Comparison of Free and Immobilized L-asparaginase Synthesized by Gamma-Irradiated Penicillium cyclopium

• Published in: Polish journal of microbiology 2016-01, Vol.65 (1), p.43-50
• Main Authors: El-Refai, Heba A, Shafei, Mona S, Mostafa, Hanan, El-Refai, Abdel-Monem H, Araby, Eman M, El-Beih, Fawkia M, Easa, Saadia M, Gomaa, Sanaa K
• Format: Article
• Language: English
• Subjects: Acetone; Amberlite (trademark); Ammonia; Asparaginase; Asparaginase - genetics; Asparaginase - metabolism; Cancer therapies; Cellulose; Enzymatic activity; Enzyme activity; Enzymes; Enzymes, Immobilized - metabolism; Free form; Fungi; Gamma irradiation; Gene Expression Regulation, Enzymologic - radiation effects; Gene Expression Regulation, Fungal - radiation effects; High temperature; Hydrogen-Ion Concentration; Immobilization; Immobilized enzymes; Irradiation; Kinetics; L-asparaginase; Leukemia; Metals; Mutation; Penicillium; Penicillium - enzymology; Penicillium - radiation effects; pH effects; Polyvinyl alcohol; Precipitation (Meteorology); Proteins; Radiation dosage; Thermal stability
• ISSN: 1733-1331; 2544-4646
• DOI: 10.5604/17331331.1197274

Gamma irradiation is used on Penicillium cyclopium in order to obtain mutant cells of high L-asparaginase productivity. Using gamma irradiation dose of 4 KGy, P. cyclopium cells yielded L-asparaginase with extracellular enzyme activity of 210.8 ± 3 U/ml, and specific activity of 752.5 ± 1.5 U/mg protein, which are 1.75 and 1.53 times, respectively, the activity of the wild strain. The enzyme was partially purified by 40-60% acetone precipitation. L-asparaginase was immobilized onto Amberlite IR-120 by ionic binding. Both free and immobilized enzymes exhibited maximum activity at pH 8 and 40 degrees C. The immobilization process improved the enzyme thermal stability significantly. The immobilized enzyme remained 100% active at temperatures up to 60 degrees C, while the free asparaginase was less tolerant to high temperatures. The immobilized enzyme was more stable at pH 9.0 for 50 min, retaining 70% of its relative activity. The maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) of the free form were significantly changed after immobilization. The K(m) value for immobilized L-asparaginase was about 1.3 times higher than that of free enzyme. The ions K+, Ba2+ and Na+ showed stimulatory effect on enzyme activity with percentages of 110%, 109% and 106% respectively.