Fermentation of starch for enhanced alkaline protease production by constructing an alkalophilic Bacillus pumilus strain

Abstract.

A new engineering strain, Bacillus pumilus c172-14 (pBX 96), was obtained by introducing the pBX 96 plasmid, which carries the α-amylase amy gene, into the host strain of alkalophilic Bacillus pumilus c172 via transformation. The newly constructed strain was found to express the amy gene and could use starch instead of glucose or starch hydrolysate as carbon source for its fermentation of alkaline protease. The pBX 96 plasmid in the new host was found to be segregationally and structurally stable. The expression of amy gene did not affect the host strain's resistance to bacteriophages. Moreover, the level of alkaline protease was improved significantly compared with the parent strain. The constructed strain gave a maximum alkaline protease activity of 14,014 U/ml in shaking flask after 48 h cultivation when growing in a medium containing 6% corn meal, 4% soybean flour, 0.4% Na₂HPO₄, 0.03% KH₂PO₄, 0.02% MgCl₂, 0.3% CaCl₂, 0.25% Na₂CO₃, 0.1% glucose, and 20 µg/ml kanamycin (pH 7.0). The optimal pH value and temperature of the alkaline protease were 11.0 and 40°C, respectively. This enzyme was stable over a pH range of 8–11. Its residual activity remained at 100% when treated under a temperature of less than 45°C for 30 min. The corresponding residual activity reduced to 65% of its optimal value at 60°C for 30 min. The alkaline protease was a kind of serine protease, which was demonstrated by the complete inactivation by PMSF (1 mM). This newly constructed strain will be useful in the alkaline protease industry.