Batch extractive fermentation of alkaline protease using aqueous two-phase system

Abstract

Alkaline proteases are very important industrial enzymes. They are used widely in detergent, cleaning stains and soils containing proteins, food, pharmaceutical, leather and film industries, as well as in waste processing companies. But problems still occurred regarding low production yield, product inhibition, and complexity of recovery system which inevitably result in high production cost. Extractive fermentation using aqueous two-phase system (ATPs), in current study was Polyethylene glycol/ potassium phosphate system, is a promising alternative to the conventional process, since it provides a non-denaturing natural environment for biomolecules, and stabilizes cells. PEG 1000 was not found suitable for B. subtilis TISTR 25 extractive fermentation, in contrast to PEG 4000, 6000 and 10 000. The higher PEG molecular weight, the lower total alkaline protease production obtained. The effect of PEG molecular weight on phase diagram was investigated and the results did not show significant variations. However, that of ATP with PEG1000 was found further off from the origin, in addition, its bottom phase contained measurable amounts of PEG while only trace amounts was found in other PEG systems. The shape of cell was changed from rod type to elongated rod type when fermentations were carried on in ATPs of PEG 4000, 6000 and 10000 with the same compositions of 18 %(w/w) of potassium phosphate and 12% (w/w) of PEG. Next, effects of potassium phosphate, and PEG 4000 concentrations on extractive fermentation were studied. It was discovered that increase in concentrations of PEG 4000 and potassium phosphate caused decreasing alkaline protease activity. The effect of volume ratio on extractive fermentation was also investigated. The results indicated that the higher volume ratio, the lower alkaline protease activity in the top phase. With the lower volume ratio, the shape of cells was found shorter than that in the conventional fermentation. The most suitable system for alkaline protease production by B.subtilis TISTR25 was PEG4000 10.2% (พ/พ), and potassium phosphate 9.36 % (w/w) which gave 4.38, and 56.29 unit/mg for purification factor, and specific alkaline protease in the top phase, respectively.