The purpose of the present study was to find a suitable carrier and optimum conditions for the production of maltosyl (α1→6) cyclodextrins (G2-CDs) using immobilized Bacillus acidopullulyticus Pullulanase. A sepiolite treated with acid with a pore size of 600-700Å was the most suitable carrier for the immobilization of pullulanase. The suitable concentration of acetate bufferr and optimum pH for the immobilization was 20 mM and 4, respectively. The temperature did not affect the immobilization within the range of 10-40°C. The optimum amount of adsorbed protein was 30-40 mg/g of carrier . Properties of pullulanase immobilized on the sepiolite carrier under the above condition were investigated. The optimum pH was 4.5 for both immobilized and native enzyme . The optimum temperature of immobilized, enzyme was 60°C, while that of native enzyme was 70°C. The stability of both immobilized and native enzyme was retained within the pH range of 4-6. The G2-CDs synthesizing activities of a column reactor (8.0i.d.×19.9 cm) packed with immobilized pullulanase fell to approximately50% of the initial conversion ratio after 74 days of continuous use at 60°C at a flow rate of SV=0 .072 hr-1 using maltose and cyclodextrin solution [70% (w/w), maltose : cyclodextrins=4 :1; pH5] as the substrate.

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Differential scanning calorimetry (DSC) was used to estimate the intracellular water content during the gelatinization of starches inside cotyledon cells prepared from adzuki, faba and kidney beans. DSC thermograms and their characteristics of cotyledon cells in excess water were compared with those of isolated starches in various water contents. DSC thermograms of cotyledon cells in excess water (85-88%) showed an endothermic transition with a peak temperature, which trailed a shoulder or another peak at higher temperatures. The melting temperature of cotyledon cells in excess water were 95.7, 94.5 and 96.9t for adzuki, faba and kidney beans, respectively. The water content of isolated starches which showed the same melting temperature as the cotyledon cells in excess water were 56, 58 and 61% for adzuki, faba and kidney beans, respectively. The gelatinization enthalpies of cotyledon cells and the proportion of higher-temperature peak in thermograms in excess water were identical with those of isolated starches at water contents of 56-58%. From these results, the intracellular water content during the gelatinization of starches inside cotyledon cells of legumes in excess water were estimated to be 56-61%.

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The learning performance ability of the egg yolk sialyloligosaccharide fraction was investigated. The egg yolk sialyloligosaccharide fraction was administrated to rats aged 14 to 21 days. Goalreaching time and success ratio of goal reaching were measured using maze test on 42-49 days old rats. The results showed that the group which were administered egg yolk sialyloligosaccharides fraction had higher success ratio of goal reaching than control group, and goal reaching time was also reduced significantly as compared to control . The results of this study suggest that the sialyloligosaccharide derivatives might play an important role to improve the learning performance of infants .

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A strain of Plesiomonas capable of producing intracellular thermostable maltose phosphorylase (MP) and trehalose phosphorylase (TP), which were useful for the production of trehalose from maltose, was isolated from mud of a Japanese seashore. The isolate (SH-35) grew well among the ranges of pH and temperature at pH 6-9 and 15-45°C with optima at pH 7.0 and 37°C by shaking cultivation, though the maximum yield of these enzymes were obtained at pH 7.5 and 34°C for MP and pH 8.0 and 37°C for TP with medium containing maltose as a carbon source and the mixture of Polypepton-S, yeast extract and urea as nitrogen sources. Optimum pH and temperature for producing trehalose were pH 7-8 and 55-60°C in the presence of 10-40% (w/w) maltose and 5-50 mM inorganic phosphate, and about 60% (as dry basis) of maltose was converted into trehalose by the simultaneous action of both enzymes before and after extraction from cells under the best conditions. These microbial and enzymatic characteristics are consistent with the industrial production of trehalose from maltose.

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The treatment of Leuconostoc mesenteroides B-512F dextransucrase with 1.25 mNi o-phthalaldehyde (OPA) inactivated the enzyme almost completely within 30 min. The addition of substrates dextran T-10 or sucrose retarded the enzyme inactivation by OPA. The addition of sucrose-monocaprate, an analog of sucrose, also retarded the inactivation. Differential modification of dextransucrase by OPA was conducted in the presence of sucrose, dextran T-10, or sucrosemonocaprate. Modified enzymes were digested by trypsin, and the resultant peptides were separated by HPLC. All of the peptides containing lysine residues protected by the ligands had homologies to the catalytic domain of streptococcal glucosyltransferases (GTFs), and these regions in GTFs were apart from the catalytic aspartic acid. Two peptides which contained sucrose-protected lysine were located in the direction of the amino terminal from the catalytic aspartate. Those peptides were also protected from the OPA modification by dextran and there were two more dextran-protected peptides which were close to the glucan-binding domain. Hydroxyl amino acids were frequently found in those substrate-protected peptides, indicating that the peptides originated from the sugar-affinity sites.

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An octasaccharide [XGO8, composed of n-xylose, D-galactose, and D-glucose (3: 1: 4) ] purified from the (1→4) β-D-glucanase-digest of xyloglucan of tamarind (Tamarindus indica L .) seed, was coupled with bovine serum albumin (BSA), and used as an immunogen. The polyclonal antibodies raised in rabbits interacted with XGO8-BSA conjugates and of other oligosaccharide fragments, and also with the original xyloglucan. Hapten inhibition studies using various oligosaccharides showed that the antibodies recognize β-D-galactosyl- (1→2) -α-D-xylosyl, and also α-D-xylosyl side chains attached to the β- (14) -n-glucosyl backbone chain in the xyloglucan. The antibodies were used for histological localization of the xyloglucan in plant tissues, by either the fluorescence or the peroxidase-labeled immunostaining method. By the latter method it was shown that, in the soybean cotyledon, the location of the xyloglucan is restricted to the primary cell-walls .

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Extracellular β-xylosidase ([EC 3.2.1.37]: xylan 1, 4-β-xylosidase) was purified from a culture filtrate of Aspergillus aculeatus No. F-50 by column chromatography using DEAE-Sephadex A-50, Sephacry 5-200, and DEAE-Toyopearl 650M columns, and preparative isoelectric focusing. The purified enzyme was homogeneous on SDS-polyacrylamide gel electrophoresis . The molecular weight was about 105, 000 by SDS-PAGE and its p1 value was 4.3. The optimum pH and temperature for the /3 -xylosidase activities were 2.0 and 70°C, respectively. The β-xylosidase was stable for 30 min at 50°C and stable between pH 3 and 7. The enzyme activity was strongly inhibited by Cu²⁺, Mn²⁺, and Hg²⁺. The enzyme hydrolyzed xylooligosaccharides, xylobiose through xylopentaose, to form xylose. The β-xylosidase showed potent activity towards larchwood xylan .

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The β-D-mannosidase (β-D-mannoside mannohydrolase, [EC 3 .2.1.25]) from culture filtrate of Aspeygillus aculeatus was purified with ammonium sulfate precipitation and column chromatographies with DEAE-Sephadex A-50, Sephacryl S-200, and preparative isoelectric focusing. The enzyme has a molecular weight of 130, 000 (SDS-PAGE) and an isoelectric point of pH 4 .0. The enzyme shows maximum activity at pH 2.0 and at 70°C, and is stable for 30 min at 50°C and between pH 4 to 7.Enzyme activity was completely inhibited by Ag⁺, Pb²⁺, and Hg²⁺ The β-mannosidase has high specificity towards p-nitrophenyl fl-D-mannopyranoside and mannooligosaccharides .

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The glucansucrases synthesize glucan by a two-site mechanism in which the glucose and the growingg glucan are covalently attached to the active site. The glucose is transferred to the reducing end of the growing glucan chain by an insertion mechanism in which glucose is inserted between the enzyme and the growing chain. The chain is released from the active-site by acceptor reactions . When the acceptor is a glucan chain, a branch linkage is formed. When the acceptor is a low molecular weight carbohydrate, the glucan chain is released with the acceptor attached to the reducing end, and a low molecular weight acceptor product is produced with a glucose residue attached to the acceptor . With many acceptors, the acceptor product is an acceptor itself and a series of acceptor-products are produced. The structure of the acceptor product depends on the structure of the acceptor and the particular glucansucrase. Acceptors divert glucose away from the synthesis of glucan and terminate glucan synthesis. The amount and the number of acceptor products varies, depending on the concentration ratio of acceptor to sucrose. At low acceptor to sucrose ratios, the yield of acceptor-products is low and at high ratios, the yields are high, and the amount of dextran is higher at low ratios and lower at high ratios. With acceptors that form a homologous series of acceptor-products (e .g., maltose), a low ratio gives a relatively large number of acceptor-products (≥10) and a high ratio gives a small number of acceptor-products (1-2) in high yields with respect to sucrose.

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This paper from the 1994 award address of the Society is composed of following four research topics that were carried out at the Bioconversion Laboratory, National Food Research Institute, during 1986 and 1991. A summary of these is as follows . Cellulase system of Cellvibrio gilvus was studied at enzymatic and DNA levels . A unique xylanase which hydrolyzed a series of pNP-cellooligosaccharides into pNP and respective cellooligosaccharides was purified. Shotgun cloning of the cellulase system using 4-methylumberriferyl cellobioside led to the isolation of a fl-glucosidase gene . The G+C content of the gene was very high (69.4%) and four amino acids (Ala, Arg, Gly, Pro) comprised 37% of the total amino acids . However, the deduced amino acid sequence has high homology with those of other β-glucosidases . Extensive treatment of wheat bran hemicellulose by Cellulase Onozuka RS (a commercial crude enzyme preparation from Trichoderma viride) produced an oligosaccharide fraction with a yield of 30%. This fraction was selectively utilized by Bifidobacteria, and its administration to rats increased the percentage of Bifidobacteria in the intestinal microflora from 1% to 40%. Structure of the main component of this oligosaccharide fraction was determined to be β-D-Xylp- (1°C→4) [α-L -Araf- (1→2) ] [α-L-Araf- (1→3) ] -β-D-Xylp- (1→4) -β-D-Xylp- (1→4) -D-Xvlp. Cellobiose phosphorylase was purified from C. gilvus cells and its catalytic properties were studied in detail. Different from sucrose phosphorylase, the enzyme was found to follow the sequential mechanism. It selectively utilized f3-glucose as an acceptor to form cellobiose . Inhibition observed with glucose at higher concentrations was extensively analyzed and a model for competitive substrate inhibition was proposed. The enzyme can utilize several glucose derivatives at its C-2 or C-6 positions . By the combined action of sucrose phosphorylase, xylose isomerase and cellobiose phosphorylase, sucrose was effectively (75% yield) converted into cellobiose under the presence of small amount of inorganic phosphate. Sucrose phosphorylase was purified from Leuconostoc mesenteroides cells and its transfer reactions were studied. The enzyme showed the highest specificity to xylulose as an acceptor to form α-D-glucopyranosyl- (1→2) β-D-xylulofuranoside (Glc-Xul). More than 90% of xylulose was converted into Glc-Xul when 300 mivi sucrose and 100 mii xylulose were incubated with the enzyme. Glc Xul is 70% as sweet as sucrose and has physicochemical properties close to those of sucrose. However, it was neither utilized by dextransucrase to form dextran nor by Streptococcus sobrinus cells to produce acid. This sugar is expected to be a sucrose substitute that is resistant to dental plaque formation.

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A β-glucosidase was intensively purified with high recovery from a commercial preparation of Aspergillus niger by consecutive column chromatography. The enzyme was an acidic protein with a p1 of 3.8, and split cellotiose to produce specifically β-D-glucose. Substrate specificity studies demon strated that the purified enzyme required absolutely the C-4 configuration of the terminal, non-reducing-β-D-glucose residues in the substrate molecules.The enzyme synthesized β-glucobiose from D-glucose with high concentration by the condensation reaction. In this reaction, the enzyme specifically required β-glucose as donor substrate . Synthesized β-glucobiose was identified as gentiobiose by PPC and HPLC. On the other hand, β-glucotrioses were synthesized by the transglucosylation reaction when cellobiose and gentiobiose were used as substrates individually. The two main products among these β-glucotrioses were identified as 62-O-β-D-glucosyl cellobiose and gentiotriose by NMR analysis, respectively . The industrial production of gentiooligosaccharide-containing syrups was developed by using both reactions of fungal β-glucosidase. The optimum pH (pH 4.0-4 .5) and temperature (65°C) for the production of β-glucooligosaccharide were fairly close to the optimum conditions of hydrolysis reaction of cellobiose by β-glucosidase. The enzyme (1 .5 units/g-glucose) was incubated with highly concentrated glucose solution (65%, w/w) at pH 4.0 and 65°C for 72 hr, and β-glucooligosaccharide syrup (GentoseR #45) containing about 45% (w/w) of oligosaccharide was produced by the reaction of the enzyme. Furthermore, ?A-glucooligosaccharide syrup (Gentosec #80) containing more than 90% (w/w) of oligosaccharide was fractionated by the cation exchange resin column chromatography using GentoseR #45 as the raw material. These syrups had a bitter taste derived from gentiooligosaccharide, and GentoseR #45 in particular had both the bitter taste and sweet taste of glucose. GentoseR #45 showed the same viscosity as that of sucrose, and that of GentoseR #80 was a little higher than sucrose. The oligosaccharides of these syrups were stable when treated 10 min at pH 3-4 and 120°C or at pH 1-2 and 100°C, and only about 10% (w/w solid) of oligosaccharides was hydrolyzed at pH 1-2 and 120°C . These syrups showed high hygroscopicity and high moisture-retaining activity. Also, these syrups reduced the freezing point of water more than sucrose, and other properties of gentiooligosaccharide-containing syrups such as osmotic pressure and water activity were almost the same as that of sucrose . Moreover, gentiooligosaccharide-containing syrups were not digested by pancreatic a-amylase, and the maximum no-effect level values of both syrups were estimated to be more than 0 .3 g/kg. β-Glucooligosaccharides were selectively utilized by Bifidobacterium spp., Lactobacillus spp. except L. fermentum and L. salivarius group, and Mitsuokella multiacidus group, however, almost all of Eubacterium spp., Fusobacterium spp. and Clostridium peffringens group could not utilize the saccharides. The administration of β-glucooligosaccharides (4 g daily for 10 days) promoted the growth of Bifidobacteria and lowered fecal pH, in vivo. Furthermore, the volunteers never had diarrhea or flatulence and the consistency of feces was improved. From these results, it was presumed that gentiooligosaccharide-containing syrups might be utilized as brand-new oligosaccharides for the improvement of intestinal microflora and widely used for food processing and other additives.

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Facilitative transport of glucose across the plasma membrane of mammalian cells is mediated by members of the glucose transporter (GLUT) proteins which possess 12 membrane-spanning helical segments. These proteins show tissue- and cell-specific expression and exhibit distinct kinetic and regulatory properties that reflect their specific functional roles. Six members of the GLUT family have already been described. This review summarizes recent advances concerning structure and function of the GLUT protein. Structure function relationship of the HepG2/erythrocyte-type glucose transporter (GLUT1) has been studied by in vitro site-directed mutagenesis. In a highly-conserved QQXSGXNXXXYY in transmembrane helix 7, Gln²⁸² has been shown to be involved in a recognition of the outside-specific ligand, such as ATB-BMPA. Tyr²⁹³→I1e mutant GLUT1 has been shown to be locked into an outward-facing conformation and Tyr²⁹³ in the edge of this motif is involved in closing the exofacial site in the transport catalysis process. These observations suggested that transmembrane helix 7 has an important role in ligand recognition as well as in transport catalysis . Mutagenesis at Pro³⁸⁵ in transmembrane helix 10 has been carried out and reduction in transport and exofacial ATB-BMPA photolabeling occur as the result of substitution with isoleucine, suggesting that retention of flexibility in this region is required for the transport catalysis process. Mutation in either Trp388 or Trp412 residue resulted in reduced transport activity while only Trp388 mutant expressed in oocytes showed a marked reduction in the affinity for cytochalasin B. Examinations by C-terminally truncated transporters revealed that the infracellular C-terminus of GLUTI also contributes to the glucose transport process .

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