We studied the crucial components which elevate the expression of recombinant novel glutaminase free Lasparaginase II (rL-asp II) from Bacillus subtilis WB800N. The Plackett-Burman tool identified sucrose, NH4Cl, NaH2- PO4 and MgSO4 as the significant influencing factors (p<0.05). Further investigations showed that artificial neural network- genetic algorithm (ANN-GA) was more effective than central composite design (CCD) in optimizing the influencing factors. The maximum rL-asp II expression was found to be 389.56 IU/ml and 525.98 IU/ml using CCD (R2=90.4%) and ANN-GA (R2=96.2%), respectively. The validation experiments were carried out in a 3 L batch bioreactor where kinetic modelling of the obtained data was done. The rL-asp II expressed effectively inhibiting the polyacrylamide formation in vitro where no solidification was observed, when 2ml of purified rL-asp II used even after 60 min of incubation. This is the first study to report highest production of rL-asp II in B. subtilis WB800N (525.98 IU/ ml) till date by combining statistical designs with consecutive intermittent addition of IPTG in batch reactor.