RESUMO
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.
Assuntos
Amilose/metabolismo , Bacillus/enzimologia , Ciclodextrinas/química , Ciclodextrinas/metabolismo , Geobacillus stearothermophilus/enzimologia , Oligossacarídeos/biossíntese , Aspergillus oryzae/enzimologia , Bacillus/classificação , Bacillus/genética , Bacillus/isolamento & purificação , Bacillus/metabolismo , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Meios de Cultura/farmacologia , Ciclização , Ciclodextrinas/isolamento & purificação , Glucosiltransferases/metabolismo , Glicosídeo Hidrolases/metabolismo , Peso Molecular , Ressonância Magnética Nuclear Biomolecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Espectrometria de Massas por Ionização por Electrospray , alfa-Amilases/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Geobacillus stearothermophilus/enzimologia , Glucosiltransferases/química , Glicosídeos/química , Oligossacarídeos/química , Polissacarídeos/química , Aspergillus niger/enzimologia , Bacillus/enzimologia , Catálise , Cromatografia Líquida de Alta Pressão , Glucana 1,4-alfa-Glucosidase/metabolismo , Glicosilação , Hidrólise , Espectrometria de Massas , Metilação , alfa-Glucosidases/metabolismo , beta-Amilase/metabolismoRESUMO
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.
Assuntos
Muramidase/química , Oligossacarídeos/síntese química , Animais , Sequência de Carboidratos , Catálise , Embrião de Galinha , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Clara de Ovo , Glicosilação , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Peso Molecular , Oligossacarídeos/química , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
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.