Engineering of barley α-amylase

Protein engineering of barley alpha-amylase addressed the roles of Ca2+ in activity and inhibition by barley alpha-amylase/subtilisin inhibitor (BASI), multiple attack in polysaccharide hydrolysis, secondary starch binding sites, and BASI hot spots in AMY2 recognition. AMY1/AMY2 isozyme chimeras facilitated assignment of function to specific regions of the structure. An AMY1 fusion with a starch binding domain and AMY1 mutants in the substrate binding cleft gave degree of multiple attack of 0.9-3.3, compared to 1.9 for wild-type. About 40% of the secondary attacks, succeeding the initial endo-attack, produced DP5-10 maltooligosaccharides in similar proportion for all enzyme variants, whereas shorter products, comprising about 25%, varied depending on the mutation. Secondary binding sites were important in both multiple attack and starch granule hydrolysis. Surface plasmon resonance and inhibition analyses indicated the importance of fully hydrated Ca2+ at the AMY2/BASI interface to strengthen the complex. Engineering of intermolecular contacts in BASI modulated the affinity for AMY2 and the target enzyme specificity.