Anil Kumar Meghavarnam
Bangalore University, India
Title: Production, Purification and Anticancer properties of therapeutic enzyme L-asparaginase from Fusarium culmorum ASP-87
Biography
Biography: Anil Kumar Meghavarnam
Abstract
Three hundred and sixty tropical soil fungi were screened for L-asparaginase production by dye based rapid screening method. A species of Fusarium showed appreciable amount of enzyme activity and identified as Fusarium culmorum by 18s rRNA sequencing. Optimization studies under submerged fermentation (SmF) revealed that 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. The enzyme was purified to homogeneity by ion exchange followed by gel filtration chromatography, to 14.03 fold with a specific activity of 16.66 U/mg of protein with 2.6% yield. The molecular mass was 90 kDa. The optimal pH and temperature of purified enzyme was 8.0 and 40°C. The enzyme retained 90% of activity at pH 8 upto72 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. It was 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 different solid substrates were used among which, soya bean meal in combination with wheat bran and 0.1% ammonium chloride 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.