and purified, however, the enzyme failed to catalyze these reactions with sufficient yield. When lysozyme enzyme was examined for catalyzing the same reactions, it failed similarly to catalyze the aldol reaction. However, the enzyme was capable of catalyzing the synthesis of β-amino carbonyl compounds through the one-pot three-component Mannich reaction at 35 °C in water/acetone (1:1) as a green solvent within just 3 to 16 hours in high yields. The results have shown that one of the characteristic features of the present method is the high chemoselectivity of cyclohexanone toward imines (prepared in situ) in preference to aldehydes. To study the enzyme's conformational changes, intrinsic fluorescence and circular dichroism in far-UV region were examined that showed a decrease in the secondary structure of α-helix so the active sites that were located on this structure are no longer able to continue the reaction. The biological activity of the bacterium Micrococcus luteus and turbidimetric assay also were examined for enzyme activity. The results clearly showed enzymatic changes after several rounds of reusing the enzyme. In addition, Michaelis–Menten kinetic parameters, KM, kcat, and the substrate specificity parameter kcat/KM were calculated for its comparison with the kinetics of the enzyme's natural reaction This suggests that the influence of substrate affinity and hydrophobicity is of minor importance and that other factors were involved. The lysozyme enzyme was employed as an efficient, inexpensive enzyme that could be easily separated, recovered, and reused for up to 4 rounds
  9. Keywords:
  10. Biocatalyst ; N-Acetylmuramide Glycanhydrolase ; Intein ; Mannich Reaction ; Carbon-Carbon Forming Reaction ; Lysozyime ; Protein Splicing

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