Exploration of soil and marine sources for Microbes producing Asparaginase

Abstract

A systematic study to explore the soil and marine sources for L-asparaginase producing eukaryotes was carried out. The study included identification of the isolate, optimization of process parameters; purification and characterization of the enzyme and evaluation of in vitro anti-leukemic activity. In screening 79 isolates were found to produce L-asparaginase. Two isolates with good extracellular asparaginase activity were selected for developing as an alternative resource to bacterial L-asparaginase. Optimization was carried out by classical and statistical methods. Plackett Burman design was used for preliminary screening of factors and Response Surface Methodology was employed for optimizing their levels. The production of enzyme was regulated by nitrogen sources and asparagine. Enzyme from the best isolate was subjected to purification, characterization and in vitro studies. Purification steps included ammonium sulfate precipitation followed by separation using Ion exchange and gel filtration chromatography. A purity fold 86.75 was achieved. The molecular weight of the purified enzyme by SDS-PAGE technique was found to be 35.26 kDa. The purified sample had optimum activity at pH 8.0 and 40°C. The purified sample was stable at pH range of 7-11.0 and temperature ranging from 20 to 50°C. The enzyme was inhibited by serine protease inhibitors, cysteine protease inhibitors and ferrous ions. The relative activity of the enzyme using L-glutamine as substrate was found to be 7%. The Km and Vmax of the enzyme was found to be 277.78 µM substrate and 40.39 µM/min, which indicate the better substrate specificity. The sample was found to have IC50 (77.42IU/mL) against MOLT-4. The isolates were identified as Aspergillus terreus NCIM 1357 and Aspergillus cervinus NCIM 1356 by ARI, Pune, India and were submitted to NCIM. The above results were encouraging and worth pursuing for further development of the fungal isolates as an alternative resource for therapeutic L-asparaginase.