Investigations of a Useful .ALPHA.-Glycosidase for the Enzymatic Synthesis of Rare Sugar Oligosaccharides

Abstract

Numerous substances that contain oligosaccharides in their structures are known to possess various physiological functions in living organisms. In many cases, these oligo-saccharides may contain uncommon monosaccharides, which are typically designated as rare sugars. From a physiological viewpoint, many researchers have attempted the synthesis of these oligosaccharides which contain rare sugars. Methodologies for oligosaccharide synthesis often require complicated processes such as regioselective protection and deprotection of the hydroxyl groups andor separation of the and anomers of the glycosylation products. In recent synthetic studies of these oligosaccha-rides, considerable attention has been given to the use of enzymesspecifically, exotype carbohydrate hydrolases, such as glycosidases, due to their availability. In contrast to chemical reactions, enzymatic reactions are simple, involve mild conditions, and possess precise positional and anomeric selectivity in the formation of the glycosidic linkages. Several types of glycosidases have demonstrated efficient catalytic activities in the synthesis of oligosac-charides using donor glycosides and acceptor sugars via transglycosylation. In general, glycosidases are known to possess strict specificities for the glycon structures of the glycosidic substrates, and accordingly, various types of glycosidases are classified based on these specificities. Among the many and glycosidases that have been isolated from plants, animals, insects, and microorganisms , most show activities towards glycosides that consist of common sugars, which exist in large quantities as components of the biomass. Consequently, the type of oligo-saccharides that can be constructed by the glycosidase catalyzed transglycosylation is rather limited. To employ the glycosidasecatalyzed transglycosylation in the construction of oligosaccharides that consist of rare sugars, we have undertaken the development of glycosidases that show broad or unique glycon specificity by recognizing various types of sugars as substrates. For this purpose, we undertook the following studies: 1) investigations of unknown glycon specificity of the enzymes by using various types of rare sugar containing glycosides as sub-strates, 2) studies in the change of glycon specificity of the enzymes by sitespecific mutagenesis, 3) from microorganisms in nature, a search for new glycosidases that possess unique and desirable glycon specificities. Herein, we report the results of the first two studies. Abstract: Construction of various rare sugar oligosaccharides by glycosidase-catalyzed transglycosylation reaction may require α-glycosidases that possess unique glycon specificity. In order to obtain such α-glycosidase, we carried out two studies to: 1) investigate unknown glycon specificities of several α-glycosidases using various types of rare sugar containing glycosides as substrates, and 2) change the glycon specificities of the α-glucosidase from Geobacillus stearothermophilus by site-specific mutagenesis. Through the former studies, several α-glycosidases were found to possess hydrolytic activities towards specific glycon monodeoxy analogs of p-nitrophenyl (pNP) α-D-glycopyranosides. Using Aspergillus niger α-glucosidase that showed activity towards 2-deoxy glucoside and jack bean α-mannosidase that showed activity towards 6-deoxy mannoside (α-D-rhamnoside), the glycon 2-deoxy derivative of isomaltoside (ethyl 2-deoxy-α-D-arabino-hexopyranosyl-1,6-β-D-thioglucopyranoside) and α-D-rhamnodisaccharide derivative (ethyl α-D-rhamnopyranosyl-1,2-α-D-thiorhamnopyranoside) were prepared by their transglycosylation reaction in good yields. For the latter studies , fifteen mutant enzymes of Geobacillus stearothermophilus α-glucosidase were prepared and their hydro-lytic activities towards the maltose, eight diastereomers of pNP α-D-aldohexopyranoside, and possible monodeoxy-and mono-O-methyl analogs of pNP α-D-gluco,-manno and-galactopyranosides were elucidated. For these mutant enzymes, there were differences between the specificities for pNP α-D-glucopyranoside and those for maltose, while significant changes were not confirmed in the specificity for other pNP α-D-aldohexopyranosides or the partially modified analogs of pNP α-D-glycopyranosides.