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Thermostable trehalose synthase, which catalyzes the conversion of maltose into trehalose by intramolecular transglucosylation, was purified from a cell-free extract of the thermophilic bacterium Thermus aquaticus ATCC 33923 to an electrophoretically homogeneity by successive column chromatographies. The purified enzyme had a molecular weight of 105,000 by SDS-polyacrylamide gel electrophoresis and a pI of 4.6 by gel isoelectrofocusing. The N-terminal amino acid of the enzyme was methionine. The optimum pH and temperature were pH 6.5 and 65°C, respectively. The enzyme was stable from pH 5.5 to 9.5 and up to 80°C for 60min. The trehalose synthase from Thermus aquaticus is more thermoactive and thermostable than that from Pimelobacter sp. R48. The yield of trehalose from maltose by the enzyme was independent of the substrate concentration, and tended to increase at lower temperatures. The maximum yield of trehalose from maltose by the enzyme reached 80-82% at 30-40°C. The activity was inhibited by Cu(2+) , Hg(2+), Zn(2+), and Tris.

Highly branched α-glucan molecules exhibit low digestibility for α-amylase and glucoamylase, and abundant in α-(1→3)-, α-(1→6)-glucosidic linkages and α-(1→6)-linked branch points where another glucosyl chain is initiated through an α-(1→3)-linkage. From a culture supernatant of Paenibacillus sp. PP710, we purified α-glucosidase (AGL) and α-amylase (AMY), which were involved in the production of highly branched α-glucan from maltodextrin. AGL catalyzed the transglucosylation reaction of a glucosyl residue to a nonreducing-end glucosyl residue by α-1,6-, α-1,4-, and α-1,3-linkages. AMY catalyzed the hydrolysis of the α-1,4-linkage and the intermolecular or intramolecular transfer of maltooligosaccharide like cyclodextrin glucanotransferase (CGTase). It also catalyzed the transfer of an α-1,4-glucosyl chain to a C3- or C4-hydroxyl group in the α-1,4- or α-1,6-linked nonreducing-end residue or the α-1,6-linked residue located in the other chains. Hence AMY was regarded as a novel enzyme. We think that the mechanism of formation of highly branched α-glucan from maltodextrin is as follows: α-1,6- and α-1,3-linked residues are generated by the transglucosylation of AGL at the nonreducing ends of glucosyl chains. Then AMY catalyzes the transfer of α-1,4-chains to C3- or C4-hydroxyl groups in the α-1,4- or α-1,6-linked residues generated by AGL. Thus the concerted reactions of both AGL and AMY are necessary to produce the highly branched α-glucan from maltodextrin.

The gene encoding for a novel hydrolase, cyclic α-maltosyl-(1→6)-maltose [CMM, cyclo-{→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→}] hydrolase (CMMase), was cloned from the genomic library of Arthrobacter globiformis M6 and designated cmmF. The gene consisted of 1,353 bp encoding a protein of 450 amino-acids with a calculated molecular mass of 49,344 Da. The deduced amino-acid sequence showed similarities to cyclodextrinase, maltogenic amylase and neopullulanase. On the other hand, the complete sequence of the α-glucosidase gene (cmmB), which encodes an enzyme involved in the degradation of CMM, revealed that the gene consisted of 1,704 bp encoding a protein of 567 amino-acids with a calculated molecular mass of 63,014 Da. The four conserved regions common in the α-amylase family enzymes were also found in CMMase and α-glucosidase, indicating that these enzymes should be assigned to this family. The DNA sequence of 5,675 bp analyzed in this study contained another four open reading frames (ORFs), designated cmmC, cmmD, cmmE and cmmG, downstream of cmmB. CmmC, cmmD and cmmE were expected to encode proteins concerned with incorporation of CMM via cell membrane. CmmG was expected to encode a transcriptional regulator protein. CMMase gene, α-glucosidase gene and another four ORFs formed a gene cluster together with 6-α-maltosyltrasferase gene (cmmA) which encodes a CMM-forming enzyme, namely cmmABCDEFG. The results of gene analysis suggested that A. globiformis M6 has a unique starch utilization pathway via CMM.

Cyclic nigerosylnigerose (CNN) is produced enzymatically from starch by the combined action of 6-alpha-glucosyltransferase and 3-alpha-isomaltosyltransferase. In our previous study, alpha-1,6-branching chains found in the structure of amylopectin and glycogen were shown to be favorable for CNN formation by the two enzymes. Therefore, we examined whether the introduction of alpha-1,6-branch points into starch using the action of branching enzyme (BE) could improve the yield of CNN from starch. Thermostable BE from Geobacillus stearothermophilus TC-91 was prepared as a purified recombinant protein. Pretreatment of amylose with BE considerably increased the CNN yield from 5% to 38%. When BE acted on tapioca starch, the CNN yield was elevated from 47% to 60%. Conversely, BE treatment of waxy corn starch containing very little amylose resulted in a negligible increase in CNN yield. In addition, BE exerted a beneficial effect when starch with a lower degree of hydrolysis was used as a substrate. The present results indicate that the addition of alpha-1,6-glucosidic linkages to starch using BE is an effective strategy to improve the yield of CNN from starch.

Lactosucrose (LS, 4(G)-beta-D-galactosylsucrose) is a non-digestible oligosaccharide, and the consumption of LS selectively increases the proportion of intestinal bifidobacteria. We examined in this study the hypolipidemic potential of LS. An oral triolein tolerance test on rats indicated that LS reduced the elevation of serum triglyceride (TG) and free fatty acids (FFA). Furthermore, LS inhibited the enzymatic digestion of triolein by pancreatic lipase in vitro. NMR spectroscopy showed that LS formed an intermolecular complex with triolein. The long-term consumption of a diet containing 5% LS for 8 weeks significantly decreased the weight of abdominal adipose tissue when compared with that of the control group. Thus, LS may reduce adipose tissue accumulation by inhibiting intestinal lipid absorption via a direct interaction with TG.

Cyclic maltosyl-maltose [CMM, cyclo-[--6)-alpha-D-Glcp-(1--4)-alpha-D-Glcp-(1--6)-alpha-D-Glcp-(1--4)-alpha-D-Glcp-(1--]], a novel cyclic tetrasaccharide, has a unique structure. Its four glucose residues are joined by alternate alpha-1,4 and alpha-1,6 linkages. CMM is synthesized from starch by the action of 6-alpha-maltosyltransferase from Arthrobacter globiformis M6. Recently, we determined the mechanism of extracellular synthesis of CMM, but the degrading pathway of the saccharide remains unknown. Hence we tried to identify the enzymes involved in the degradation of CMM to glucose from the cell-free extract of the strain, and identified CMM hydrolase (CMMase) and alpha-glucosidase as the responsible enzymes. The molecular mass of CMMase was determined to be 48.6 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and 136 kDa by gel filtration column chromatography. The optimal pH and temperature for CMMase activity were 6.5 and 30 degrees C. The enzyme remained stable from pH 5.5 to 8.0 and up to 25 degrees C. CMMase hydrolyzed CMM to maltose via maltosyl-maltose as intermediates, but it did not hydrolyze CMM to glucose, suggesting that it is a novel hydrolase that hydrolyzes the alpha-1,6-linkage of CMM. The molecular mass of alpha-glucosidase was determined to be 60.1 kDa by SDS-PAGE and 69.5 kDa by gel filtration column chromatography. The optimal pH and temperature for alpha-glucosidase activity were 7.0 and 35 degrees C. The enzyme remained stable from pH 7.0 to 9.5 and up to 35 degrees C. alpha-Glucosidase degraded maltosyl-maltose to glucose via panose and maltose as intermediates, but it did not degrade CMM. Furthermore, when CMMase and alpha-glucosidase existed simultaneously in a reaction mixture containing CMM, glucose was detected as the final product. It was found that CMM was degraded to glucose by the synergistic action of CMMase and alpha-glucosidase.

In this study, we examined the effects of dietary lactosucrose (LS, a non-digestible oligosaccharide) on the IgE response in mice immunized with ovalbumin (OVA)/alum. In addition to IgG1 and IgG2a responses, the anti-OVA IgE response in mice fed LS diets was dose-dependently suppressed, as compared with the control mice, while the serum total IgG levels were comparable. Moreover, dietary LS feeding inhibited antigen-specific IgE and IgG1 productions even after a second immunization. Regarding with cytokine production, when stimulated in vitro with OVA, splenocytes obtained from LS-fed mice produced a similar level of IFN-gamma, and lower levels of IL-4 and IL-5, as compared with the control mice. But IL-10 production by OVA-stimulated splenocytes was augmented in LS-fed mice, suggesting that IL-10 producing cells are responsible for the immunoregulatory effect of LS. Our findings indicate the further possibility that dietary LS supplementation can be used to prevent IgE-mediated allergic diseases.

Lactosucrose (4G-β-D-galactosylsucrose, LS) is suggested to be an oligosaccharide required for the proliferation of Bifidobacteria in the intestine. We have examined the dietary effects of LS on the intestinal immune function of mice. BALB/c mice were fed with 2 and 5% LS for 4 weeks, and the intestinal mucosal immune responses were determined. In the 2 and 5% LS fed groups, the amounts of IgA in feces and in cecum contents were significantly increased. In addition, IgA, transforming growth factor-β1 (TGF-β1) and interleukin-6 (IL-6) secretion by Peyer’s patch (PP) cells were enhanced in LS fed mice. In LS fed mice, pH in the cecum was decreased. LS, in addition, suppressed serum IgG1. These results suggest that LS supplementation changes the intestinal environment of microflora, and indirectly enhances the immune function in the gut, and suppresses the systemic immune response to the dominant type 2 helper T (Th2).

Production of a novel cyclomaltopentaose cyclized by an alpha-1,6-linkage, [ICG5; 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--}], from starch was performed using isocyclomaltooligosaccharide glucanotransferase (IGTase) derived from Bacillus circulans AM7. The optimal conditions for ICG5-production from partially hydrolyzed starch were as follows: substrate concentration, 1.0% (w/v); pH, 5.5; temperature, 45 degrees C; reaction time, 24 h, IGTase, 1.0 unit/g-dry solid (DS); isoamylase, 2,500 units/g-DS. The yield of ICG5 reached 25.9% under optimal conditions. ICG5-production was achieved from partially hydrolyzed starch using a crude enzyme preparation containing IGTase. Finally, ICG5 was obtained in a yield of 17.9% (99.3% purity, 2,681 g-DS). A digestive test with a human salivary amylase, an artificial gastric juice, a pancreatic amylase, and small intestinal enzymes showed that ICG5 was an indigestible oligosaccharide.

We examined the dietary effects of cyclic nigerosylnigerose (CNN), a dietary indigestible oligosaccharide with four D-glucopyranosyl residues linked by alternating alpha-(1-->3)- and alpha-(1-->6) glucosidic linkages, on the intestinal immune function of mice, and the effects were compared with those of alpha-(1-->3)-linked oligosaccharide (nigerooligosaccharides, NOS) or alpha-(1-->6)-linked oligosaccharide (isomaltooligosaccharides, IMO). BALB/c mice were fed with 1-5% CNN, 5% IMO, or 12.5% NOS for 4 weeks, and the intestinal mucosal immune responses were determined. In the 1-5% CNN fed groups, the amounts of IgA in feces increased significantly. In addition, IgA, transforming growth factor-beta1 (TGF-beta1), and interleukin-6 (IL-6) secretion by Peyer's patch (PP) cells were enhanced in CNN fed mice. In the 5% CNN group, pH in the cecum decreased, and the amounts of lactic acid and butyric acid increased. These findings were not observed in the NOS- or IMO-fed group of mice. They suggest that CNN supplementation changes the intestinal environment of microflora and indirectly enhances the immune function in the gut.

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 α-(1→6)-linkage, cyclo -{→6)-α- d -Glc p -(1→4)-α- d -Glc p -(1→4)-α- d -Glc p -(1→4)-α- d -Glc p -(1→4)-α- d -Glc p -(1→}. The other minor product was cyclomaltohexaose cyclized by an α-(1→6)-linkage, cyclo -{→6)-α- d -Glc p -(1→4)-α- d -Glc p -(1→4)-α- d -Glc p -(1→4)-α- d -Glc p -(1→4)-α- d -Glc p -(1→4)-α- d -Glc p -(1→}. We propose the names isocyclomaltopentaose (ICG5) and isocyclomaltohexaose (ICG6) for these novel cyclic maltooligosaccharides having one α-(1→6)-linkage. ICG5 was digested by α-amylase derived from Aspergillus oryzae , cyclomaltodextrin glucanotransferase (CGTase) from Bacillus stearothermophilus , and maltogenic α-amylase. On the other hand, ICG6 was digested by CGTase from B. stearothermophilus and B. circulans , and maltogenic α-amylase. This is the first report of enzymatically produced cyclomaltopentaose and cyclomaltohexaose, which have an α-(1→6)-linkage in their molecules.

We investigated the acceptor specificity of a thermostable trehalose phosphorylase from Thermoanaerobacter brockii ATCC 35047 (TbTP) was examined using beta-D-glucose-1-phosphate (beta-G1P) as a glucosyl donor and oligosaccharides as the acceptor. Oligosaccharides with a reducing-end glucose residue as the C-6 substituent (e.g., isomaltose, gentiobiose, melibiose, isomaltotriose, and isopanose) were found to be successful acceptors. The transfer products of isomaltose, gentiobiose, and melibiose were isolated and characterized as 6-O-alpha-D-glucopyranosyl trehalose (alpha-GlcTre), 6-O-beta-D-glucopyranosyl trehalose (beta-GlcTre), and 6-O-alpha-D-galactopyranosyl trehalose (alpha-GalTre), respectively. To produce alpha-GalTre, a novel nonreducing trisaccharide, the reaction conditions of alpha-GalTre were examined using trehalose as a glucosyl donor. As a result, the yield of alpha-GalTre reached 40.5%.

Malto-oligosyltrehalose synthase (EC 5.4.99.15, MTSase) catalyzes the conversion of α-1,4-glucan to glycosyltrehalose by forming an α,α-1,1-glucosidic linkage on the reducing side of the α-1,4-glucan. This enzyme also slightly hydrolyses the glucan and releases glucose from the reducing end of the glucan. We mutated the gene of MTSase from Sulfolobus acidocaldarius ATCC 33909 and expressed the mutated gene in E. coli. The mutants of Asp228, Glu255 or Asp443 corresponding to the catalytic residues of the α-amylase family enzymes showed no enzymatic activity. The transglycosylation activity of the mutants of Lys390 or Lys445 decreased, but the hydrolytic activity of the mutants increased in comparison to the wild-type enzyme. The substitution of Lys390 or Lys445 for a bulky residue, tryptophan, caused the loss of the transglycosylation activity of the enzyme, and provided a novel hydrolase reacting on the reducing side of the α-1,4-glucan.

Our previous study has shown that a soluble hesperidin derivative, glucosyl hesperidin (G-hesperidin), preferentially lowers serum triglyceride (TG) level in hypertriglyceridemic subjects through the improvement of very low-density lipoprotein (VLDL) metabolic abnormality. G-Hesperidin has also been found to decrease an elevated serum apolipoprotein B (apo B) level in the hypertriglyceridemic subjects, suggesting a possibility that this compound suppresses excess VLDL secretion in the liver. In the present study, to gain a better understanding of possible mechanisms by which G-hesperidin lowers serum TG, we examined whether this derivative affects apo B secretion from HepG2 human hepatoma cells, a model of hepatic VLDL secretion. As a result, G-hesperidin significantly reduced apo B secretion from the oleate-stimulated HepG2 cells. Furthermore, G-hesperidin significantly suppressed apo B secretion only in the oleate-stimulated cells and failed to act on the cells incubated without oleate. In the oleate-stimulated cells, G-hesperidin significantly decreased cellular cholesteryl ester (CE), although it had no effect on cellular TG or free cholesterol amounts. Moreover, the oleate-stimulated cells had a decrease in cellular apo B amounts by G-hesperidin exposure. These findings indicate that G-hesperidin down-regulates the assembly of apo B-containing lipoproteins via the reduction of CE synthesis augmented with oleate and results in suppressing excess apo B secretion from the cells. This effect is speculated to be associated with the improvement of VLDL metabolic abnormality in hypertriglyceridemic subjects and considered as a mechanism of lowering serum TG.

We examined the digestibility of cyclic tetrasaccharide (CTS; cyclo{→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→}). CTS was resistant to two types of α-amylase and to artificial gastric juice. Enzymes in the rat small intestinal mucosa slightly hydrolyzed CTS. CTS was not utilized by 22 strains of human intestinal bacteria. CTS was not fermented by rat cecal content. In the single administration to rats, more than 90% of the administered CTS was recovered in the feces. From the above results, CTS was confirmed as an indigestible and unfermentable saccharide. As a result of 4 weeks’ feeding to rats, CTS was effective in the suppression of body fat accumulation and lowering of serum triglyceride of the rats. These effects of CTS depended on the amount of CTS in the diet.

Although hesperidin lowers serum total cholesterol (TC) or triglyceride (TG) in animal models, its effect in humans remains unclear. Using a soluble hesperidin derivative, glucosyl hesperidin (G-hesperidin), as a hesperidin source, we examined the efficacy on hyperlipidemic subjects. G-Hesperidin was administered to the subjects at 100 or 500 mg/d for 6 wk. The percentage of subjects who had a change in serum cholesterol levels was less than 20%. However, 45-55% of the total subjects showed a reduction in serum TG level. The subjects were classified into normal (TC230mg/dL, TG150mg/dL), high-TC (TC230 mg/dL, TG150 mg/dL) and high-TG (TG150 mg/dL) types. While serum cholesterol levels scarcely changed in any phenotype, TG level was significantly reduced by administration in the high-TG type. In this phenotype, serum apolipoprotein (apo) C-II and E levels decreased by the administration, but non-apo B. G-Hesperidin also raised low-density lipoprotein (LDL)-cholesterol/apo B in the high-TG type. These results indicate that G-hesperidin preferentially lowers serum TG in hypertriglyceridemic subjects and that this effect is possibly caused by the facilitation of catabolism of TG-rich lipoproteins and may contribute to the reduction of small dense LDL.

不飽和脂肪酸の加熱分解と揮発性アルデヒド生成に及ぼすトレハロース添加の影響について検討した.(1) トレハロース添加系の不飽和脂肪酸残存量は,a-リノレン酸では66.6mg,リノール酸では77.7mg,オレイン酸では85.3mgであり,糖質無添加系より高い値であった.糖アルコールであるマルチトール添加系の不飽和脂肪酸残存量も,トレハロース添加系と同様,糖質無添加系より高い値であった.(2) a-リノレン酸加熱後の気相中揮発性アルデヒド濃度は,トレハロース添加系およびマルチトール添加系で著しく減少した.またリノール酸からの2,4-デカジェナール生成においても,トレハロース添加系およびマルチトール添加系で気相中濃度が著しく減少した.以上の結果から,トレハロースは,加熱による不飽和脂肪酸分解と揮発性アルデヒド生成を抑制することが明らかになった.

A nonreducing trisaccharide was formed in a reaction mixture containing trehalose and the cell-free extract of a thermophilic anaerobe, Thermoanaerobium brockii ATCC 35047. This saccharide was isolated from the mixture by trehalase digestion, alkaline treatment and preparative HPLC. From the results of partial acid hydrolysis, methylation analysis, and 13C-NMR analysis, the chemical structure of the saccharide was determined to be 2-O-α-D-glucopyranosyl α-D-glucopyranosyl α-D-glucopyrano- side (selaginose). Two enzymes related to Selaginose synthesis were partially purified by DEAEToyopearl column chromatography from the cell-free extract of T brockii. One was a trehalose phosphorylase (EC 2.4.1.64), which catalyzes the reversible phosphorolysis of trehalose into β-glucose-1-phosphate and glucose. The other synthesized kojibiose from β-glucose 1-phosphate and glucose. The latter enzyme was considered to be a novel phosphorylase. The hypothetical mechanism of selaginose synthesis was proposed as follows: trehalose+Pi→glucose+β-glucose 1-phosphate, β-glucose 1-phosphate +trehalose→selaginose+Pi.

The thermophilic anaerobe Thermoanaerobium brockii ATCC 35047 produces a novel phosphorylase, kojibiose phosphorylase, which catalyzes the reversible phosphorolysis of kojibiose to form β-glucose 1-phosphate and D-glucose. The enzyme was purified from a cell-free extract to an electrophoretically homogeneous state by successive column chromatography on DEAE-Toyopearl 650S, CM-Toyopearl 6505, Hydroxyapatite, Ultrogel AcA44, Mono Q, and Butyl-Toyopearl 650 M . The enzyme had a molecular weight of 83, 000 by SDS-polyacrylamide gel electrophoresis and a pl of 4 .3 to 4.4 by gel isoelectrofocusing. The enzyme showed the highest activity at pH 5 .5 and 65°C, and was stable from pH 5.5 to 9.7 and up to 65°C. The enzyme activity was inhibited by Hg2+ and Pb2+0. The Km values for kojibiose, Pi, glucose, and β-glucose 1-phosphate were 0 .77, 0.85, 3.52, and 0.77 mM, respectively.

魚肉加熱時のトリメチルアミン(TMA)生成に及ぼすトレハロース添加の影響について検討した.1. サバ肉ミンチにトレハロースを添加して加熱した場合の気相中のTMA濃度は,糖質無添加系の64%であった.マルトースおよびネオトレハロース添加系では,それぞれ84%および90%であり,グルコース,フルクトースおよびスクロース添加による影響は認められなかった.2. サバ肉加熱後にトレハロースを添加した場合,またはTMA水溶液にトレハロースを添加して加温した場合には,気相中TMA濃度は糖質無添加系と差がなかった.3. 加熱によるサバ肉中のトリメチルアミンオキシド(TMAO)減少量は,トレハロース添加系では糖質無添加系の40%であり,TMA生成量も糖質無添加系の37%と低かった.以上の結果から,トレハロースは,加熱によるTMAOからのTMA生成を抑制することが明らかになった.

A thermostable trehalose phosphorylase (EC 2.4.1.64) from a thermophilic anaerobe, Thermoanaero bium brockii ATCC 35047, was purified to an electrophoretically homogeneous state by successive column chromatographies on DEAE-Toyopearl 6505, Butyl-Toyopearl 650 M and Ultrogel AcA 44 . The molecular weight of the enzyme was estimated to be 190, 000 Da by gel filtration, and 88, 000 Da by SDS-polyacrylamide gel electrophoresis. The enzyme showed the highest activity in the range of pH 7.0 to 7.5 for phosphorolysis, and pH 6.0 to 7.0 for synthesis . The optimum temperature of the enzyme was 70°C in both directions. The enzyme was stable from pH 6.0 to 9.0, and up to 60°C . This result showed that our trehalose phosphorylase was the most thermostable in those reported to date . Activity was inhibited by Cue, Hg2+, Mg2+, Mn2+, Pb2+, and Zn2+ The Kms for trehalose, Pi, D-glucose and R-D-glucose 1-phosphate were 0.97, 0.57, 2.4 and 0.75 mM, respectively.

トレハロースを含むとされる天然食品及び発酵食品中のトレハロース含量について,ガスクロマトグラフィーにより分析を行った.1) トレハロースはキノコ類,パン酵母に比較的多く含まれており,固形物当たりのトレハロース含量は,キノコ類で10～23%,パン酵母で7～11%であった.2) トレハロースは,酒類(日本酒,ビール,ワイン),味醂,大豆製品,エビ類に,海藻類ではモズク,ヒジキに検出され,各々のトレハロース含量は,30～240ppm,260 ppm, 5～150ppm, 5～5000ppm, 4ppm, 2700ppmであった.以上の結果より,トレハロースは,キノコ類,酵母類に多く含まれていたが,その他の食品にも幅広く含まれており,トレハロースは,日常的な食事において広く摂取されている糖質であると推察される.

種々の野菜のSOD様活性にあたえるトレハロースの影響を検討し,次の結果を得た.1. 人参の固形分と等量のトレハロースを含む人参粉末を調製し,40℃で7日間保存した.この粉末のSOD様活性残存率は,トレハロースを加えない人参粉末の活性残存率より高いことがNBT法とESR法で確認された.残存SOD様活性は,トレハロース混合割合に依存し,トレハロースの割合が高いほど残存活性が高い傾向にあった.トレハロースを含む人参粉末は,グルコース,ソルビトール,マンニトール,マルトース,あるいは,スクロースなどの種々の糖質を含む粉末と比較して,最もSOD様活性の安定化効果が高かった.人参の他,なす,きゅうり,きゃべつ,ほうれん草,かぼちゃ,大根,あるいは,玉ねぎの粉末においてもトレハロースによりSOD様活性の安定化効果が認められ,野菜中に含まれる多様なSOD様活性物質成分に対して,トレハロースに安定化効果があると考えられた.2. 野菜中のどの様なSOD様活性物質に対して安定化効果があるのかを推定する目的で,SOD,および,各種抗酸化物質をトレハロース水溶液中で保存し,SOD様活性の安定化効果を調べた.西洋わさび由来のSODは,トレハロース濃度が高いほど活性残存率が高く,トレハロース溶液中で保存したときの活性残存率が最も高く,次いでスクロース,グルコースおよびマルトース,ソルビトールおよびマルチトールの順であった.αグルコシルヘスペリジン,αグルコシルルチン,没食子酸,あるいは,カテキンを,水中,あるいは,濃度40%w/vのトレハロース溶液中で保存した場合,いずれも,トレハロース溶液中の方が抗酸化物質のSOD様活性の残存率が高かった,トレハロースは,野菜中のSODと抗酸化物質の両方を安定化すると思われた.

The optimum conditions for the production of trehalose from starch were investigated using two thermostable enzymes, maltooligosyl trehalose synthase (MTSase) and maltooligosyl trehalose trehalohydrolase (MTHase), from Sulfolobus acidocaldarius ATCC 33909. The optimum pH was 5.5 and the optimum temperature was 55—57°C using isoamylase from Pseudomonas amyloderamosa as a debranching enzyme. The addition of CGTase to the reaction mixture during the saccharification process caused an increase in trehalose and a decrease in maltose and maltotriose. Isoamylase was better than pullulanase as a debranching enzyme. The yield of trehalose was independent of the type of starch used. Under optimum conditions, the yield of trehalose from corn starch at 30% concentration was more than 82%.

Cyclomaltodextrin glucanotransferase from Bacillus stearothermophilus produced a series of glycosyl-trehaloses through the transglycosylation reaction with cyclomaltohexaose as the glycosyl donor and trehalose as its acceptor. After beta-amylase treatment, five species of glycosyl-trehaloses were isolated by column chromatography. After chemical and enzymatic analyses, it was concluded that these oligosaccharides were alpha-maltosyl alpha-D-glucopyranoside, alpha-maltotriosyl alpha-D-glucopyranoside, alpha-maltosyl alpha-maltoside, alpha-maltotriosyl alpha-maltoside, and alpha-maltotriosyl alpha-maltotrioside. These were not hydrolyzed by salivary amylase, artificial gastric juice, or pancreatic amylase, however they were hydrolyzed by enzymes of the small intestine.

Glycoside hydrolase (GH) family 65 contains phosphorylases acting on maltose (Glc-α1,4-Glc), kojibiose (Glc-α1,2-Glc), trehalose (Glc-α1,α1,-Glc), and nigerose (Glc-α1,3-Glc). These phosphorylases can efficiently catalyze the reverse reactions with high specificities, and thus can be applied to the practical synthesis of α-glucosyl oligosaccharides. Here, we determined the crystal structures of kojibiose phosphorylase from Caldicellulosiruptor saccharolyticus in complex with glucose and phosphate and in complex with kojibiose and sulfate, providing the first structural insights into the substrate recognition of a glycoside hydrolase family 65 enzyme. The loop 3 region comprising the active site of kojibiose phosphorylase is significantly longer than the active sites of other enzymes, and three residues around this loop, Trp391, Glu392, and Thr417, recognize kojibiose. Various mutants mimicking the residue conservation patterns of other phosphorylases were constructed by mutation at these three residues. Activity measurements of the mutants against four substrates indicated that Trp391 and Glu392, especially the latter, are required for the kojibiose activity. Database Structural data are available in the Protein Data Bank database under accession numbers 3WIQ and 3WIR. Structured digital abstract CsKP and CsKP bind by x-ray crystallography (View interaction) .

A novel enzyme, trehalose synthase, was purified from a cell-free extract of Pimelobacter sp. R48 to an electrophoretically homogeneous state by successive chromatographies on DEAE-Toyopearl 650, Butyl-Toyopearl 650, and Mono Q HR5/5 columns. The molecular weight of the enzyme was estimated to be 62,000 by SDS-polyacrylamide gel electrophoresis, and the enzyme had a pI of 4.6 by gel isoelectrofocusing. The enzyme catalyzed the conversion of maltose into trehalose by intramolecular transglucosylation. The enzyme also converted into maltose but was inactive on other saccharides. The N-terminal amino acid of the enzyme was serine. The optimum pH and temperature were pH7.5 and 20 degrees C, respectively. The enzyme was stable in the range of pH 6.0-9.0 and up to 30 degrees C for 60 min. The rate of conversion of maltose into trehalose was independent of the maltose concentration. The maximum yield of trehalose from maltose were 81.8%, 80.9%, and 76.7% at 5, 15, and 25 degrees C, respectively. The activity was inhibited by Cu2+, Hg2+, Ni2+, Zn2+, and Tris.

A novel enzyme, maltooligosyl trehalose trehalohydrolase from Arthrobacter sp. Q36 was purified from a cell-free extract to an electrophoretically pure state by successive column chromatography on Sepabeads FP-DA13, Butyl-Toyopearl 650M, DEAE-Toyopearl 650S, and Toyopearl HW-55S. The enzyme specifically catalyzed the hydrolysis of the alpha-1,4-glucosidic linkage that bound the maltooligosyl and trehalose moieties of maltooligosyl trehalose. The Km of the enzyme for maltosyl trehalose, maltotriosyl trehalose, maltotetraosyl trehalose, and maltopentaosyl trehalose was 5.5 mM, 4.6 mM, 7.0 mM, and 4.2 mM, respectively. The enzyme had a molecular mass of 62,000 by SDS-polyacrylamide gel electrophoresis and a pI of 4.1 by gel isoelectrofocusing. The N-terminal amino acid of the enzyme was threonine. The enzyme showed the highest activity at pH 6.5 and 45 degrees C, and was stable from pH 5.0 to 10.0 and up to 45 degrees C. The activity was inhibited by Hg2+, Cu2+, Fe2+, and Zn2+.

One kojibiose phoshorylase (KP) homolog gene was cloned from Caldicellulosiruptor saccharolyticus ATCC43494. Recombinant KP from C. saccharolyticus (Cs-KP) expressed in Escherichia coli showed highest activity at pH 6.0 at 85 °C, and was stable from pH 3.5 to 10.0 and up to 85 °C for phosphorolysis. Cs-KP showed higher productivity of kojioligosaccharides of DP ≧ 4 than KP from Thermoanaerobacter brockii ATCC35047.

B. stearothermophzlus由来のCGTaseを高濃度のα-CDに作用させることによりアミロース粒子が生成することを見出し,その生成条件ならびにその諸性質を調べ,以下の結果を得た.1) アミロース粒子の生成は,主に反応温度,基質濃度に影響され,α-CD濃度35%,反応温度30℃で最高の生成率96%を示した.2) 他の細菌起源のCGTaseにおいてもアミロース粒子の生成が認められた.3) 得られるアミロース粒子は3～20gumの球状で,そのλmaxは560～580nmを示し,数平均分子量は5500～7000で,グルコース・ユニットとして34～43個からなるアミロースであった.4) 本物質はX線回折により澱粉のB形X線回折図を示し,偏光十字が観察されることから規則性のある結晶構造を有するアミロースであることがわかった.

A non-reducing disaccharide was synthesized by trehalose phosphorylase from a thermophilic anaerobe, Thermoanaerobacter brockii, using trehalose as a glucosyl donor and D-galactose as an acceptor.This disaccharide was efficiently isolated from the reaction mixture by glucose eliminationusing baker's yeast, trehalase digestion, alkaline treatment and preparative HPLC. The chemicalstructure of the disaccharide was determined to be α-D-galactopyranosyl α-D-glucopyranoside byacid hydrolysis, methylation analysis, and by 13C-NMR spectrometry. This saccharide was onlyminimally hydrolyzed by α-galactosidase, but not hydrolyzed by α- or β-glucosidase, β-galactosidase and trehalase.

The bacterial strain PP710, isolated from soil and identified as Paenibacillus species, produced a low-digestibility alpha-glucan containing a large amylase-resistant portion. This alpha-glucan was obtained in high yields from maltodextrin (dextrose equivalent 3) by using the condensed culture supernatant of the strain as the enzyme preparation. The water-soluble dietary fiber content of the low-digestibility alpha-glucan was 80.2%, and showed resistance to a rat intestinal enzyme preparation. The alpha-glucan was found to be a novel highly branched alpha-glucan by acid hydrolysis, NMR analysis, gel permeation chromatography, methylation analysis, and enzymatic digestion.

The gene for a novel glucanotransferase, isocyclomaltooligosaccharide glucanotransferase (IgtY), involved in the synthesis of a cyclomaltopentaose cyclized by an alpha-1,6-linkage [ICG5; 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-->}] from starch, was cloned from the genome of B. circulans AM7. The IgtY gene, designated igtY, consisted of 2,985 bp encoding a signal peptide of 35 amino acids and a mature protein of 960 amino acids with a calculated molecular mass of 102,071 Da. The deduced amino-acid sequence showed similarities to 6-alpha-maltosyltransferase, alpha-amylase, and cyclomaltodextrin glucanotransferase. The four conserved regions common in the alpha-amylase family enzymes were also found in this enzyme, indicating that this enzyme should be assigned to this family. The DNA sequence of 8,325-bp analyzed in this study contained two open reading frames (ORFs) downstream of igtY. The first ORF, designated igtZ, formed a gene cluster, igtYZ. The amino-acid sequence deduced from igtZ exhibited no similarity to any proteins with known or unknown functions. IgtZ was expressed in Escherichia coli, and the enzyme was purified. The enzyme acted on maltooligosaccharides that have a degree of polymerization (DP) of 4 or more, amylose, and soluble starch to produce glucose and maltooligosaccharides up to DP5 by a hydrolysis reaction. The enzyme (IgtZ), which has a novel amino-acid sequence, should be assigned to alpha-amylase. It is notable that both IgtY and IgtZ have a tandem sequence similar to a carbohydrate-binding module belonging to a family 25. These two enzymes jointly acted on raw starch, and efficiently generated ICG5.

Glucosyl hesperidin (G-hesperidin) is a water-soluble derivative of hesperidin. We compared the absorption and metabolism of G-hesperidin with those of hesperidin in rats. After oral administration of G-hesperidin or hesperidin to rats, hesperetin was detected in sera hydrolyzed with beta-glucuronidase, but it was not detectable in unhydrolyzed sera. Serum hesperetin was found more rapidly in rats administered G-hesperidin than in those administered hesperidin. The area under the concentration-time curve for hesperetin in the sera of rats administered G-hesperidin was approximately 3.7-fold greater than that of rats administered hesperidin. In the urine of both administration groups, hesperetin and its glucuronide were found. Urinary excretion of metabolites was higher in rats administered G-hesperidin than in those administered hesperidin. These results indicate that G-hesperidin presents the same metabolic profile as hesperidin. Moreover, it was concluded that G-hesperidin is absorbed more rapidly and efficiently than hesperidin, because of its high water solubility.

Chimeric phosphorylases were constructed of the kojibiose phosphorylase (KP) gene and the trehalose phosphorylase (TP) gene from Thermoanaerobacter brockii. Four chimeric enzymes had KP activity, and another had TP activity. Chimera V-III showed not TP, but KP activity, although only 125 amino acid residues in 785 residues of chimera V-III were from that of KP. Chimera V-III had 1% of the specific activity of the wild-type KP. Furthermore, the temperature profile and kinetic parameters of chimera V-III were remarkably changed as compared to those of the wild-type KP. The results of the molecular mass of chimera V-III using GPC (76,000 Da) strongly suggested that the chimera V-III protein exists as a monomer in solution, whereas wild-type KP and TP are hexamer and dimer structures, respectively. The result of the substrate specificity for phosphorolysis was that the chimera acted on nigerose, sophorose and laminaribiose, in addition to kojibiose. Furthermore, chimera V-III was also able to act on sophorose and laminaribiose in the absence of inorganic phosphate, and produced two trisaccharides, beta-D-glucosyl-(1-->6)-laminaribiose and laminaritriose, from laminaribiose.

To examine the serum triglyceride (TG)-lowering effect of a soluble hesperidin derivative, glucosyl hesperidin (G-hesperidin), and its mechanisms, we carried out a G-hesperidin administration test in hypertriglyceridemic subjects. G-Hesperidin was administered to the subjects at 500 mg/d for 24 wk. In this study, the subjects were classified into high-TG type (TG > 150 mg/dL), borderline-TG type (TG 110-150 mg/dL) and normal-TG type (TG < 110 mg/dL) on the basis of their initial serum TG values. Among these phenotypes, serum TG level significantly decreased in the high-TG type during the G-hesperidin administration period. It was also observed that elevated values of serum remnant-like particle cholesterol (RLP-C), apolipoprotein (apo) B, apo C-II, apo C-III and apo E occurred in the high-TG type and that these serum levels were significantly reduced by G-hesperidin administration. Moreover, polyacrylamide gel electrophoresis analysis of serum lipoproteins revealed that the very low-density lipoprotein (VLDL)/low-density lipoprotein (LDL) ratio and LDL migration index of the high-TG type were remarkably higher than those of the other phenotypes but that their high values were significantly reduced by the administration. These results indicate that G-hesperidin preferentially lowers serum TG in hypertriglyceridemic subjects and that this effect is possibly caused by the improvement of VLDL metabolic abnormality, leading to the reduction of small dense LDL.

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.

The glucosyl transfer reaction of kojibiose phosphorylase (KP; EC 2.4.1.230) was examined using glycerol or myo-inositol as an acceptor. In the case of glycerol, KP produced two main transfer products: saccharides A and B. The structure of saccharide A was O-alpha-D-glucopyranosyl-(1-->1)-glycerol and that of saccharide B was O-alpha-D-glucopyranosyl-(1-->2)-O-alpha-D-glucopyranosyl-(1-->1)-glycerol. These results show that KP transferred a glucose residue to the hydroxyl group at position 1 of glycerol. On the other hand, when myo-inositol was used as an acceptor, KP produced four transfer products: saccharides 1-4. The structures of saccharides 1 and 2 were O-alpha-D-glucopyranosyl-(1-->1)- and O-alpha-D-glucopyranosyl-(1-->5)-myo-inositol, respectively; those of saccharides 3 and 4 were O-alpha-D-glucopyranosyl-(1-->2)-O-alpha-D-glucopyranosyl-(1-->1)- and O-alpha-D-glucopyranosyl-(1-->2)-O-alpha-D-glucopyranosyl-(1-->5)-myo-inositol, respectively. KP transferred a glucose residue to the hydroxyl group at position 1 or 5 of myo-inositol. On the basis of the structures of their glucosyl transfer products, glycerol and myo-inositol were found to have a common structure with three hydroxyl groups corresponding to the hydroxyl group of the glucose molecule at positions 2, 3 and 4. The conformation of these three hydroxyl groups in the structure is equatorial. This structure is the substrate recognition site of KP. It has been suggested that KP strictly recognizes the structures of glycerol and myo-inositol, and catalyzes the transfer reaction of a glucose residue to the hydroxyl group at position 1 in glycerol, and at position 1 or 5 in myo-inositol, corresponding to position 2 in glucose.

The influence of saccharides on the formation of acrylamide (AcA) was investigated. The reducing saccharides reacted with asaparagine to form AcA, but the non-reducing saccharides, except sucrose, gave no AcA. AcA formation from a mixture containing glucose and asaparagaine was suppressed by the non-reducing saccharides, especially trehalose (76% suppression) and neotrehalose (75% suppression). Glucose is heat-degraded into pyruvaldehyde and 5-hydroxymethyl-2-furfural in the water system. The degradation products react with asparagines to generate AcA. Trehalose appears to inhibit not only the formation of these intermediates and asparagines for AcA, but also the AcA formation from these intermediates.

A bacterial strain M6, isolated from soil and identified as Arthrobacter globiformis, produced a novel nonreducing oligosaccharide. The nonreducing oligosaccharide was produced from starch using a culture supernatant of the strain as enzyme preparation. The oligosaccharide was purified as a crystal preparation after alkaline treatment and deionization of the reaction mixture. The structure of the oligosaccharide was determined by methylation analysis, mass spectrometry, and (1)H and (13)C NMR spectroscopy, and it was demonstrated that the oligosaccharide had a cyclic structure consisting of four glucose residues joined by alternate alpha-(1--4)- and alpha-(1--6)-linkages. The cyclic tetrasaccharide, cyclo-{--6)-alpha-D-Glcp(1--4)-alpha-D-Glcp(1--6)-alpha-D-Glcp(1--4)-alpha-D-Glcp(1--}, was found to be a novel oligosaccharide, and was tentatively called cyclic maltosyl-maltose (CMM). CMM was not hydrolyzed by various amylases, such as alpha-amylase, beta-amylase, glucoamylase, isoamylase, pullulanase, maltogenic alpha-amylase, and alpha-glucosidase, but hydrolyzed by isomalto-dextranase to give rise to isomaltose. This is the first report of the cyclic tetrasaccharide, which has alternate alpha-(1--4)- and alpha-(1--6)-glucosidic linkages.

A gene encoding a trehalose phosphorylase was cloned from Thermoanaerobacter brockii ATCC 35047. The gene encodes a polypeptide of 774 amino acid residues. The deduced amino acid sequence was homologous to bacterial maltose phosphorylases and a trehalose 6-phosphate phosphorylase catalyzing anomer-inverting reactions. On the other hand, no homology was found between the T. brockii enzyme and an anomer-retaining trehalose phosphorylase from Grifola frondosa.

Glucosyl-L-sorbose, -maltose, and -sucrose were synthesized using kojibiose phosphorylase (KPase) from Thermoanaerobacter brockii ATCC35047 with beta-D-glucose-1-phosphate (beta-G1P) as a glucosyl donor. One disaccharide and two trisaccharides thus synthesized were isolated by Toyopearl HW-40S column chromatography. The results of KPase digestion, methylation analysis, and 13C-NMR studies indicated that these oligosaccharides were alpha-D-glucopyranosyl-(1-->5)-alpha-L-sorbopyranose, alpha-D-glucopyranosyl-(1-->2)-alpha-D-glucopyranosyl-(1-->4)-D-glucopyranose (4-alpha-D-kojibiosyl-glucose), and alpha-D-glucopyranosyl-(1-->2)-alpha-D-glucopyranosyl-(1-->2)-beta-D-fructofuranoside, which are all novel oligosaccharides. Glucosyl-L-sorbose was partially hydrolyzed to glucose and L-sorbose by alpha-glucosidases, while glucosyl-sucrose and glucosyl-maltose were not hydrolyzed by glucoamylase, alpha-glucosidases, or CGTase.