Phosphoryl oligosaccharides (POs) have been prepared from potato starch hydrolysate. We inves-tigated the action pattern of neopullulanase (EC 3.2.1.135) on the components of PO-1 fraction, which is the main part of POs. Neopullulanase hydrolyzed PO-1 and produced 33-phosphoryl maltotetraose from the oligosaccharides attached to the phosphoryl groups at C-3, and 62-phosphoryl maltotriose and 62-phosphoryl maltose from the oligosaccharides attached to the phosphoryl groups at C-6. Neopullulanase hydrolyzed the glucosyl linkage at the reducing site up to one glucosyl residue from the 6-phosphoryl residue and up to two glucosyl residues from the 3-phosphoryl residue. The reaction specificity of neopullulanase was compared with bacterial liquefying α-amylase (BLA), Taka-amylase A (TAA), bacterial saccharifying α-amylase (BSA), human saliva α-amylase (HSA), and porcine pancreas α-amylase (PPA) (EC 3.2.1.1) using the components of PO-1 fraction as the substrate. The specificity of neopullulanase on PO-1 fraction was quite different from those of the a-amylases; PPA, HSA, TAA, and BLA.

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In a previous study, we prepared phosphoryl oligosaccharides (POs) from potato starch hydrolysate and investigated their inhibitory effect on the formation of a calcium phosphate precipitate. In this study, we investigated the enhancement of the inhibitory effect using the coupling reaction of cyclomaltodextrin glucanotransferase (CGTase) (EC 2.4.1.19). In this treatment, the effect of the POs was obviously improved and conversion of the PO structure was observed. By the coupling reaction of CGTase, the fraction of POs with one phosphoryl group decreased, and the fraction with at least two phosphoryl groups increased to 33% from 10%. Phosphoryl maltopentaose was the main substrate for the coupling reaction of the enzyme. The products (CGT-POs) obtained after CGTase reaction were fractionated by ion-exchange chromatography into three fractions, CGT-PO1, CGT-P02a, and CGT-P02b. The fractions of CGT-P02a and CGT-P02b were the main components of the CGT-POs having an inhibitory effect on calcium phosphate formation. The fraction CGT-P02a was maltooligosaccharide with an average degree of polymerization (DP) of 7.89, to which two phosphoryl groups were attached. The fraction CGT-P02b was maltooligosaccharide with an average DP of 11.06, to which three phosphoryl groups were attached. In addition, the fraction CGT-P02b was more effective than fraction CGT-P02a since the threshold value of the concentration of CGT-P02b to exhibit the inhibitory effect was 7-fold lower than that of CGT-P02a.

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The phosphoryl oligosaccharide-1 (PO1) fraction prepared from potato starch hydrolysate was conjugated with ovalbumin (OVA) through the Maillard reaction. The conjugate was prepared by treatment at 50°C and 65% relative humidity. The inhibitory effect of the conjugate on the formation of a calcium phosphate precipitate was examined. The optimum treatment period (7 days) for expression of the inhibitory effect of PO1 on the formation of calcium phosphate was determined. In this period, PO1 molecule attached about 10 mol to 1 mol of OVA. The average molecular weight of the OVA-PO1 conjugate rose from 45 kD to 53 kD. The isoelectric point of the conjugate also changed from 4.5 to 4.0. The structures of the OVA-PO1 conjugate hardly changed in comparison with the properties of native OVA. On the other hand, OVA-G6P conjugate formed more rapidly than the OVA-PO1 conjugate under these conditions. However, the OVA-G6P conjugate quickly developed brown color, while coloring of the OVA-POl conjugate was minimal. These results indicate that making a conjugate with OVA is a noteworthy way to enhance the inhibitory effect of PO1 on the formation of a calcium phosphate and that the OVA-POl conjugate may be a better food additive than OVA-G6P conjugate.

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Aspergillus niger α-glucosidase was crystallized by acetone addition after ammonium sulfate fractionation and chromatographies on DEAE-Sepharose CL-6B and Bio-Gel P-150 (1st and 2nd) columns. The crystalline α-glucosidase, which was a glycoprotein containing 27% carbohydrate as glucose, gave a single band on polyacrylamide disc gel electrophoresis. The molecular weight was estimated to be about 12.0 × 10⁴ by SDS-polyacrylamide disc gel electrophoresis. However, the enzyme consisted of two components (Mr, 4.2 × 10⁴ and 9.0 × 10⁴ ) separable by reversed-phase HPLC causing irreversible inactivation. Their N-terminal sequences were analyzed to be LXPAPSQ and SQDYISL. The optimum pH was 4.5. In the initial reaction stage, phenyl α-maltoside was cleaved into α-glucose and phenyl α-glucoside. The ratios of the maximum velocities (Km values, in parentheses, mM of nonreducing terminal) for maltose, malto-triose, -tetraose, -pentaose, -hexaose, -heptaose and -octaose, maltodextrin (DP= 17), kojibiose, nigerose, isomaltose, phenyl α-glucoside, phenyl α-maltoside, panose and soluble starch were estimated to be 100 (1.30), 122 (1.20), 105 (1.32), 106 (2.09), 108 (3.85), 107 (5.64), 107 (6.02), 103 (20.8), 34.4 (6.76), 78.5 (20.0), 45.4 (9.80), 9.42 (1.82), 110 (1.49), 41.1 (4.88) and 111 (7.14), respectively. The enzyme also hydrolyzed α-glucans such as soluble starch, however, the rates of cleavage were very slow.

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The hydrolysis of various β-1, 4-galactosyl-maltooligosaccharides (β-1, 4-Gal-Gn) by the action of an exo-maltotetraohydrolase (EC 3.2.1.60, G4-amylase) from Pseudomonas stutzeri was examined to obtain information on action mode and kinetic parameters. The enzyme split 2 points of the. α-1, 4-glucosidic linkage in β-1, 4-galactosyl-maltotetraose (β-1, 4-Gal-G4) and 3 points in β-1, 4-Gal-G5, -Gal-G6 and -Gal-G7 to form various molar ratios of β-1, 4-Gal-G2, -Gal-G3 and -Gal-G4, respectively, and larger ko/Km values were observed with larger β-1, 4-Gal-Gn as substrates. The G4-amylase catalyzed the formation of p-nitrophenyl β-1, 4-D-galactosyl-α-maltopentaoside (β-1, 4-Gal-G5P) by its transfer action with a yield of 3.3, 11.3 and 6.8% by weight, and coincided well with the hydrolytic pattern of β-1, 4-Gal-Gn (n=5-7), from the mixture of p-nitrophenyl α-D-glucopyranoside (GP) and β-1, 4-Gal-G5, -Gal-G6 or -Gal-G7, respectively. The G4-amylase synthesized β-1, 4-Gal-G5P with a yield of 6.1% by weight of the mixture of GP with crude Gal-Gn containing primarily Gal-G6 and Gal-G7, of which about 10% was the respective β-1, 6 isomer. By an analogous method, the preparation of p-nitropheny16-0-benzyl-α-maltopentaoside (BG5P) was also examined with the mixture of GP and methyl 6-O-benzyl-α-maltooligosaccharides (BGn-Me) containing primarily BG6-Me and BG7-Me. In contrast to the action on Gal-Gn, the enzyme split 1 point of the α-1, 4-glucosidic linkage in BGn-Me, and the BG4 formed was readily transferred to the 4-position of GP by the α-1, 4-glucosidic linkage with a yield of 10.2% (w/w) . By these methods, the complicated procedures to obtain pure p-nitrophenyl α-malto-pentaoside (G5P) were eliminated from the conventional production methods of Gal-G5P and BG5P.

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Two kinds of cross-linked starch were prepared from corn starch by treatment with epichlorohydrin, and they were then extrusion-cooked. Some properties of the extrudates were investigated by scanning electron microscopy, X-ray diffractmetry and differential scanning calorimetry (DSC). The cross-linked starches exhibited the crystalline structures and some thermal transition, however, they did not increase their viscosities in amylograms. The cross-linked starches could be perfectly molten by extrusion cooking and solidified immediatly after extrusion. The cross-linked starch treated with epichlorohydrin of 3% to corn starch in weight (3% cross-linked starch) was most stably extruded under the following conditions: added water, 20%; die temperature, 150-160°C; and die pressure, 32-36 kg/cm². The 3% cross-linked starch extrudate showed a flat X-ray diffraction pattern unlike the corn starch extrudate. The measurement of DSC clarified that all of extrudates showed almost no thermal transition or retrogradation. The solubility of the extrudate was negligible. It was thus concluded that an insoluble molten starch material could be prepared by extruding epichlorohydrin-cross-linked starch.

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The moisture content at which freezable water first appeared was 0.43 mg/mg-solids as measured by DSC. The freezable water content increased linearly above 0.43 mg/mg-solids. When sufficient water molecules were present in the starch (2.5 mg/mg-solids), only one phase transition peak which can be considered starch gelatinization was observed. As the moisture content decreased, the area of gelatinization peak decreased, but the peak temperature remained constant at about 78°C. The gelatinization peak disappeared between 0.25 and 0.50 mg/mg-solids. Under the moisture content of 1.5 mg/mg-solids, another phase transition peak which can be considered the melting of starch crystallites appeared. The peak temperature increased with the decrease in moisture content. The range of moisture content at which the gelatinization peak disappeared corresponded to that at which the freezable water appeared in sweet potato starch. It was concluded that the gelatinization of sweet potato starch requires freezable water.

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Glycogen-filled vacuoles (glycogenosomes) were ubiquitously observed in hepatocytes of masu salmon (Oncorhynchus masou), mimicking the cytology of human glycogen storage disease type II (GSD II) with an inherited deficiency of lysosomal acid α-glucosidase (AAG). We re-examined the relationship between α-glucosidase activities and appearance of glycogenosomes in the fish. The fish liver AAG activity measured at pH 4.5 with glycogen as the substrate was comparable to that of mouse liver. However, we observed that neutral α-glucosidase (NAG) activity measured at pH 6.5 was significantly lower than that in mouse liver. These results suggest that the formation of glycogeno-somes is associated with NAG deficiency, similar to the late-onset adult form of GSD II.

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Various lipid-coated glycoside hydrolases were prepared, which were soluble in most organic solvents such as isopropyl ether, isooctane and benzene but insoluble in aqueous solution. They could act as efficient catalysts for transglycosylation of α-D-or β-D-mannoside, N-acetyl-β-D-glucosaminide, β-D-glucoside or β-D-galactoside to hydrophobic acceptor alcohols in dry isopropyl ether. When a native β-D-galactosidase was used in the aqueous solution containing acetonitrile for the same reaction (a conventional method), the yield of the transgalactosylation was low due to the predominant process of hydrolysis reaction. The enzyme activity for glycosylation depends on the coating of lipids as well as the orgin or type of enzyme. These lipid-coated glycoside hydrolases could also act as an efficient catalyst for transglycosylation in the two water-organic phases: both the hydrophobic lipid-coated enzyme and alcohols were solubilized in isopropyl ether and mixed with an aqueous solution of glycosyl donors such as lactose and cellobiose.

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Galα1-3Gal is an important constituent in glycoconjugates. This disaccharide is also known as one of the epitopes which binds with anti-pig antibodies in discordant xenotransplantation. In this study, Galαl-3GalαpNp was synthesized regioselectively by transglycosylation reaction using p-nitrophenyl a-galactopyranoside (pNp-α-Gal) as a donor and an acceptor with the aid of α-galactosidase from coffee beans. The p-nitrophenyl group in Galal-3GalapNp was selectively removed by CAN after reduction to the p-acetamidophenyl group. Ga1β-3G1cNAc and Galfll-3Ga1NAc are also important constituents of mucin or complex types of glycoprotein. Galβ-3G1cNAc was synthesized regio-selectively by transglycosylation reaction using p-nitrophenyl β-galactopyranoside (pNp-β-Gal) as a donor and G1cNAc as an acceptor with the aid of β-galactosidase from Xanthomonas manihotis. A gene encoding β-galactosidase from Bacillus circulans which had hydrolysis specificity for β1-3 linkage was manipulated in Escherichia coil. Galβ1-3G1cNAc and Galβ-3Ga1NAc were synthesized regio-selectively by transglycosylation reaction using this enzyme.

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