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Microb Cell Fact. 2011 Dec 28;10:114. doi: 10.1186/1475-2859-10-114.

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.

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  • 1Center of Excellence, Department of Microbiology, Dr, Ram Manohar Lohia Avadh University, Faizabad-224001, UP, India.



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.


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 Mg2+ (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, MgSO4 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), MgSO4 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.


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.

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