Isolation and characterization of two positional isomers of novel heterogeneous branched cyclomaltohexaoses (alpha-cyclodextrins) having a D-galactobiosyl residue on the side chain.

Transgalactosylated products, 2-O-alpha-D-galactobiosyl-cyclomaltohexaoses (alpha-cyclodextrins, alphaCDs), were synthesized by alpha-galactosidase from coffee bean using melibiose and alphaCD as a donor substrate and an acceptor, respectively. Two positional isomers of 2-O-alpha-galactobiosyl-alphaCDs were isolated and purified by HPLC, and their structures were elucidated by FABMS and NMR spectroscopies, as well as by an enzymatic degradation method. The chromatographic behavior of these novel galactosylated alphaCDs was compared on three HPLC columns with different separation modes.

Enzymatic Synthesis of Mannosyl-cyclodextrin by α-Mannosidase from Jack Bean.

Mannosylated derivatives of cyclodextrins (CDs), mannosyl-α, ß, and γCD were synthesized from a mixture of mannose and α, ß, and γCD by the reverse action of α-mannosidase from jack bean, respectively. Their structures were analyzed by FAB-MS and 13C-NMR spectroscopies, and they were identified as 6-O-α-d-mannosyl-α, ß, and γCD. The optimum conditions for the production of 6-O-α-d-mannosyl-αCD by α-mannosidase were examined. Optimum pH and temperature were pH 4.5 and 60°C, respectively. Yield of mannosyl-αCD increased with increasing mannose concentration and reached more than 35% (mol/mol) at the concentration of 2 m mannose and 0.4 m αCD.

Synthesis of glycosyl glycerol by cyclodextrin glucanotransferases.

Glycerol was transglycosylated by cyclodextrin glucanotransferases using starch as a donor substrate. Among the enzymes tested, those from Geobacillus stearothermophilus and Thermoanaerobacter sp. were suitable for the transglycosylation. Several products were isolated and their structures were elucidated. They were composed of glucose and a series of a-1,4-linked maltooligosyl residues bound with glycerol. O-alpha-D-Glucosyl-(1-->1)-glycerol and O-alpha-D-glucosyl-(1-->2)-glycerol were identified as the major and minor components of the smallest transfer products, respectively. O-alpha-D-Glucosyl-(1-->4)-O-alpha-D-glucosyl-(1-->1)-glycerol was also identified as a main dimer product. Reducing sugars were produced in extremely low amounts. The optimum temperatures for the transglycosylation by G. stearothermophilus and Thermoanaerobacter enzymes were approximately 60 degrees C and 80 degrees C, respectively. The reaction of 30% (w/v) glycerol and 20% (w/v) soluble starch was optimum for efficient transglycosylation. Maltosyl and maltotriosyl glycerols inhibited porcine pancreas a-amylase significantly, whereas the monomer, glucosyl glycerol, exhibited much weaker inhibition.

Preparation of branched cyclomaltoheptaose with 3-O-α-L-fucopyranosyl-α-D-mannopyranose and changes in fucosylation of HCT116 cells treated with the fucose-modified cyclomaltoheptaose.

From a mixture of 4-nitrophenyl α-L-fucopyranoside and D-mannopyranose, 3-O-α-L-fucopyranosyl-D-mannopyranose was synthesised through the transferring action of α-fucosidase (Sumizyme PHY). 6(I),6(IV)-Di-O-(3-O-α-L-fucopyranosyl-α-D-mannopyranosyl)-cyclomaltoheptaose {8, 6(I),6(IV)-di-O-[α-L-Fuc-(1→3)-α-D-Man]-ßCD} was chemically synthesised using the trichloroacetimidate method. The structures were confirmed by MS and NMR spectroscopy. A cell-based assay using the fucosyl ßCD derivatives, including the newly synthesised 8, showed that derivatives with two branches of the α-L-Fuc or α-L-Fuc-(1→3)-α-D-Man residues possessed slight growth-promoting effects and lower toxicity in HCT116 cells compared to those with one branch. These compounds may be useful as drug carriers in targeted drug delivery systems.

Reverse reaction of Aspergillus niger APC-9319 alpha-galactosidase in a supersaturated substrate solution: production of alpha-linked galactooligosaccharide (alpha-GOS).

The alpha-galactosidase that effectively catalyzes a reverse reaction of galactose, Aspergillus niger APC-9319 alpha-galactosidase, was screened from industrial enzyme preparations for food processing containing alpha-galactosidase activity. Reverse reaction of A. niger APC-9319 alpha-galactosidase was performed using a supersaturated solution (90% galactose [w/v]). A. niger APC-9319 alpha-galactosidase was not inhibited even in high substrate concentration, and effectively catalyzed the reverse reaction. The yield of the reaction product, alpha-linked galactooligosaccharide (alpha-GOS), increased greatly as the initial concentration of galactose increased to 90% (w/v), and was more than 50%. Furthermore, the half life of enzyme activity was about three times as long as that using 60% galactose (w/v). alpha-GOS (1.4 g) was prepared from galactose (3.0 g) by reverse reaction of A. niger APC-9319 alpha-galactosidase. The alpha-GOS contained 58% alpha-galactobiose (alpha-Gal2), 28% alpha-galactotriose, and 14% oligosaccharides larger than alpha-galactotriose. The main component of positional isomers in alpha-Gal2 was alpha-1,6Gal2.

Synthesis of novel heterobranched beta-cyclodextrins having beta-D-N-acetylglucosaminyl-maltotriose on the side chain.

From a mixture of N-acetylglucosaminyl-beta-cyclodextrin (GlcNAc-betaCD) and lactose, beta-D-galactosyl-GlcNAc-betaCD (Gal-GlcNAc-betaCD) was synthesized by the transfer action of beta-galactosidase. GlcNAc-maltotriose (Glc3) and Gal-GlcNAc-Glc3 were produced with hydrolysis of GlcNAc-betaCD by cyclodextrin glycosyltransferase, and Gal-GlcNAc-betaCD by bacterial saccharifying alpha-amylase respectively. Finally, GlcNAc-Glc3-betaCD and Gal-GlcNAc-Glc3-betaCD were synthesized in 5.2% and 3.5% yield when Klebsiella pneumoniae pullulanase was incubated with the mixture of GlcNAc-Glc(3) and betaCD, or Gal-GlcNAc-Glc3 and betaCD respectively. The structures of GlcNAc-Glc3-betaCD and Gal-GlcNAc-Glc3-betaCD were analyzed by FAB-MS and NMR spectroscopy and identified as 6-O-alpha-(6(3)-O-beta-D-N-acetylglucosaminyl-maltotriosyl)-betaCD, and 6-O-alpha-(4-O-beta-D-galactopyranosyl-6(3)-O-beta-D-N-acetylglucosaminyl-maltotriosyl)-betaCD respectively.

Allergic airway eosinophilia is suppressed in ovalbumin-sensitized Brown Norway rats fed raffinose and alpha-linked galactooligosaccharide.

We recently found that dietary raffinose suppressed allergic airway eosinophilia in ovalbumin-sensitized Brown Norway rats. Using this model in the present study, we compared the efficacy of other oligosaccharides with that of raffinose. Brown Norway rats were immunized s.c. with ovalbumin on d 0 and exposed to aerosolized ovalbumin on d 20; broncho-alveolar lavage fluid was obtained on d 21. In Expt. 1, rats were fed a control diet or diets supplemented with different oligosaccharides (50 g/kg diet, raffinose, alpha-linked galactooligosaccharide, fructooligosaccharide, and xylooligosaccharide). The number of eosinophils in the fluid was significantly lower in rats fed raffinose and alpha-linked galactooligosaccharide diets than in those fed the control diet. Dietary fructooligosaccharide and xylooligosaccharide did not affect airway eosinophilia. In Expt. 2, i.p. administration of raffinose and alpha-linked galactooligosaccharide, but not fructooligosaccharide and xylooligosaccharide, suppressed airway eosinophilia in rats fed the control diet. In Expt. 3, suppression of airway eosinophilia by dietary alpha-linked galactooligosaccharide occurred in cecectomized rats administered neomycin. Reduced levels of interleukin (IL)-4 and IL-5 mRNA in lung tissue were associated with the suppression of airway eosinophilia. We propose that indigestible oligosaccharides differ in their suppressive effect on allergic airway eosinophilia in ovalbumin-sensitized Brown Norway rats and that the effect appears not to be mediated by intestinal microflora.

Purification and characterization of a new type of alpha-glucosidase from Paecilomyces lilacinus that has transglucosylation activity to produce alpha-1,3- and alpha-1,2-linked oligosaccharides.

A fungus producing an alpha-glucosidase that synthesizes alpha-1,3- and alpha-1,2-linked glucooligosaccharides by transglucosylation was isolated and identified as Paecilomyces lilacinus. The cell-bound enzyme responsible for the synthesis was extracted by suspension of mycelia with 0.1 M phosphate buffer (pH 8.0), and the extract was purified. The molecular weight and the isoelectric point were estimated to be 54,000 and 9.1, respectively. The enzyme was most active at pH 5.0 and 65 degres C. The enzyme hydrolyzed maltose, nigerose, and kojibiose. The enzyme also hydrolyzed soluble starch and amylose with the rate toward maltose. p-Nitro-phenyl alpha-glucoside and isomaltose were not good substrates. The enzyme had high transglucosylation activity to synthesize oligosaccharides containing alpha-1,3- and alpha-1,2-linkages. At an early stage of the reaction, considerable maltotriose, 4-O-alpha-nigerosyl-D-glucose, and 4-O-alpha-kojibiosyl-D-glucose were synthesized. Afterwards, nigerose and kojibiose were accumulated gradually with glucose as an acceptor.

Preparation and characterization of novel branched beta-cyclodextrins having beta-D-galactose residues on the non-reducing terminal of the side chains and their specific interactions with peanut (Arachis hypogaea) agglutinin.

Six novel branched beta-cyclodextrins (betaCDs) having beta-D-galactose residues on the non-reducing terminal of the sugar side chains, namely 6(1),6(4)-di-O-(beta-D-galactosyl)-betaCD (10), 6-O-(beta-D-galactosyl)-betaCD (11), 6(1),6(4)-di-O-(beta-lactosyl)-betaCD (14), 6-O-(beta-lactosyl)-betaCD (15), 6(1),6(4)-di-O-(4'-O-beta-D-galactosyl-beta-lactosyl)-betaCD (18), and 6-O-(4'-O-beta-D-galactosyl-beta-lactosyl)-betaCD (19), were chemically synthesized using the trichloroacetimidate method. The reaction products were separated by HPLC on an amino column into dibranched and monobranched betaCDs. Their structures were confirmed by mass spectrometry (MS) and two-dimensional (2D) NMR spectroscopic analysis. To study the length of the sugar side chains attached to the CD ring, which leads to differences in the functions of the branched CDs, interactions of these compounds with peanut (Arachis hypogaea) agglutinin (PNA) were investigated using an optical biosensor and an inhibition assay based on hemagglutination. The results showed that all branched betaCDs interacted with PNA, and the binding affinity was 18>14>10 and 19>15>11 when the derivatives were compared on the basis of side chain length.

Inhibition of beta-fructofuranosidases and alpha-glucosidases by synthetic thio-fructofuranoside.

A synthetic beta-thio-fructofuranoside of mercaptoethanol inhibited not only beta-fructofuranosidases but also alpha-glucosidases. The compound was hardly hydrolyzed by the glycosidases. The thio-fructoside competitively inhibited beta-fructofuranosidases from Aspergillus niger, Candida sp., and Saccharomyces cerevisiae, but not Arthrobacter beta-fructofuranosidase at all. Sucrase activity of rat intestinal sucrase/isomaltase complex was also suppressed in the presence of the thio-fructoside. The thio-fructoside showed noncompetitive inhibition toward maltase activity of the rat intestinal enzyme complex and Saccharomyces sp. alpha-glucosidase. Inhibition against the Bacillus stearothermophilus alpha-glucosidase, Rhizopus glucoamylase, and porcine kidney trehalase were more slight than that against these two alpha-glucosidases.