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Extracellular L-Asparaginase Production in Candida Utilis: Production and Activity Determination Conditions

Mahdinia, Ehsan | 2013

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  1. Type of Document: M.Sc. Thesis
  2. Language: Farsi
  3. Document No: 44872 (06)
  4. University: Sharif University of Technology
  5. Department: Chemical and Petroleum Engineering
  6. Advisor(s): Vosoughi, Manouchehr; Alemzadeh, Iran
  7. Abstract:
  8. L-Asparaginase has two major uses. For decades it has been known and used as an antitumor agent. Besides, it has been introduced as a food processing aid in the last trench against acrylamide formation in foods. Here, we have produced extracellular L-Asparaginase from Candida utilis and studied the parameters surrounding its production. Thus, two species of yeast were cross-examined: Saccharomyces cerevisiae (BBRC 30006) and Candida utilis (ATCC 9950). Moreover, the most suitable method for detecting and determining L-Asparaginase activity was investigated and selected for this purpose which is via L-Aspartate determination with Hydroxylamine. Furthermore, six deferent parameters of Thermodynamics, Temperature, Carbon Source, Kinetics, pH and Agitation, affecting the process of activity detection and determination were studied and the best conditions were determined: Temperature of 40°C, Carbone source of Fructose, pHs above 5/5. It was observed that the substrate is spontaneously hydrolyzed in solution and the rate at which this happens is vigorously increased with temperature. It was also seen that the reaction of L-Aspartic Acid with Hydroxylamine is time consuming, independent of L-Aspartate concentration and is fiercely interrupted by pHs below 5/5. Extracellular periplasmic L-Asparaginase was successfully produced in the Candida utilis strain. The enzyme was successfully extracted and its level of activity was determined. Effect of seven different parameters (best conditions) of Incubation Time (16 hours), Carbon Source (Fructose), Buffer (Phosphate pH=7/0), Nitrogen Source (YNB ), Inoculation Ratio (0/5%), Stability during Refrigeration (Up to 120 hours) and Extracting Agent (2-Mercaptoethanol) on the enzyme production were studied. The highest level of enzyme activity was observed at 40°C equal to 400U/mL
  9. Keywords:
  10. Extracellular L-Asparaginase ; Acryle Amide ; Candida Utilis Yeast ; Fad Processing Aid ; Extracellular Enzyme ; Anticancer Drugs

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