Kyoko Koizumi

Estimation of the distributions of chain length of amylopectins by high-performance liquid chromatography with pulsed amperometric detection☆

Abstract High-performance anion-exchange chromatography for a detailed estimation of the distribution of chain length of amylopectins was developed using a pulsed amperometric detector under alkaline conditions. As maltosaccharides having different degrees of polymerization, which were produced by debranching of amylopectin with isoamylase, exhibit different pulsed amperometric detector responses, the individual maltosaccharides (degree of polymerization 6–17) were isolated by high-performance liquid chromatography on an amino column and an octadecylsilane column to use as quantification standards. By the use of this high-performance anion-exchange chromatography the chain length distributions of some typical amylopectins were characterized in detail.

High-performance liquid chromatographic separation of carbohydrates on graphitized carbon columns

Graphitized carbon columns (GCC) for high-performance liquid chromatography are relatively new and have a unique ability to resolve isomeric and closely related compounds. The retention mechanism of carbohydrates on GCC is mainly based on adsorption and the flat surface of GCC packings brings about unique selectivity, but also includes hydrophobic interactions. The chromatographic behaviour of monosaccharides, disaccharides, cyclodextrins (CDs), branched CDs, oligosaccharide alditols, chito-oligosaccharides, N-linked oligosaccharides and glycopeptides has been studied, and it has become apparent that the elution patterns are based on the size and planarity of the molecule (position and configuration of linkage).

High-performance anion-exchange chromatography of homogeneous D-gluco-oligosaccharides and -polysaccharides (polymerization degree >50) with pulsed amperometric detection

Abstract High-performance anion-exchange chromatography under alkaline conditions with pulsed amperometric detection was applied to the analyses of (1 → 2)-, (1 → 3)-, (1 → 4)- and (1 → 6)-linked homogeneous α- or β- d -gluco-oligosaccharides and -polysaccharides up to a degree of polymerization (DP) of ⩾ 50. Each series of homogeneous d -gluco-oligomers and -polymers showed a linear relationship between log k′ and DP in isocratic elution using 150 mM sodium hydroxide solution containing 100 mM sodium acetate as the eluent. An effective separation of individual members of an homologous series of linear glucans was achieved using gradient elution, accomplished by maintaining the sodium hydroxide concentration at 150 mM and increasing the sodium acetate concentration during the analysis. The detector response per HCOH group in d -gluco-oligomers (DP 2–7) was almost the same.

High-performance liquid chromatography of mono- and oligo-saccharides on a graphitized carbon column

Abstract Chromatographic behavior of several mono- and di-saccharides cyclomaltaoses on Hypercarb, a graphitized carbon column, was investigated. Monosaccharides were weakly retained on this column and were eluted with water within 1.8–3.6 min at 1 mL/min of flow rate and 30°. Nevertheless, each peak of d -xylose, d -glucose, d -galactose, and l -fucose was split into anomer peaks. Disaccharides were adequately eluted with 15:85 methanol-water or 4:96 acetonitrile-water, and each showed two peaks for the anomers. These peaks each coalesced into a single peak upon the addition of m m sodium hydroxide of the eluent. Simultaneous separation of nine glucodisaccharides was achieved on this column by gradient elution with m m sodium hydroxide solution containing 1.5–5.0% acetonitrile. Detection was by a pulsed amperometric detector. Cyclomaltohexaose (αCD), cyclomaltoheptaose (βCD), cyclomaltooctaose (γCD), and their glucosyl derivatives (G-αCD, G-βCD and G-γCD) were analyzed by using 13–15% aq. acetonitrile as the eluent. Interestingly their retention times were increased with increases in temperature, and the sharpness of their peaks was also shown to be enhanced. Furthermore, positional isomers of glucosyl-inositol and of dimaltosyl-βCD, neither of which can be separated on conventional bonded phases, were well resolved on this column.

Analyses of homogeneous d-gluco-oligosaccharides and -polysaccharides (degree of polymerization up to about 35) by high-performance liquid chromatography and thin-layer chromatography

Abstract Conditions for the separation of (1→2)-, (1→3)-, (1→4)- and (1→6)-linked homogeneous d -gluco-oligosaccharides and -polysaccharides were investigated by high-performance liquid chromatography on a 3-μm chemically modified amine column (ERC-NH-1171) and by thin-layer chromatography (TLC) on three kinds of silica gel plates. Saccharide samples were prepared by partial hydrolyses or partial acetolyses of cyclosophoraose [cyclic (1→2)-β- d -glucan], curdlan [(1→3)-β- d -glucan], amylose [(1→4)-α- d -glucan], cellulose [(1→4)-β- d -glucan], dextran [(1→6)-α- d -glucan], and luteose [(1→6)-β- d -glucan]. Each series of saccharides, other than β- d -(1→3)- and β- d -(1→4)-linked ones, whose soluble higher oligomers could not be obtained, was well resolved from glucose to the polymer having a degree of polymerization (DP) of about 35 on the amine column by using simple isocratic elution with acetonitrile—water. TLC analyses were performed on silica gel 70 and silica gel 60 TLC plates with a concentration zone using n -butanol—ethanol—water as the developing solvent, and on Si 50000 HPTLC plates using n -butanol—pyridine—water. These methods made it possible to separate simultaneously each series of homogeneous saccharides of DP up to 20–30.

Isolation and characterization of branched cyclodextrins

Abstract Three branched cyclodextrins (CDs) were isolated by high-performance liquid chromatography (l.c.) from the mother liquors of a large-scale preparation of the unbranched CDs with Bacillus ohbensis cyclomaltodextrin glucanotransferase. Evidence from chromatographic behavior on three l.c. columns of different separation modes, fragmentation analysis, 13C-n.m.r. spectroscopy, methylation analysis, and fast-atom bombardment-mass spectrometry (f.a.b.—m.s.) indicated that these compounds were 6-O-α- d -glucopyranosylcyclomaltohexaose (1), 6-O-α- d -glucopyranosylcyclomaltohepataose (2), and 6,6‴-di-O-α- d -glucopyranosylcyclomaltoheptaose (3).

Comparative Study of the Cyclization Reactions of Three Bacterial Cyclomaltodextrin Glucanotransferases

ABSTRACT The actions of cyclomaltodextrin glucanotransferases (CGTase; EC2.4.1.19 ) from alkalophilic Bacillus sp. strain A2-5a (A2-5a CGTase), Bacillus macerans (BmacCGTase), and Bacillus stearothermophilus (BsteCGTase) on amylose were investigated. All three enzymes produced large cyclic α-1,4-glucans (cycloamyloses) at the early stage of the reaction, but these were subsequently converted into smaller cycloamyloses. However, the rates of this conversion differed among the three enzymes. The product specificity of each CGTase in the cyclization reaction was determined by measuring the amount of each cycloamylose from CD6 to CD31 (CDn, a cycloamylose with a degree of polymerization of n). A2-5a CGTase produced 10 times more CD7, while Bmac CGTase produced 34 times more CD6 than other cycloamyloses. Bste CGTase produced 12 and 3 times more CD6 and CD7 than other cycloamyloses, respectively. The substrate specificities of the linearization reactions of CD6, CD7, CD8, and larger cycloamyloses (a mixture of CD22 to CD50) were investigated, and we found that CD7 and CD8 are extremely poor substrates for both hydrolytic and transglycosidic linearization (coupling) reactions while larger cycloamyloses are linearized at a much higher rate. By repeating these cyclization and linearization reactions, the larger cycloamyloses initially produced are converted into smaller cycloamyloses and finally into mainly CD6, CD7, and CD8. These three enzymes also differ in their hydrolytic activities, which seem to accelerate the conversion of larger cycloamyloses into smaller cycloamyloses.

Isolation and characterization of novel heterogeneous branched cyclomalto-oligosaccharides (cyclodextrins) produced by transgalactosylation with α-galactosidase from coffee bean

Transgalactosylated derivatives of cyclomalto-hexaose (alpha CD), -heptaose (beta CD), and -octaose (gamma CD) were synthesized by alpha-galactosidase from coffee bean using melibiose as a donor and alpha CD, beta CD or gamma CD as an acceptor. Mono- and di-O-alpha-D- galactosylated CDs were isolated and purified by HPLC. Their structures were elucidated by fast-atom bombardment mass spectrometry (FABMS) and 13C NMR spectroscopy. For structural determination of positional isomers of 6(1),6n-di-O-alpha-D-galactosyl-CDs, digestion products with cyclodextrin glucanotransferase were analyzed by HPLC and FABMS.

Synthesis of branched cyclomalto-oligosaccharides using Pseudomonas isoamylase

Branched cyclomalto-oligosaccharides (cyclodextrins) were synthesised from cyclomalto-oligosaccharides and maltose or maltotriose through the reverse action of Pseudomonas isoamylase. The reaction rate was greater with maltotriose than with maltose, and with increasing size of the cyclomalto-oligosaccharide (cG6 less than cG7 less than cG8). Maltotriose is effective as both a side-chain donor and acceptor, and three isomers of 6-O-alpha-maltotriosylmaltotriose (branched G6) were formed through mutual condensation, but maltose was effective only as a side-chain donor. Each branched cyclomalto-oligosaccharide and G6 was purified by liquid chromatography, and their structures were determined by chemical, enzymic, and 13C-n.m.r. spectroscopic analyses.

Determination of cyclic glucans by anion-exchange chromatography with pulsed amperometric detection

Abstract Anion-exchange chromatography with pulsed amperometric detection was applied to the determination of cyclodextrins ( CD s), branched CD s and cyclosophoraoses. These cyclic glucans with degree of polymerization 6–25 were well resolved in each series by using simple isocratic elution with 150 m M sodium hydroxide solution containing 140–200 m M sodium acetate. The separation mode was not only simple anion exchange, but also involved some hydrophobic interactions and, moreover, inclusion interactions also seemed to take part in the retention. The detector response per glucose unit of these cyclic glucans was almost the same, regardless of the molecular weight and linkage form. The limit of determination of the cyclic glucans was 5–10 pmol and the detection limit was 2.5–5 pmol with a signal-to-noise ratio of 3.