Savitha Janakiraman
Bangalore University, India
Biography
Three hundred and sixty soil fungi were screened for L-asparaginase production by rapid screening method. Fusarium culmorum showed appreciable amount of enzyme activity. Optimization studies under submerged fermentation (SmF) revealed that the production of enzyme was maximum at day 4, pH 7.5, temperature 30°C and at 1% substrate concentration. Addition of 0.2% citric acid, 0.5% ammonium chloride, 0.002% calcium chloride enhanced the production by 6 fold. L-asparaginase was purified by ammonium sulphate precipitation, gel filtration and ion exchange chromatography. Fourteen fold increase with specific activity of 16.66 U/mg protein with 2.6% yield. The molecular weight of the enzyme was estimated to be 90 kDa. The optimal pH and temperature of purified enzyme was 8.0 and 40°C. The enzyme retained 90% activity at pH 8 after 72 hrs and 50% activity at 60°C for 60 min. The Km and Vmax of purified enzyme was 3.57mM and 0.5μmol/ml/min at 37°C respectively, activated by Mn2+ and Tween 80, inhibited by Cu2+ and EDTA. Production of L-asparaginase was also carried out under solid state fermentation (SSF). Sixty five substrates were used, soya bean meal enhanced the production by 10 fold. Soya bean meal in combination with wheat bran and 0.1% ammonium chloride further enhanced the production by 14 fold. The purified L-asparaginase showed cytotoxic effect on human leukemic cell line (Jurkat) with IC50 value of 90μg/ml. The enzyme did not elicit any immunogenic effects on human lymphocytes. The enzyme induced apoptotic cell death by arresting the growth of cells at G2-M phase.
Abstract
Abstract : Studies on L-Asparaginase from Fusarium culmorum for commercial exploitation