The determinants of α-amylase pH-activity profiles

Abstract

The glycosyl hydrolases present a large family of enzymes that are of great significance for industry. Consequently, there is considerable interest in engineering the enzymes in this family for optimal performance under a range of very diverse conditions. Until recently, tailoring glycosyl hydrolases for specific industrial processes mainly involved stability engineering, but lately there has also been considerable interest in engineering their pH-activity profiles. We mutated four neutral residues (N190, F290, N326 and Q360) in the chimeric Bacillus Ba2 α-amylase to both charged and neutral amino acids. The results show that the pH-activity profile of the Ba2 α-amylase can be changed by inserting charged residues close to the active site. The changes in the pH-activity profile for these neutral → charged mutations do not, however, correlate with the predictions from calculations of the pΚa values of the active site residues. More surprisingly, the neutral → neutral mutations change the pH-activity profile as much as the neutral → charged mutations. From these results, it is concluded that factors other than electrostatics, presumably the dynamic aspects of the active site, are important for the shape of the pH-activity profiles of the α-amylases.