Comparative one-factor-at-a-time, response surface (statistical) and bench-scale bioreactor level optimization of thermoalkaline protease production from a psychrotrophic Pseudomonas putida SKG-1 isolate

BACKGROUND: Production of alkaline protease from various bacterial strains using statistical methods is customary now-a-days. The present work is first attempt for the production optimization of a solvent stable thermoalkaline protease by a psychrotrophic Pseudomonas putida isolate using conventional, response surface methods, and fermentor level optimization. RESULTS: The pre-screening medium amended with optimized (w/v) 1.0% glucose, 2.0% gelatin and 0.5% yeast extract, produced 278 U protease ml(-1 )at 72 h incubation. Enzyme production increased to 431 Uml(-1 )when Mg(2+ )(0.01%, w/v) was supplemented. Optimization of physical factors further enhanced protease to 514 Uml(-1 )at pH 9.0, 25°C and 200 rpm within 60 h. The combined effect of conventionally optimized variables (glucose, yeast extract, MgSO(4 )and pH), thereafter predicted by response surface methodology yielded 617 U protease ml(-1 )at glucose 1.25% (w/v), yeast extract 0.5% (w/v), MgSO(4 )0.01% (w/v) and pH 8.8. Bench-scale bioreactor level optimization resulted in enhanced production of 882 U protease ml(-1 )at 0.8 vvm aeration and 150 rpm agitation during only 48 h incubation. CONCLUSIONS: The optimization of fermentation variables using conventional, statistical approaches and aeration/agitation at fermentor level resulted in ~13.5 folds increase (882 Uml(-1)) in protease production compared to un-optimized conditions (65 Uml(-1)). This is the highest level of thermoalkaline protease reported so far by any psychrotrophic bacterium.