Optimization for Production & Purification of Asparaginase Enzyme

Dashtban Kenari, Laleh | 2009

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  1. Type of Document: M.Sc. Thesis
  2. Language: Farsi
  3. Document No: 40019 (06)
  4. University: Sharif University of Technology
  5. Department: Chemical and Petroleum Engineering
  6. Advisor(s): Alemzadeh, Iran; Maghsodi, Vida
  7. Abstract:
  8. The enzyme L-Asparaginase (L-asparagine amidohydrolase, EC has been thoroughly researched by many researchers worldwide because of its immensely useful medical applications. It can catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia that is effective for the cure of leukemia, especially for puerile Acute Lymphocytic Leukemia [ALL] and Lymphosarcomas. The present study discusses the studies carried out for the optimal production of the enzyme from Escherichia coli ATCC 11303. For extraction of the intracellular enzyme, Sonication is more useful method for cell disruption than alkali lysis and enzymatic method. We improved Luria Bertani (LB) media (tryptone 1% w/v, yeast extract 0.5% w/v and NaCl 1% w/v) to obtain maximum enzyme activity. The results showed that, inoculum age of 18 hours and inoculum size of 10% was found to be the most favorable for optimal enzyme production and lactose, yeast extract and KH2PO4 was the best carbon, nitrogen and ion sources, respectively. We used statistical methods in order to survey the effect of every influential parameter on production and their interactions. Response Surface Methodology helps us optimizing effect of various variables with minimum number of experiments. By this method, values of lactose, tryptone, yeast extract, KH2PO4 and L-asparagine concentration to obtain the maximum enzyme activity were investigated. The results showed that the enzyme had its maximum activity in 1.08% lactose, 1.79% tryptone, 1.6% yeast extract, 2% KH2PO4and 0.19% L-asparagine concentration, which is 1.03 U/ml enzyme. We increase the purity of enzyme by using an ammonium sulfate fractionation procedure. Maximum enzyme activity was in 40% saturation of ammonium sulfate and the purity fraction was 1.36. Experimental results showed that E. coli cells treated with 9.4% (w/v) K2HPO4 and 14% (v/v) Triton X-100 at 25oC for 4 hr released near 70% of enzyme
  9. Keywords:
  10. Escherichia Coli Bacteria ; Response Surface Methodology ; Purification ; Optimization ; L-Asparginase Enzyme

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