Norio Shiomi

Properties of Fructosyltransferases Involved in the Synthesis of Fructan in Liliaceous Plants

Summary We isolated nine fructo-oligosaccharides from asparagus roots and onion bulbs, and identified them aauthor F (1-s-D-fructofuranosyl) n sucrose n = 1, 2 and 3, 6 G (1-s-D-fructofuranosyl) n sucrose n = 1, 2 and 3 and l F (1-β-d-fructofuranosyl) m -6 G (1-s-D-fructofuranosyl) n Sucrose m = 1, n = 1; m = 2, n = 1; and m = 1,n = 2. The nine fructo-oligosaccharides were synthesized in vitro from sucrose by enzyme preparations ofasparagus roots and onion bulbs. Three different types of fructosyltransferases are suggested to be involved in the synthesis of the saccharides. The fructosyltransferases were highly purified and their substrate specificities were investigated indetail. They were confirmed to be sucrose : sucrose 1 F -s-D-fructosyltransferase (SST, EC 2.4.1.99), 1 F (1-s-D-fructosyl) n sucrose : 1 F (1-β-d-fructosyl) m sucrose 6 G -s-D-fructosyltransferase (6 G -FT) and 1F(1-s-D-fructosyl) n sucrose: 1 F (1-s-D-fructosyl) m sucrose 1 F -s-D-fructosyltransferase (1 F -FT). From the properties and substrate specificities of SST, 6 G -FT and 1 F -FT, a scheme for the biosyntheticpathways of fructans in asparagus and onion plants was proposed.

Two Novel Oligosaccharides Formed by 1F-Fructosyltransferase Purified from Roots of Asparagus (Asparagus officinalis L.)

Two novel oligosaccharides, tetra-and penta-saccharides were synthesized by fructosyl transfer from 1-kestose to 4G-β-D-galactopyranosylsucrose with a purified 1F-fructosyltransferase of asparagus roots and identified as 1F-β-D-fructofuranosyl-4G-β-D-galactopyranosylsucrose, O-β-D-fructofuranosyl-(2→1)-β-D-fructofurano-syl-O-[β-D-galactopyranosyl-(1→4)]-α-D-glucopyranoside and 1F(1-β-D-fructofuranosyl)2-4G-β-D-galactopyranosylsucrose, [O-β-D-fructofuranosyl-(2→1)]2-β-D-fructofuranosyl-O-[β-D-galactopyranosyl-(1→4)]-α-D-glucopyranoside, respectively. Both oligosaccharides were scarcely hydrolyzed by carbohydrase from rat small intestine. Human intestinal bacterial growth by 1F-β-D-fructofuranosyl-4G-β-D-galactopyranosylsucrose was compared with that by the tetrasaccharides, stachyose and nystose. Bifidobacteria utilized 1F-β-D-fructofuranosyl-4G-β-D-galactopyranosylsucrose to the same extent as stachyose or nystose. On the other hand, the unfavorable bacteria, Clostridium perfringens, Escherichia coli and Enterococcus faecalis, that produce mutagenic substances did not use the synthetic oligosaccharide.

NMR analysis of tri- and tetrasaccharides from asparagus

Detailed analysis of the 1H and 13C NMR spectra of five fructo‐oligosaccharides (1–5) isolated from Asparagus officinalis L. (Liliaceae) was carried out. Tetrasaccharides 1, 2 and 3 consist of one unit of glucose and three units of fructose, whereas trisaccharides 4 and 5 consist of one unit of glucose and two units of fructose. The 1H NMR signals of these tetrasaccharides were severely overlapped around δH 3.6–4.0 ppm and the 13C NMR signals of the corresponding signals of three fructose residues were close to each other. Therefore, it was difficult to make assignments of those signals using conventional NMR methods. Overcoming these signal‐overlapping problems required the application of some special NMR techniques. The spectral analysis was started from the anomeric proton of the glucose, since each compound had only one glucose residue and its 1H NMR spectrum showed a separate characteristic anomeric proton signal. After the 1H and 13C signals of the glucose had been assigned, the fructose residues attached to the C‐1 and/or C‐6 positions of glucose were determined from the HMBC spectrum, which also revealed further fructosyl fructose linkages. The assignments of each signal of C‐5 and C‐6 of the fructose residues overlapped in 2D spectra were achieved by the 1D INAPT method. Unresolved correlation peaks of methylene protons in HMQC could be separated by the CH2‐selected HSQC method, which gave sufficient 13C resolution to separate each CH2 signal, resulting in the determination of the chemical shift of the methylene protons. Copyright

Effect of temperature on soluble invertase activity, and glucose, fructose and sucrose status of onion bulbs (Allium cepa) in store

The activity of soluble invertase, and the variation in glucose, fructose and sucrose contents in onion bulbs (Allium cepa) during long-term storage at 10°C and 20°C were investigated. Invertase activity increased progressively after 8 weeks to 0.084 and 0.092 nkat/g fresh weight (FW), then sharply to 0.29 and 0.35 nkat/g FW at 20°C and 10°C, respectively, and remained high during 5 weeks. Then, activity decreased abruptly to 0.039 and 0.041 nkat/g, and remained low during the last 8 weeks and close to that observed initially. Glucose increased to 17.73 and 14.62 mg/g FW after 4 weeks at 20°C and 10°C, respectively, then decreased sharply between week 5 and week 7 to 4.13 and 4.91 mg/g FW, respectively, and remained rather stable ranging from 9 and 10 mg/g FW at both temperatures. Fructose showed a similar pattern and was 14.8 and 21.68 mg/g FW at 20°C and 10°C, respectively. Between week 10 and week 24, fructose ranged from 5 and 6 mg/g FW, and from 6 and 7 mg/g FW at 20°C and 10°C, respectively. Sucrose increased to 19.63 and 14.43 mg/g FW at 20°C and 10°C, respectively, decreased during 3 weeks, and then increased randomly from 5.69 to 9.42 mg/g FW at 20°C, but remained in a steady state at 10°C ranging 5.03±0.78 mg/g FW. During the last 6 weeks, the sucrose content was higher at 20°C than at 10°C. The fructose–glucose ratio varied during the first 8 weeks but remained at a steady level during the last 16 weeks. The (glucose+fructose)/sucrose ratio increased randomly at 10°C, whereas at 20°C the ratio increased during 10 weeks then decreased progressively during the final 14 weeks.

Characterization of recombinant β-fructofuranosidase from Bifidobacterium adolescentis G1

BackgroundWe have previously reported on purification and characterization of β-fructofuranosidase (β-FFase) from Bifidobacterium adolescentis G1. This enzyme showed high activity of hydrolysis on fructo-oligosaccharides with a low degree of polymerization. Recently, genome sequences of B. longum NCC2705 and B. adolescentis ATCC 15703 were determined, and cscA gene in the both genome sequences encoding β-FFase was predicted. Here, cloning of cscA gene encoding putative β-FFase from B. adolescentis G1, its expression in E. coli and properties of the recombinant protein are described.ResultsUsing the information of cscA gene from Bifidobacterium adolescentis ATCC 15703, cscA gene from B. adolescentis G1 was cloned and sequenced. The N-terminal amino acid sequence of purified β-FFase from B. adolescentis G1 was identical to the deduced amino acid sequences of cscA gene from B. adolescentis G1. To confirm the translated product of the cscA gene, the recombinant protein was expressed in Escherichia coli. Molecular mass of the purified recombinant enzyme was estimated to be about 66,000 by SDS-PAGE and 60,300 by MALDI TOF-MS. The optimum pH of the enzyme was 5.7 and the enzyme was stable at pH 5.0-8.6. The thermostability of the enzyme was up to 50°C. The Km (mM), Vmax (μmol/mg of protein/min), k0 (sec-1) and k0/Km(mM-1 sec-1) for 1-kestose, neokestose, nystose, fructosylnystose, sucrose and inulin were 1.7, 107, 107.5, 63.2, and 1.7, 142, 142.7, 83.9, and 3.9, 152, 152.8, 39.2, and 2.2, 75, 75.4, 34.3, and 38, 79, 79.4, 2.1, and 25.9, 77, 77.4, 3.0, respectively. The hydrolytic activity was strongly inhibited by AgNO3, SDS, and HgCl2.ConclusionThe recombinant enzyme had similar specificity to the native enzyme, high affinity for 1-kestose, and low affinity for sucrose and inulin, although properties of the recombinant enzyme showed slight difference from those of the native one previously described.

Chilling effect on soluble sugars, respiration rate, total phenolics, peroxidase activity and dormancy of onion bulbs

Besides onions being one of the most cultivated and consumed vegetables, during storage onion bulbs are still affected by many physiological, biochemical and technological factors which can influence their quality. Respiration rate (RRO2), soluble sugars (SS), total phenolics (TP), and peroxidase (POD) activity were measured in inner bud tissues during a dormancy break of onion bulbs treated four weeks at 0oC and stored in the dark at 20oC. Control bulbs were stored simultaneously in the same condition. Breakage of dormancy was checked by the appearance of first green internal leaves by cutting longitudinally 30 bulbs. After eight weeks, RRO2 of sprouted onions was 52% higher than that of freshly harvested and dormant bulbs. One week after cooling SS decreased from 15 to 9 mg g-1 fresh weight, and then peaked from 9 to 19 mg g-1 after three weeks. For control bulbs, a similar peak was observed after six weeks. For inner buds of cold-treated onions, a slight increase of TP (from 0.17 to 0.2 mg g-1; fresh weight) was observed during the first two weeks of cooling, and then a decrease to 0.11 mg g-1 was observed after eight weeks. For inner buds of control bulbs, TP also increased slightly from 0.17 to 0.2 mg g-1 after five weeks, and decreased to 0.15 mg g-1 after seven weeks when bulbs began to sprout. POD activity showed a similar pattern in relation to TP. For cold-treated bulbs, POD activity increased to 1.7 U g-1 fresh weight after two weeks, and decreased to 1.1 U g-1 during the last four weeks. For control samples, POD activity was stable during 4 weeks and decreased progressively by 29% during the last four weeks. This decrease in POD activity coincided with the decrease in TP, and coincided with onset of sprouting. With cold treatment, first sprouts were observed during the third week, while total sprouting was observed after eight weeks. In comparison, only 20% of the control bulbs sprouted after the period of 8 weeks.

Synthesis and structural analysis of five novel oligosaccharides prepared by glucosyltransfer from β-D-glucose 1-phosphate to isokestose and nystose using Thermoanaerobacter brockii kojibiose phosphorylase

Five novel oligosaccharides (tetra-, penta- and hexa-saccharides) were synthesized by glucosyltransfer from beta-D-glucose 1-phosphate to isokestose (O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-alpha-D-glucopyranoside) or nystose (O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-alpha-D-glucopyranoside) using Thermoanaerobacter brockii kojibiose phosphorylase. The oligosaccharides were identified as 2(2-alpha-D-glucopyranosyl)(m)isokestose; [O-alpha-D-glucopyranosyl-(1-->2)](m)-O-[beta-D-fructofuranosyl-(2-->1)](2)-alpha-D-glucopyranoside: m=1, 2, and 3, and 2(2-alpha-D-glucopyranosyl)(n)nystose; [O-alpha-D-glucopyranosyl-(1-->2)](n)-O-[beta-D-fructofuranosyl-(2-->1)](3)-alpha-D-glucopyranoside: n=1 and 2 using gas liquid chromatography analysis of the methyl derivatives, and MALDI-TOF-MS and NMR measurements of the newly formed oligosaccharides. 1H, 13C NMR signals of each saccharide were assigned using 2D-NMR techniques, including COSY, HSQC, HSQC-TOCSY, HMBC, CH(2)-selected E-HSQC, and CH(2)-selected E-HSQC-TOCSY.

Serum glucose and insulin response in rats administered with sucrose or starch containing adenosine, inosine or cytosine.

Blood glucose and insulin responses and gastric emptying were examined in rats intubated with sucrose or soluble starch that contained adenosine, inosine and cytosine. The increase in serum glucose and insulin levels in the rats following loading with sucrose (2.5 g/kg of body weight) or soluble starch (1.875 g/kg of body weight) was significantly reduced by the administration of adenosine, inosine and cytosine (0.0625-0.125 g/kg of body weight). The gastric emptying rates were only marginally affected by the nucleoside administration. The activities of sucrase, maltase, isomaltase and glucoamylase in a crude preparation from the small intestinal mucosa of rats were mildly inhibited by the nucleosides. The decrease in blood glucose and insulin levels may have been in response to a decrease in glucose absorption caused by the inhibiting effect of the nucleosides on the mucosal enzymes that digest sucrose, maltose, and malto- and isomalto-oligosaccharides.

Cloning and functional characterization of a fructan 1-exohydrolase (1-FEH) in edible burdock (Arctium lappa L.)

BackgroundWe have previously reported on the variation of total fructooligosaccharides (FOS), total inulooligosaccharides (IOS) and inulin in the roots of burdock stored at different temperatures. During storage at 0°C, an increase of FOS as a result of the hydrolysis of inulin was observed. Moreover, we suggested that an increase of IOS would likely be due to the synthesis of the IOS by fructosyltransfer from 1-kestose to accumulated fructose and elongated fructose oligomers which can act as acceptors for fructan:fructan 1-fructosyltransferase (1-FFT). However, enzymes such as inulinase or fructan 1-exohydorolase (1-FEH) involved in inulin degradation in burdock roots are still not known. Here, we report the isolation and functional analysis of a gene encoding burdock 1-FEH.ResultsA cDNA, named aleh1, was obtained by the RACE method following PCR with degenerate primers designed based on amino-acid sequences of FEHs from other plants. The aleh1 encoded a polypeptide of 581 amino acids. The relative molecular mass and isoelectric point (pI) of the deduced polypeptide were calculated to be 65,666 and 4.86. A recombinant protein of aleh1 was produced in Pichia pastoris, and was purified by ion exchange chromatography with DEAE-Sepharose CL-6B, hydrophobic chromatography with Toyopearl HW55S and gel filtration chromatography with Toyopearl HW55S. Purified recombinant protein showed hydrolyzing activity against β-2, 1 type fructans such as 1-kestose, nystose, fructosylnystose and inulin. On the other hand, sucrose, neokestose, 6-kestose and high DP levan were poor substrates.The purified recombinant protein released fructose from sugars extracted from burdock roots. These results indicated that aleh1 encoded 1-FEH.

Isolation and structural confirmation of the oligosaccharides containing α-D-fructofuranoside linkages isolated from fermented beverage of plant extracts.

Fermented beverage of plant extracts was prepared from the extracts of approximately 50 types of vegetables and fruits. Natural fermentation was carried out mainly by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp. and Pichia spp.). Two oligosaccharides containing an α-fructofuranoside linkage were detected in this beverage and isolated using carbon-Celite column chromatography and preparative HPLC. The structural confirmation of the saccharides was determined by methylation analysis, MALDI-TOF-MS, and NMR measurements. These saccharides were identified as α-D-fructofuranosyl-(2→6)-D-glucopyranose, which was isolated from a natural source for the first time, and a novel saccharide β-D-fructopyranosyl-(2→6)-α-D-fructofuranosyl-(2↔1)-α-D-glucopyranoside.