Alpha-amylase production in aqueous two-phase systems by Bacillus subtilis

The production of a-amylase by Bacillus subtilis was studied in polyethylene glycol (PEG 10 000)/dextran (505 000) aqueous two-phase systems at various concentrations. An increase in the PEG concentration from 7 to 25% (w/w) in the aqueous two-phase system resulted in an increase in the phase volume ratio with a concomitant decrease in the partition coefficient (K) and recovery of a-amylase in the top phase. However, varying dextran concentrations from 2.5 to 10% (w/w) decreased both the phase volume ratio and the partition coefficient of a-amylase. At dextran concentrations lower than 2.5% (w/w) the phases could not separate. The purification ratio was found to increase with increasing PEG concentration up to 9% (w/w), while it was slightly decreased at higher concentrations. The PEG 9% (w/w) and dextran 2.5% (w/w) system was found to be optimum for cultivation of Bacillus subtilis, where more than 95% of the produced a-amylase was selectively partitioned to the upper phase giving a purification factor of 2.3. In this system the a-amylase activity in the top phase, which reached 93 U/ml after 48 h of cultivation, was 1.2 times higher in comparison to the homogeneous medium. It was observed that Bacillus subtilis secreted 84% of the total a-amylase produced within 24 h, while the respective time exceeded 33 h in the homogeneous medium. The bacterial cells were microscopically observed to partition totally to the bottom phase in the system used. The dextran-rich bottom’s phase affinity for cells was exploited in the development of a semicontinuous cell recycle system, in which the bottom phase was reused and the cell-free top phase was withdrawn. In the first two uses a-amylase activity in the top phase exceeded 90 U/ml, while in the third use it was reduced to 84 U/ml. Concluding the use of the PEG 9% (w/w)/dextran 2.5% (w/w) aqueous two-phase system enabled the recirculation of the bacterial cells and the reduction of the required time of cultivation, thus contributing significantly to the reduction of the cost of the fermentation.