ABSTRACT
A bacterial strain AM7, isolated from soil and identified as Bacillus circulans, produced two kinds of novel cyclic oligosaccharides. The cyclic oligosaccharides were produced from amylose using a culture supernatant of the strain as the enzyme preparation. The major product was a cyclomaltopentaose cyclized by an alpha-(1-->6)-linkage, cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}. The other minor product was cyclomaltohexaose cyclized by an alpha-(1-->6)-linkage, cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}. We propose the names isocyclomaltopentaose (ICG5) and isocyclomaltohexaose (ICG6) for these novel cyclic maltooligosaccharides having one alpha-(1-->6)-linkage. ICG5 was digested by alpha-amylase derived from Aspergillus oryzae, cyclomaltodextrin glucanotransferase (CGTase) from Bacillus stearothermophilus, and maltogenic alpha-amylase. On the other hand, ICG6 was digested by CGTase from B. stearothermophilus and B. circulans, and maltogenic alpha-amylase. This is the first report of enzymatically produced cyclomaltopentaose and cyclomaltohexaose, which have an alpha-(1-->6)-linkage in their molecules.
Subject(s)
Amylose/metabolism , Bacillus/enzymology , Cyclodextrins/chemistry , Cyclodextrins/metabolism , Geobacillus stearothermophilus/enzymology , Oligosaccharides/biosynthesis , Aspergillus oryzae/enzymology , Bacillus/classification , Bacillus/genetics , Bacillus/isolation & purification , Bacillus/metabolism , Carbohydrate Sequence , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Culture Media/pharmacology , Cyclization , Cyclodextrins/isolation & purification , Glucosyltransferases/metabolism , Glycoside Hydrolases/metabolism , Molecular Weight , Nuclear Magnetic Resonance, Biomolecular , Oligosaccharides/chemistry , Oligosaccharides/metabolism , Spectrometry, Mass, Electrospray Ionization , alpha-Amylases/metabolismABSTRACT
Transglycosylation reactions to cyclic tetrasaccharide (CTS, cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]) and its derivatives were investigated. An enzyme, 6-alpha-glucosyltransferase, which is involved in CTS synthesis from starch, from Bacillus globisporus C11 produced 4-O-alpha-glucosyl-CTS (4G-CTS) from a mixture containing CTS and maltopentaose. Another enzyme, 3-alpha-isomaltosyltransferase, synthesized 3-O-alpha-isomaltosyl-CTS (3IM-CTS) from CTS and panose. Two novel branched CTSs, 3-O-alpha-isomaltosyl-4-O-alpha-glucosyl-CTS (3IM-4G-CTS) and 3-O-alpha-isomaltosyl-(4-O-alpha-glucosyl)-CTS [3IM-(4G)-CTS], were synthesized by the isomaltosyl transfer of IMT into 4G-CTS. IMT also produced a novel saccharide, 3-O-alpha-isomaltosyl-3-O-alpha-isomaltosyl-CTS (3IM-3IM-CTS) from 3IM-CTS. It was confirmed that the oligosaccharides, including 4G-CTS, 3IM-CTS, 3IM-4G-CTS, 3IM-(4G)-CTS and 3IM-3IM-CTS, remaining in the reaction mixture during the production of CTS from starch were the transfer products of 6GT and IMT into CTS.
ABSTRACT
Production of cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->] (CTS, cyclic tetrasaccharide) from starch was attempted using 1,6-alpha-glucosyltransferase (6GT) and 1,3-alpha-isomaltosyltransferase (IMT) from Bacillus globisporus C11. The optimal conditions for production from partially hydrolyzed starch were as follows: substrate concentration, 3%; pH 6-7; temperature, 30 degrees C; 6GT, 1 unit/g-dry solid (DS); IMT, 10 units/g-DS. The production of CTS was demonstrated and 544 g of CTS hydrate crystal powders were obtained from 3500 g of partially hydrolyzed starch. Two major by-products were also isolated from the reaction mixture and identified as the branched derivatives of CTSs, 4-O-alpha-D-glucopyranosyl-CTS and 3-O-alpha-isomaltosyl-CTS.
ABSTRACT
Egg white lysozyme was found to catalyze the transfer of N-acetylglucosamine to cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->] (CTS). Structural analysis showed that the transfer product was 3-O-beta-N-acetylglucosaminyl CTS, cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-[beta-GlcNAc-(1-->3)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. This branched saccharide is anticipated to be a model compound of the sugar chains of glycoproteins.
Subject(s)
Muramidase/chemistry , Oligosaccharides/chemical synthesis , Animals , Carbohydrate Sequence , Catalysis , Chick Embryo , Chromatography, Affinity , Chromatography, High Pressure Liquid , Egg White , Glycosylation , Indicators and Reagents , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Molecular Weight , Oligosaccharides/chemistry , Spectrometry, Mass, Fast Atom BombardmentABSTRACT
Cyclomaltodextrin glucanotransferase (EC 2.4.1.19, abbreviated as CGTase) derived from Bacillus stearothermophilus produced a series of transfer products from a mixture of cyclomaltohexaose and cyclic tetrasaccharide (cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->], CTS). Of the transfer products, only two components, saccharides A and D, remained and accumulated after digestion with glucoamylase. The total combined yield of the saccharides reached 63.4% of total sugars, and enzymatic and instrumental analyses revealed the structures of both saccharides. Saccharide A was identified as 4-mono-O-alpha-glucosyl-CTS, [-->6)-[alpha-D-Glcp-(1-->4)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->], and sachharide D was 4,4'-di-O-alpha-glucosyl-CTS, [-->6)-[alpha-D-Glcp-(1-->4)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-[alpha-D-Glcp-(1-->4)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. These structures led us to conclude that the glycosyltransfer catalyzed by CGTase was specific to the C4-OH of the 6-linked glucopyranosyl residues in CTS.