초록
<P><B>Background</B></P><P>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 <I>Pseudomonas putida </I>isolate using conventional, response surface methods, and fermentor level optimization.</P><P><B>Results</B></P><P>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<SUP>-1 </SUP>at 72 h incubation. Enzyme production increased to 431 Uml<SUP>-1 </SUP>when Mg<SUP>2+ </SUP>(0.01%, w/v) was supplemented. Optimization of physical factors further enhanced protease to 514 Uml<SUP>-1 </SUP>at pH 9.0, 25°C and 200 rpm within 60 h. The combined effect of conventionally optimized variables (glucose, yeast extract, MgSO<SUB>4 </SUB>and pH), thereafter predicted by response surface methodology yielded 617 U protease ml<SUP>-1 </SUP>at glucose 1.25% (w/v), yeast extract 0.5% (w/v), MgSO<SUB>4 </SUB>0.01% (w/v) and pH 8.8. Bench-scale bioreactor level optimization resulted in enhanced production of 882 U protease ml<SUP>-1 </SUP>at 0.8 vvm aeration and 150 rpm agitation during only 48 h incubation.</P><P><B>Conclusions</B></P><P>The optimization of fermentation variables using conventional, statistical approaches and aeration/agitation at fermentor level resulted in ~13.5 folds increase (882 Uml<SUP>-1</SUP>) in protease production compared to un-optimized conditions (65 Uml<SUP>-1</SUP>). This is the highest level of thermoalkaline protease reported so far by any psychrotrophic bacterium.</P>