The determinants of alpha-amylase pH-activity profiles

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 ran ge of very diverse conditions. Until recently, tailoring glycosyl hydrolase s for specific industrial processes mainly involved stability engineering, but lately there has also been considerable interest in engineering their p H-activity profiles. We mutated four neutral residues (N190, F290, N326 and Q360) in the chimeric Bacillus Ba2 alpha -amylase to both charged and neut ral amino acids. The results show that the pH-activity profile of the Ba2 a lpha -amylase can be changed by inserting charged residues close to the act ive site. The changes in the pH-activity profile for these neutral --> char ged mutations do not, however, correlate with the predictions from calculat ions of the pK(a) values of the active site residues. More surprisingly, th e neutral --> neutral mutations change the pH-activity profile as much as t he neutral --> charged mutations. From these results, it is concluded that factors other than electrostatics, presumably the dynamic aspects of the ac tive site, are important for the shape of the pH-activity profiles of the a lpha -amylases.