Production, purification and kinetic characterization of glutaminase free anti-leukemic L-asparaginase with low endotoxin level from novel soil isolate

Abstract

Anti-leukemic enzyme L-asparaginase despite having significant applicability in medicine, holds side effects attributed to glutaminase activity and endotoxin content. Glutaminase activity proves to be toxic to non-tumor cells as glutamine is an essential amino acid. Endotoxin illicit the production of vasoactive amines and induce septic shock. Hence there is a need for glutaminase free L-asparaginase with minimum endotoxin level. The report aims at the development of a downstream process for purification of glutaminase free L-asparaginase and subsequent endotoxin removal. Producing bacteria were isolated from various soil samples and screened initially for asparaginase and glutaminase activity. The glutaminase free L-asparaginase producing bacteria were identified as Bacillus altitudinis. Production of L-asparaginase was optimized. The optimum medium comprised of comprising Lactose (1.5 g/L), NaCl (1.2 g/L), Yeast extract (5 g/L), L-asparagine (20 g/L) with pH 7.0 and incubation time of 18 h. Kinetic parameters Km and Vmax were computed to be 9.09x10−2M and 0.09 M/S. L-asparaginase Purification was achieved with a specific activity of 800 U/mg of enzyme. Molecular weight of the purified L-asparaginase was determined to be around 35 KDa using SDS-PAGE. The developed process also brought down the endotoxin content below the FDA recommended level. The endotoxin content of the purified enzyme was determined to be 0.015EU/mL.