Several soil isolates from 1 g of soil sample were isolated and screened for the production of L-asparaginase. Primary screening was performed using rapid plate assay; dye indicator studies were conducted, and phenol red with 0.005% concentration was found to be optimum. The secondary screening was carried out using the Nesslerization method. The bacteria screened for L-asparaginase production with no glutaminase activity was identified as Bacillus subtilis. Crude L-asparaginase enzyme was partially purified 1.57 folds of purity and 110 U/mg of specific activity. The glutaminase-free L-asparaginase activity was also confirmed using LC-MS analysis. The presence of mass peaks at 147.0 in the reaction mixture suggested an absence of glutaminase activity. An optimized medium obtained comprised of Dextrose 1.5 g/L, K2HPO4 1.2 g/L, L-asparagine 15 g/L, and Tryptone 5 g/L. The highest L-asparaginase activity was observed at 6.0 pH and 30 °C. Kinetic parameters associated with biomass and L-asparaginase production were also studied. The computed values were µm 0.104 h-1, Xm 6g/L P0 1.7U/mL Pm 8.2 U/mL YX/S 4 g-cell/g-glucose µPm 0.35 h-1 qp 5.46 U/g/h YP/x 13.6667 U/g-cell. The novel bacterial isolates showed promise as a potential glutaminase-free L-asparaginase producer, which can prove to be of industrial applications.
Keywords: Bacillus subtilis; L-asparaginase; glutaminase; phenol red.