BACKGROUND: Owing to the importance of glutaminase in biotech product production, its production with isolated Bacillus subtilis RSP-GLU (MTCC 9727) was investigated. Fermentation factors play an important role in product enhancement. Hence, glutaminase production was optimized using an artificial neural network (ANN) coupled genetic algorithm (GA). RESULTS: A '6-12-1' topology ANN was constructed to identify the nonlinear relationship between fermentation factors and enzyme yield. ANN-predicted values were optimized for glutaminase production using a GA. The overall mean absolute predictive error (MAPE) and the mean square errors (MSE) were observed to be 0.00125 and 1.77 and 0.002 and 3.06 for training and testing, respectively. The goodness of neural network prediction (coefficient of R-2) was found to be 0.996. The maximum interactive impact on glutaminase production was noted with rpm versus medium volume. The use of ANN-GA hybrid methodology resulted in a significant improvement (47%) in glutaminase yield. CONCLUSION: Five different optimum fermentation conditions out of 500 revealed maximum enzyme production. Glutaminase enzyme production in this Bacillus subtilis RSP-GLU is strongly influenced by aeration of the fermentation. A hybrid ANN-GA effectively identifies the different fermentation conditions for optimum production of enzyme in a given large set of conditions. (C) 2009 Society of Chemical Industry