α-Glucosidase (EC 3.2.1.20), an exo-glycosylase to hydrolyze α-glucosidic linkage, is characterized by the variety of substrate specificity. Enzyme also catalyzes the transglucosylation, on which industrial interests focus due to the production of valuable glucooligosaccharides. α-Glucosidase is a physiologically important enzyme in most of organisms (microorganisms, insects, plants and animals including human). Therefore, there are many types of α-glucosidases to display unique functions, in which we are interested. This report describes the recently analyzed unique functions of α-glucosidases by mainly focusing on honeybee α-glucosidase isoenzymes, dextran glucosidase, multiple forms of rice α-glucosidases, and Escherichia coli α-xylosidase.

The mechanisms involved in the biosynthesis of six polysaccharides is described in the following order: (1) Introduction to the first purported biosynthesis of polysaccharides, glycogen and starch by phosphorylases; (2) biosynthesis of Salmonella O-antigen polysaccharide; (3) biosynthesis of bacterial cell wall polysaccharide, peptido-murein; (4) biosynthesis of dextran by B-512FMC dextransucrase; (5) biosynthesis of bacterial cellulose and xanthan; (6) biosynthesis of starch in starch granules. The structures of the six polysaccharides are quite diverse. There are four β-linked hetero-polysaccharides (2), (3), and (5), and two α-linked homo-polysaccharides (4) and (6). The first five are biosynthesized by prokaryote bacteria and the sixth polysaccharide (starch) was shown to be biosynthesized by eight different eukaryotic plant sources. All six of the polysaccharides have been shown to be biosynthesized by a common mechanism in which the monomer or repeating unit is added to the reducing-end of a growing polysaccharide chain in a two catalytic-site insertion mechanism. The β-linked polysaccharides are covalently α-linked to a lipid pyrophosphate, bactoprenol pyrophosphate, at the active-site of the synthesizing enzymes; the α-linked polysaccharides are β-linked directly to the synthesizing enzymes. When the monomer or repeating unit is inserted between the growing polysaccharide and the lipid pyrophosphate or the enzyme, the configuration of the linkage of the polysaccharide is inverted, giving the correct stereochemistry for the specific polysaccharide. Eventually, the polysaccharides are released from the active-sites by an acceptor reaction with water or with another carbohydrate.

A β-galactosidase (EC 3.2.1.23, β-D-galactoside galactohydrolase), commonly known as lactase is known to catalyze not only hydrolyze β-D-galactoside linkage of lactose or other β-galactosides into monosaccharides, glucose and galactose but also has transgalactosylation activity to synthesize galacto-oligosaccharides. Both reaction activities are well characterized and applied in many food industries. Although β-galactosidases are widely distributed in nature, the most thoroughly studied β-galactosidases are obtained from E. coli, and commercially used β-galactosidases are from mainly fungi and yeasts. Galacto-oligosaccharides, so-called prebiotics, have been shown to employ this growth-stimulating effect on probiotic bacteria, including bifidobacteria. Biochemical and molecular aspects of the β-galactosidase genes from different microorganisms have been studied, but the known structure and function of different β-galactosidases are limited. β-Galactosidases which belongs to the 4/7 superfamily of the glycoside hydrolase families (GHs) are currently divided into GH-1, GH-2, GH-35 and GH-42, and yet the four families are so distantly related to each other, and the hydrolytic and transgalactosylation activities of the isoenzymes appears to be different. The known 3D structures and functions of β-galactosidases from prokaryotic E. coli (mesophilic), three extremophiles like Thermus thermophilus (thermophilic), Sulfolobus solfataricus (thermophilic), Arthrobacter (psychrotrophic), and eukaryote Penicillium were compared. The sequence, homology and multiple isozymes of lactic bacterial β-galactosidases were supplemented for their phylogenetic analysis. The applications of this enzyme on the oligosaccharides and prebiotics were discussed in details.

α-Amylases occur widely in plants, animals, and microorganisms. They often act in synergy with other related and degradative enzymes and may also be regulated by proteinaceous inhibitors. Open questions exist on how α-amylases interact with polysaccharides. Several enzymes possess secondary carbohydrate binding sites situated on the surface at a certain distance of the active site cleft. The functions of such sites were studied in barley α-amylase isozymes by structure-guided mutational analysis and measurement of activity and binding parameters. Two surface sites were assigned distinct roles. One of the sites seems to participate in hydrolysis of polysaccharides by a multiple attack mechanism. Polysaccharide processing enzymes can also contain carbohydrate binding modules, e.g. starch binding domains that assist in the attack on macromolecular substrates and are useful in engineering of enzyme efficiency. The multidomain nature of these enzymes raises questions on the dynamics and structural properties in solution and in substrate complexes.

The roles of carbohydrates in human health and diseases are raising topics to develop functional foods and therapeutics. However, their complex structures and complicate synthetic chemical routes are drawbacks to understand the relationships between functions and structures of carbohydrates. Enzymatic syntheses using glycosidases and glycosyltransferases have been proposed as alternatives of the chemical synthesis. The enzymatic synthesis of glycosides is one of the oldest scientific topics, but still challenging. This review describes the recent achievements in the field of enzymatic carbohydrate synthesis using wild type and engineered glycosidases. Here we focus the methodology using retaining glycosidases which produce the same stereochemistry outcomes as that of the original substrates. Several retaining glycosidases are very useful not only to add sugar moiety to non-glycosylated natural products, such as flavonoids and ascorbic acid so on, but also to remodeling the structures of sugar parts in glycosylated compounds. Through enzyme engineering the valuable transglycosylation properties of retaining glycosidases have been improved to enhance selective transglycosylation and to increase yields by modulating hydrolysis and transglycosylation activities. The powerful enzymatic tools for the preparation of oligosaccharides are very promising for understanding the functions of carbohydrates, leading to promote human health and prevent diseases.

We found a novel inverting xylanolytic enzyme belonging to GH8, reducing end xylose-releasing exo-oligoxylanase (Rex, EC. 3.2.1.156), that hydrolyzed xylooligosaccharides (X3 or larger) to release X1 at their reducing end. Rex hydrolyzed α-X2F into X2 only in the presence of X1, clearly proving the Hehre-resynthesis hydrolysis mechanism. A library of mutant Rex at the catalytic base (Asp263) was constructed by saturation mutagenesis. Among them, D263C showed the highest level of X3 production, and D263N exhibited the fastest consumption of α-X2F. However, F− releasing activities of the mutants were much less than that of wild type. Next, Y198 of Rex that forms a hydrogen bond with the nucleophilic water was substituted with phenylalanine, causing a drastic decrease in the hydrolytic activity and a small increase in F− releasing activity from α-xylobiosyl fluoride in the presence of xylose. Y198F of Rex accumulates much more product during the glycosynthase reaction than D263C. We here conclude that an inverting glycosidase is effectively converted into glycosynthase by mutating a residue holding the nucleophilic water molecule with the general base residue while keeping the general base residue intact.

The α-amylase family constitutes the clan GH-H consisting of the three glycoside hydrolase (GH) families GH13, GH70 and GH77. The entire α-amylase family counts ~5,000 known amino acid sequences that exhibit almost 30 different enzyme specificities. Besides the other basic characteristics of the family, such as hydrolysis and/or transglycosylation of several α-glucosidic linkages and the employment of the catalytic triad in the enzymatic reaction, the α-amylase family members should contain from four up-to seven conserved sequence regions. These regions may be considered to be the ‘’ of the individual enzyme specificities and/or taxonomic sources. This article is therefore focused on showing the importance of the conserved sequence regions as the sequence fingerprints in special cases of: (i) the GH13 α-amylases from plants and archaeons; (ii) the oligo-1,6-glucosidase and neopullulanase GH13 subfamilies; and (iii) the unique GH77 amylomaltases from borreliae.

We found a new reaction of sucrose phosphorylases; transglycosylation of carboxyl group. Sucrose phosphorylases from two different sources were tested for glycosylation of carboxylic acid compounds. Streptococcus mutans sucrose phosphorylase showed remarkable transglycosylating activity, especially under acidic conditions. Leuconostoc mesenteroides sucrose phosphorylase exhibited very weak transglycosylating activity. When benzoic acid and sucrose were used as an acceptor and a donor molecule, 1-O- benzoyl α-D-glucopyranoside was produced which was identified by 1D- and 2D-NMR analyses of the purified product and its acetylated product. S. mutans sucrose phosphorylase showed broad acceptor-specificity. The sucrose phosphorylase catalyzed transglycosylation to various carboxylic compounds such as short-chain fatty acids, hydroxy acids, dicarboxylic acids, phenolic carboxylic acids, and acetic acid.

Trehalose analogue, non-reducing dissacharide of 1-α-D-glucopyranosyl α-D-galactopyranoside, was synthesized by Pyrococcus horikoshii glycosyltransferase transglycosylation reaction with sugar nucleotides and galactose. This disaccharide analogue was effective inhibitor for several disaccharidases including rat intestinal trehalase and sucrase. Trehalose was also modified by Escherichia coli β-galactosidase transglycosylation reaction with lactose to give trehalose trisaccharide analogues. These trisaccharide analogues have been supposed to be indigestible oligosaccharides exhibiting enhanced hygroscopic, cryoprotective, anti-cariogenic, and prebiotic effects. The enzymatic techniques using glycosyltransferase and glycosidase might lead to create more trehalose-based analogues with a wide variety of acceptor and donor sugars.

The high temperature jet cooking process for starch conversion has made a significant contribution in the commercial production of glucose from starch-based raw materials. However, it is still necessary to reduce manufacturing and energy costs as well as to improve quality of products. A new enzyme technology for production of glucose from granular starch using a granular starch hydrolyzing enzyme (GSHE) system has been developed. This technology replaces the traditional energy intensive process of liquefaction and saccharification. The GSHE technology offers opportunities to establish optimized feedstock for the biochemical and sweetener industries and is expected to eliminate the use of high energy during starch processing thereby providing a more cost effective glucose production process. The application of GSHE in the production of glucose from a variety of starch substrates including corn, wheat and tapioca is presented.