Optimization of the M9 medium for production of Penicillin G Acylase (PGA) from recombinant E. coli DH5 alpha carrying a plasmid encoding pac from Bacillus badius was carried out based on response surface methodology (RSM). For the first time M9 medium was used for enhancing the production of PGA from recombinant B. badius pac expressed in E. coli DH5 alpha. RSM is a useful method for studying the effect of several variables influencing the responses by varying them simultaneously and carrying out a limited number of experiments. The initial screening method of Plackett-Burman design gave rise to identification of fructose, yeast extract, and magnesium sulfate as significant medium components. A 2(3) full-factorial central composite design (CCD) was applied to further optimize concentration of each significant variable. The optimal concentration for production of PGA consisted of 10g/l fructose, 6g/l yeast extract, 1.85ml/l MgSO4 center dot 7H(2)O (100mM), 9g/l disodium hydrogen phosphate, 4g/l potassium dihydrogen phosphate, 0.75g/l sodium chloride, 2ml/l thiamine HCl (100mM), and 2ml/l calcium chloride (10mM). The model prediction of PGA activity (14.11U/ml) at optimum conditions was verified experimentally (13.94U/ml). Through statistically designed optimization, the production of PGA was found to go up from 2.2U/ml to an average of 13.94 +/- 0.03U/ml. Thus the modified M9 media has shown a 7.74-fold increase in PGA activity.