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Most starch hydrolases and related enzymes belong to the e-amylase family which contains a characteristic catalytic (fi/e)8-barrel domain. Currently known primary structures that have sequence similarities represent 18 different specificities, including starch branching enzyme. Crystal structures have been reported in three of these enzyme classes: the e-amylases, the cyclodextrin glucanotransferases, and the oligo-l,6-glucosidases. Throughout the e-amylase family, only eight amino acid residues are invariant, seven at the active site and a glycine in a short turn. However, comparison of three-dimensional models with a multiple sequence alignment suggests that the diversity in specificity arises by variation in substrate binding at the fl--+ e loops. Designed mutations thus have enhanced transferase activity and altered the oligosaccharide product patterns of e-amylases, changed the distribution of e-,/3-and 7-cyclodextrin production by cyclodextrin glucanotransferases, and shifted the relative e-1,4: e-1,6 dual-bond specificity of neopullulanase. Barley e-amylase isozyme hybrids and Bacillus e-amylases demonstrate the impact of a small domain B protruding from the (/3/e)8-scaffold on the function and stability. Prospects for rational engineering in this family include important members of plant origin, such as e-amylase, starch branching and debranching enzymes, and amylomaltase.Abbreviations: CGTase, cyclodextrin glucanotransferase; SBD, starch binding domain; TAA, takaamylase A; TIM, triose-phosphate isomerase. The mutations are described with the one-letter code, i.e.

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α-Amylase (EC 3.2.1.1) represents the best known amylolytic enzyme. It catalyzes the hydrolysis of α-1,4-glucosidic bonds in starch and related α-glucans. In general, the α-amylase is an enzyme with a broad substrate preference and product specificity. In the sequence-based classification system of all carbohydrate-active enzymes, it is one of the most frequently occurring glycoside hydrolases (GH). α-Amylase is the main representative of family GH13, but it is probably also present in the families GH57 and GH119, and possibly even in GH126. Family GH13, known generally as the main α-amylase family, forms clan GH-H together with families GH70 and GH77 that, however, contain no α-amylase. Within the family GH13, the α-amylase specificity is currently present in several subfamilies, such as GH13_1, 5, 6, 7, 15, 24, 27, 28, 36, 37, and, possibly in a few more that are not yet defined. The α-amylases classified in family GH13 employ a reaction mechanism giving retention of configuration, share 4-7 conserved sequence regions (CSRs) and catalytic machinery, and adopt the (β/α)8-barrel catalytic domain. Although the family GH57 α-amylases also employ the retaining reaction mechanism, they possess their own five CSRs and catalytic machinery, and adopt a (β/α)7-barrel fold. These family GH57 attributes are likely to be characteristic of α-amylases from the family GH119, too. With regard to family GH126, confirmation of the unambiguous presence of the α-amylase specificity may need more biochemical investigation because of an obvious, but unexpected, homology with inverting β-glucan-active hydrolases.

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