ABSTRACT
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.
Subject(s)
Asparaginase/metabolism , Gene Expression Regulation, Enzymologic/radiation effects , Gene Expression Regulation, Fungal/radiation effects , Penicillium/enzymology , Penicillium/radiation effects , Asparaginase/genetics , Enzymes, Immobilized/metabolism , Hydrogen-Ion Concentration , Kinetics , Metals , MutationABSTRACT
The formation of cyclosporin A (Cy A) by immobilized spores and mycelia of Aspergillus terreus was investigated. Different carriers were tested as immobilizing carriers, whereby Ca-alginate was selected for further experimentation. The role of alginate concentration, biomass weight, pH value of the cultivation medium, repeated utilization of the immobilized fungus as well as the supplementation of different amino acid precursors were studied. Best Cy A outputs were attained with Ca-alginate 3% (w/v), mycelial weight 15% (w/v), pH 4.5 and four repeated cycles. Similarly, the Cy A productivity was markedly accelerated in the presence of L-valine and L-valine and L-leucine mixture.
Subject(s)
Aspergillus/growth & development , Cyclosporine/metabolism , Immunosuppressive Agents/metabolism , Industrial Microbiology/methods , Alginates , Cells, Immobilized , Culture Media , Fermentation , Glucuronic Acid , Hexuronic Acids , Hydrogen-Ion Concentration , Silica Gel , Silicon Dioxide , ValineABSTRACT
A local isolate of Aspergillus terreus was selected among different microorganisms as a new cyclosporin A (Cy A) producing culture. The formation of Cy A was investigated under different fermentation conditions (including selection of the cultivation medium, fermentation time course, inoculum nature, medium volume, agitation rate, pH value). Relatively high Cy A productivities were maintained when the fermentation process was carried out using a medium composed of (g/L): glucose, 50; bactopeptone, 10; KH(2)PO(4), 5; KCl, 2.5; pH 5.3, inoculated with 2% standard inoculum of 48 h age, shaken at 200 rpm for 10 days.
