At present the α-amylase enzyme family covers 27 different enzyme specificities from hydrolases (EC 3), transferases (EC 2) and isomerases (EC 5). They belong to the three glycoside hydrolase Families 13, 70 and 77, forming a clan GH-H in the frame of the sequence-based classification of glycosidases. There is also a glycoside hydrolase Family 57 containing some α-amylases and related enzymes especially from hyperextremophiles, which however, have sequences, according to present knowledge, unrelated to those from the main Family 13. The proteins without catalytic function that exhibit clear sequence similarities to the enzymes from the α-amylase Family 13 are the main subject of this article. They are the mammalian proteins inducing transport of dibasic and neutral amino acids across cell membranes and the mammalian 4F2 heavy-chain cell surface antigens, i.e., proteins without any functional relatedness to amylolytic enzymes. Nevertheless, from the structural and evolutionary points of view, these proteins may form with the enzymes common to the α-amylase protein family. Analogous examples from other protein families (e.g. chitinases, aldo-keto reductases) are also provided.
α-Glucosidase contributing starch and glycogen metabolisms in plant and animal tissue is characterized by the variety in substrate recognition. Recent studies showed that α-glucosidases could be divided into two groups, family I and family II, in which family I enzymes belong to the α-amylase family. This paper focusing on the difference in the α-glucosidase families reviews the structural information including the catalytic amino acid residues of nucleophile and acid/base catalyst, the recognition of substrate molecule, and the intermediate in the transition state of hydrolytic reaction.
α-1, 4-Glucan lyase (EC 18.104.22.168) produces 1, 5-anhydro D-fructose from α-1, 4-linked glucose oligomers and polymers, such as maltose, maltosaccharides, starch and glycogen. It is a single polypetide enzyme with a molecular mass around 120 kDa and a pH optimum of pH 4.0-6.5. The lyase is highly specific toward α-1, 4-glucosidic bonds and shows negligible activity toward α-1, 6-glucosidic bonds. It is an exo-acting enzyme and starts its cleavage from the non-reducing end, converting the glucose units to 1, 5-anhydrofructose until the last glucose unit on the reducing end is reached or a branching point is met. Thus with maltose as substrate glucose and 1, 5-anhydrofructose are formed in an equimolar basis, while with amylopectin or glycogen as the substrate it generates 1, 5-anhydrofructose and a limit dextrin that is rich in α-1, 6-branches. At present the lyase has been purified and cloned from fungi and algae. The occurrence of 1, 5-anhydrofructcse and its further metabolite 1, 5-anhydro-D-glucitol in E. coli and mammals may indicate the operation of this alternative glycogen degradation pathway in these organisms.
Calcium independent and acid stable α-amylases for starch liquefaction were developed by protein engineering. Termamyl LCTM obtained by site-directed mutagenesis showed high calcium independence, and its performance in the absence of calcium is equal to the one with TermamylTM in the presence of 40ppm of calcium. Termamyl LCTM was further developed by random mutagenesis, and highly improved variants have been efficiently produced by recent protein engineering technologies. The development of detergent α-amylase began with microbial screening, and two α-amylases active and stable in alkaline conditions were identified. Those amylases were further developed by protein engineering (site-directed mutagenesis), resulting in variants with improved alkaline stability and calcium independence.
Saccharide is indispensable for human life. For the purpose of securing enough of it, scientists have been studying carbohydrate-active enzymes for a long time and have stored up a huge amount of information on enzymes. Many studies also have been performed on carbohydrate-active enzymes from a viewpoint of industrial use. α-Amylase and cyclodextrin glucanotransferase (CGTase), that are widely used in the saccharifying industry, are interesting materials for basic research in improving the function of enzymes. In this review, we present one of the successful experiments of introducing the raw starch-binding and -digesting ability of CGTase to an α-amylase which is widely used as an industrial enzyme. We also discuss at domain level the relationship between structure and function of the chimeric enzymes.