Application of experimental designs to optimize medium composition for production of thermostable lipase/esterase by Geobacillus thermodenitrificans AZ1

Thirty two morphologically different bacterial were isolated from different soil samples and screened for their ability to produce lipolytic enzymes. Among all isolates, the isolate coded AZ1 was selected due to its high potency to produce lipase at elevated temperature up to 65°C. Phylogenetic analysis based on 16SrDNA sequence revealed its close relationship to Geobacillus thermodenitrificans. The effect of ten culture variable on lipase production was evaluated by implementing Plackett–Burman statistical design. d-sucrose, peptone and soy bean flour were the most significant variables affecting lipase production. A pre-optimized medium based on this experiment yielded an enzyme activity of 260Umin−1ml−1. For further optimization, a fourteen trials’ multi-factorial Box–Behnken experimental design was applied to find out the optimum level of each of the significant variables. The tested variables, namely: d-sucrose (X1); peptone (X2) and soy bean flour (X3) were examined, each at three different levels coded −1, 0, +1. The optimal levels of the three components were founded to be (g/L): d-sucrose, 6.56; peptone, 6.35; and soy bean flour, 6.92, with a predicted activity of approximately 610Umin−1ml−1. According to the results of the Plackett–Burman and Box–Behnken designs the following medium composition is expected to be optimum (g/L): d-sucrose 6.56, peptone 6.35, soy bean flour 6.92, CaCl2 0.02, Y.E. 2.5, K2HPO4 1.0, MgSO4.7H2O 0.2 and Fe2 (SO4)3 0.02; pH, 8; cultivation temperature 55°C and incubation time 24h, the enzyme activity measured in the medium was approximately 593Umin−1ml−1