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Investigate Kinetic Parameters of L-Asparaginase Ii as Well as its Performance in Organic Reactions and Modifying the Enzyme Through Directed Evolution for Carrying Out Promiscuous Reactions

Noori Hosseinabadi, Zahra | 2021

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  1. Type of Document: M.Sc. Thesis
  2. Language: Farsi
  3. Document No: 54647 (03)
  4. University: Sharif University of Technology
  5. Department: Chemistry
  6. Advisor(s): Kalhor, Hamid Reza
  7. Abstract:
  8. L-asparaginase II is an amidohydrolase which hydrolyzes L-asparagine to furnish L-aspartic acid and ammonia. The enzyme has played an important role for 40 years in treating cancer in persons with juvenile leukemias or lymphomas. Furthermore, the enzyme has become important in food industries and biosensors technology.In this study, the expression of recombinant L-asparaginase II in E. Coli was optimized and the purification of this enzyme from growth culture was carried out with nickel-charged affinity resin. Afterwards, the activity, and kinetic parameters of purified recombinant L-asparaginase II including Km, kcat, Vmax, and enzyme efficiency were measured.In the next step, random mutagenesis of whole recombinant plasmid contained L-asparaginase II gene was performed on the path of directed evolution, so that the mutated enzyme can accept new substrates such as succinamic acid. Initial results indicated that one of the mutated species of the enzyme could hydrolyze succinamic acid and provide the nitrogen needed to grow in the minimal culture medium.To investigate the catalytic promiscuity in recombinant L-asparaginase II, a number of chemical reactions including hydrolysis, C-C, and C-N formation were performed with L-asparaginase II as biocatalyst, and the initial studies have indicated the enzyme has capacity to catalyze aldol reaction.Finally, to investigate the substrate promiscuity different substrates were docked to asparaginase 3D structure via computational analysis, and the molecular dynamic simulations of enzyme was performed in the presence of L-asparagine and succinamic acid. The computational results demonstrate that the asparaginase is more stable in the presence of succinamic acid compared to asparagine.
  9. Keywords:
  10. Directed Evolution ; Kinetic Parameters ; Asparaginase Enzyme ; Organic Reactions ; Recombinant Enzyme ; Substrate Walking ; Promiscuous Reactions

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