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 p K_a 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.