A lectin was isolated from the shoots of bamboo Phyllostachys pubescens by using affinity chro matography on Sephadex G-75. In hemagglutination inhibition assays, this lectin exhibited a binding specificity toward maltose and methyl α-mannoside. The N-terminal sequence was determined as Lys-Ser-Cys-Cys-Pro-Ser-Thr-Thr-Ala and identical with that of the American mistletoe toxin, ligatoxin A.

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Three polygalacturonases (PGs) were purified homogeneously in SDS-PAGE from the culture filtrate of white rot basidiomycete, Fomitopsis cytisina . By analysis of reaction products, two of three PGs were concluded to be endo-type (endoPG I and II) and the other to be exo-type (exoPG) . Molecular masses of endoPG I and II were both estimated to be 38 kDa and that of exoPG 50-60 kDa. Their molecular masses by deglycosylating with EndoH decreased by 37, 37 and 44 kDa, respectively. Isoelectric points of endoPG I, II and exoPG were 4.3, 4.2 and 4.0, respectively . Their amino acid compositions were similar to one another and furthermore, endoPG I and II were very similar in the 20 amino acids sequence of N-terminal. Their properties, including their kinetic parameters, were determined. Both endoPG I and II could act more on oligo- and polyGalUAs than hepta of DP and endoPG II could act on tri- and diGalUA. The characteristics of endoPGs from this fungus showed considerable difference from that of Stereum purpureum in spite of allogeneic white rot basidiomycete, being classified under the same Aphyllophorales as S. purpureum. ExoPG had the highest activity on nonaGalUA and, like plants exoPG, could not act on 4, 5-unsatd. oligoGalUA.

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Starch granules were prepared from tubers of Chinese arrowhead (Sagittaria trifolia L. var. edu-lis (Sieb.) Ohwi), 3 cultivars: Aokuwai (Ao), Shirokuwai (Shi) and Suitakuwai (Sui), and their 6 F, lines: Ao×Ao, Ao×Shi, Ao×Sui, Shi×Ao, Shi×Sui and Sui×Sui. The amylose content of the starches determined by amperometric iodotitrimetry were in a range of 28.2-29.9% and 26.6-31.3% by gel-permeation chromatography (GPC) of isoamylase-debranched starches. High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) of isoamylase-debranched starches showed the starches had unique amylopectin short chain length distributions of increased amounts of chains with DP 6 and 7, and decreased amounts of chains with DP 9 compared with waxy maize amylopectin. The X-ray diffractograms of the starches showed A type patterns. Among the 3 cultivars, the granular size and temperature of gelatinization by DSC of the starches were in the ascending order of Shi > Ao > Sui, and starch-granule digestibility by amyl-ase and solubility and swelling power at 70°C of the starches were in the descending order of Shi >Ao>Sui. There were positive correlations between starch-granule digestibility by amylase and either solubility at 60°C and 70°C or swelling power at 60°C and 70°C of the starches, and negative correlations between starch-granule digestibilities by amylase and temperatures of gelatinization (To and Tp) by DSC of the starches.

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Short-amylose chains released from normal corn starch with isoamylase were studied to establish an analytical method for getting directly structural information of amylopectin. The partial hydrolysates obtained without separation of amylose and amylopectin were fractionated into three fractions (fr.1, fr. 2 and fr. 3) by gel chromatography of Toyopearl HW-50S. The chain-length distribution of the fr. 3 containing shortish chains was analyzed by using HPAEC-system equipped with a pulsed amperometric detector (HPAEC-PAD). Although the shortamylose chains in the fr. 3 came from not only amylopectin but also branched amylose, it was estimated that the chains relating to branched amylose were minor in the hydrolysate. The analytical method getting directly a general information of amylopectin was conducted to typical rice starches produced in Japan as an applied experiment.

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Physiological effect of L-arabitol was investigated by feeding Sprague-Dawley male rats for four weeks with a diet containing 2.5% L-arabitol in comparison with the control diet, containing cellulose. The weights of kidney and cecum increased, while adipose tissue decreased significantly in spite of no difference in their body weights.

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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.

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The ultraviolet spectrum of 1, 5-anhydro-D-fructose (1, 5-anhydro-D-arabino-hexo-2-ulose, 1, 5-AF) showed two absorption peaks at 215 and 266 nm, and the absorption at 215 nm was higher . The molar absorptivity at 215 nm was low (34 M^-1· cm^-1) and was unsuitable for the assay. The conven tional methods for the determination for total sugar were unsuitable for 1, 5-AF because the phenolsulfuric acid method gave only 15% value of glucose and no coloration by the anthrone-sulfuric acid method. On the other hand, 1, 5-AF reduced Cu²⁺ (the method of Somogyi and Nelson or neocuproine assay) and Fe³⁺(the method of Park and Johnson) even at 35°C. The former reaction enabled the specific determination of 1, 5-AF in the presence of the other hexoses and pentoses . Neocuproine assay for reducing sugars was sensitive (0-50μg/mL) to 1, 5-AF and could perform on a small scale (sample 20μL) on a microtitre plate.

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Dextrin dextranase (DDase, EC 2.4.1.2) was extracellularly secreted in a culture medium of Acetobacter capsulatum ATCC 11894 containing both glucose and a small amount of dextrin as the essential carbon sources. The enzyme was simply purified by a high-speed refrigerated centrifugation, and immediately dialyzed against 50 mM acetate buffer (pH 4.5). The purified DDase gave a single protein band on both Native- and SDS-PAGE. The molecular mass of the purified enzyme was estimated to be about 152 kDa (SDS-PAGE). The optimum pH and temperature of the enzyme were 5.2 and 38°C, respectively. The K_m (mM) and V_max (mg dextran/mg protein/min) values for mal-tooligosaccharides (DP 3-7) and short-chain amylose (DP 17.3) were estimated to be about 10.2, 1.74; 6.41, 2.56; 3.34, 2.64; 2.59, 2.39; 1.66, 2.17; 0.12, 2.23, respectively. The conversion rate of maltodextrins into dextran increased with the increases in the DP number of donor sub-strates. The maximum yield of product dextran reached 73.9% by using short-chain amylose as a substrate. DDase showed a strong affinity on the sugars having non-reducing terminal linked with either α-1, 4- to α-1, 6-glucosidic bond. The affinity of enzyme on acceptor substrates increased with the increases in the DP number of sugars tested. Various oligosaccharides were formed, when DDase reacted on a maltose-dextran mixture. The ratio of α-1, 4- to α-1, 6-glucosidic linkages in a product dextran molecule was calculated to be about 1: 20. The average molecular mass of product dextran was estimated to be about 1270 kDa. The chemical structures of synthesized glucooligo-saccharides (DP =3-7) from a maltose-dextran mixture were 4-ο-α-isomaltosyl-D-glucose, 4-ο-α-isomaltotriosyl-D-glucose, 4-ο-α-isomaltotetraosyl-D-glucose, 4-ο-α-isomaltopentaosyl-D-glucose and 4-ο-α-isomaltohexaosyl-D-glucose. On the basis of many experimental data, we propose the possible transglucosylation actions of DDase which consist of five reaction routes.

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Malto-oligosyltrehalose synthase (EC 5.4.99.15, MTSase) is a glycosyltransferase catalyzing the conversion of maltodextrins to malto-oligosyltrehaloses by forming α, α-1, l-glucosidic linkage. The enzyme slightly catalyzes the release of glucose from maltodextrins by hydrolysis of the α-1, 4 linkage of maltodextrins on the reducing end. The amino acid sequence of MTSase from Sulfolobus acidocaldarius ATCC 33909 showed significant homologies to those of not only other bacterial MTSases but also α-amylase family enzymes. The S. acidocaldarius MTSase lost the enzyme ac tivity by site-directed mutation of Asp228, G1u255, or Asp443 of the enzyme, which corresponded to the catalytic residues of α-amylase family enzymes. The mutation of Lys390 or Lys445 brought about a decrease of the transfer activity and an increase of the hydrolysis activity of the enzyme. The two lysine residues are conserved among MTSases but not α-amylase family enzymes. The tertialy structure analysis revealed that MTSase as well as α-amylase family enzymes contained a (β/α)₈ barrel structure as the catalytic domain. The three carboxyl residues (Asp228, G1u255 and Asp443) proposed as the catalytic center of MTSase were found to be located in the active cleft and positioned similarly to those of a -amylase family enzymes. There were two loops and one he lix structures on one end of the active cleft, where a nearly closed space was formed. The lysine residues (Lys390 and Lys445) were found to be situated in the closed structure. It was suggested that several residues containing the two lysines in the closed structure were involved in catalysis of the α, α-1, 1 transfer activity of MTSase.

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Themoactinomyces vulgaris R-47 produces two a-amylases, TVA I and TVA II, both of which hydrolyze pullulan and cyclodextrins. Site-directed mutagenic and X-ray crystallographic studies ofboth enzymes were carried out. The putative catalytic residues, Asp325, G1u354, and Asp421 of TVA II were modified. The mutated enzymes retained a trace of activity, less than 0.01% of the wild-type enzyme, and the action pattern of D421N enzyme was different from wild-type and othermutated TVA II. The crystal structures of the mutated TVA II complexed with β-cyclodextrin were determined. TVA II has domains A, B and C, like other a-amylases, and also has domain N, which is not commonly found in α-amylase family enzymes. A β-cyclodextrin was bound to domain A, and was also located close to domain B. Crystals of TVA I were obtained by the vapor diffusion method in the presence of polyethylene glycol. Like TVA II, TVA Iwas composed of domain N, A, B, and C. Comparing the domains of TVA I and TVA II, the structure and position of domain N were the most different. To investigate the roles of domain N further, domains N of TVA I and TVA II were truncated, and the activities of these enzymes were drastically decreased.

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Thermococcus litoralis 4-α-glucanotransferase (GTase) belongs to family 57 of glycosyl hydro-lases and catalyzes the disproportionation and cycloamylose synthesis reactions. Using 3-ketobutylidene-β-2-chloro-4-nitrophenyl-maltopentaoside (3 KBG 5 CNP) as a donor and glucose as an acceptor, we showed that the disproportionation reaction of 4-α-glucanotransferase involves a Ping-Pong Bi Bi mechanism. Based on this reaction mechanism, we trapped the glycosyl-enzyme intermediate by treating the enzyme with 3 KBG 5 CNP in the absence of an acceptor. MALDITOFMS and PSD analysis of a peptic digest of the intermediate and great decrease in activity of the E 123 Q mutant enzyme indicated that completely conserved Glu 123 was the catalytic amino acid. Next, we have determined the structure of GTase, the first structure of the family 57 of glycosyl hydrolases, at 2.8 Å resolution. The structure of GTase is composed of three domains. N-terminal domain (domain A) contains two catalytic residues and has a novel pseudo-TIM barrel fold. C-terminal domain (domain C) is mainly made of β-strands arranged in two layered β-sheet sandwich. Domain B is located between domains A and C, and consists of α- and 3₁₀-helices. Active site cleft lies between domains A and B. The cleft is partially covered with aromatic residues to form a tunnel-like shape, which probably plays an important role in the formation of large cyclic glucans.

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The action of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) on amylose was reinvesti-gated by determining the product specificity of the cyclization reaction and the substrate specificity of the coupling and hydrolytic reactions of CGTases from alkalophilic Bacillus sp. A2-5a (A2-5a CGTase), Bacillus macerans CGTase (Bmac CGTase) and Bacillus stearothermophilus CGTase (Bste CGTase). The product specificity of the cyclization reaction of CGTase was determined by quantifying each cyclic α-1, 4-glucans with degree of polymerization (DP) from 6 to 31 (CD6 to CD31; CDn, cyclic α-1, 4-glucan with DP of n) produced at the initial reaction stage with HPAEC. All three CGTases produced CD6 to CD31 simultaneously. However, A2-5a CGTase produced more CD7, while Bmac CGTase produced more CD6 than other cyclic α-1, 4-glucans. The speci-ficity to produce CD7 or CD6 was very strict since the cyclic α-1, 4-glucan having a difference of only one glucose unit were not preferably produced by either enzyme. 4n the other hand, Bste CGTase produced more CD6 and CD7 than other cyclic α-1, 4-glucans, respectively. The substrate specificity of the coupling and hydrolytic reactions were determined by using CD6, CD7, CD8 and larger cyclic α-1, 4-glucans (mixture of CD22 to CD50). For the coupling and hydrolytic activities of all three CGTases, CD7 and CD8 were extremely poor substrate, while those activities on larger cyclic α-1, 4-glucans were higher than CD6, CD7 and CD8. The substrate specificity in the cou-pling and hydrolytic reaction could be attributed to the conversion of larger cyclic α-1, 4-glucans into smaller ones. The hydrolytic activity of CGTases on larger cyclic α-1, 4-glucans were 1/1.6 to 1/5 of the coupling activity, which indicated that the hydrolytic activity of CGTase was not so small as negligible. The hydrolytic activities of Bste CGTase were the highest among the three CGTases, while those of Bmac CGTase were the lowest, which would cause the difference in the conversion rate from larger cyclic a-1, 4-glucans into smaller ones. Based on these results, we discussed the action of CGTase on amylose.

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In Kagoshima, citric acid and shochu are produced on a commercial basis by the koji of Aspergillus groups. The black Aspergillus groups are known to produce two types of a-amylase, the acid-stable and the neutral, besides glucoamylase. In this study, we purified the acid-stable enzyme (UAA) from the acidic extract of the citric acid koji, and studied some enzymatic properties, especially the action pattern towards maltooligosaccharides, by comparison with the neutral enzyme (UNA) from the koji. Product analysis showed that UAA demonstrated a distinct cleavage pattern towards oligosaccharides more than G4, which suggests that UAA has a different subsite structure from the ordinary neutral a-amylase such as Taka-amylase A. The number of subsite is estimated to be 5, and subsite affinities of Al, A2, and A5 were evaluated to be 1.2 kcal/mol, 1.4 kcal/mol and 2.1 kcal/mol. Experiments with chemical modification suggested that UAA has a residue of Leu instead of Lys209 on Taka-amylase A, and a residue of Glu instead of His2l0. A new substrate Gal-G3-CNP was enzymatically synthesized from lactose and G3-CNP in order to discriminate between the two a -amylases, the acid-stable and the neutral.

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Endo-β-N-acetylglucosaminidase (endo-β-GlcNAc-ase) is a unique endoglycosidase that hydrolytically cleaves the N, N′-diacetylchitobiose moiety of asparagine-linked oligosaccharides of various glycoproteins. A novel endo-β-GlcNAc-ase in the culture fluid of Mucor hiemalis isolated from soil was found to have transglycosylation activity. This endo-β-GlcNAc-ase, Endo-M, couldtransfer the intact complex-type oligosaccharide from glycopeptide to suitable acceptors with an Nacetylglucosamine (GlcNAc) residue during hydrolysis of the glycopeptide. Using Endo-M, the chemo-enzymatic synthesis of a bioactive glycopeptide by chemical synthesis of N-acetylglucosaminyl peptide and enzymatic transfer of oligosaccharide was attempted. Peptide T can block HIV infection of human T cells. We added the sialo complex-type oligosaccharide to chemically synthesized N-acetylglucosaminyl Peptide T using the transglycosylation activity of Endo-M. The glycopeptide T thus produced showed a higher degree of resistance to protease digestion. We also prepared calcitonin glycopeptide by the chemo-enzymatic method described above. Calcitonin is a calcium-regulating hormone which is widely used in therapy for hyper calcemia. This glycopeptide demonstrated sufficient physiological activity. N-Acetylglucosaminyl glutamine was also a good glycoside acceptor of Endo-M. We were able to add oligosaccharides to the glutamine residues of Substance P neuropeptide and a-mating factor of yeast Saccharomyces cerevisiae using Endo-M. The glycosylated Substance P was biologically active and stable against peptidase digestion. On the other hand, the bioactivity of the glycosylated a-mating factor was lower than that of native one, although that of N-acetylglucosaminyl a-mating factor was higher. Using the transglycosylation activity of Endo-M, we prepared alkyl compound having oligosaccharide of glycoprotein. We used it as an antigen for preparing monoclonal antibody against oligosaccharide of glycoprotein.

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A recent trend has been to use commonly available foods in the prevention of disease. Such application is dependent on a food chemical and physical composition. We investigated functional ac tivity of substances derived from pentose when given in combination with various saccharides to rats. We observed that pentose-derivatives (fructosylxylosides, xylitol, some nucleosides) had a sup pressive effect on the elevation of blood glucose and insulin levels in rats given sucrose or soluble starch. The effect was due to the inhibition of disaccharide hydrolases. We also found that fructo sylxylosides and adenosine enhanced mineral (calcium and magnesium) absorption when given in combination with sucrose.

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Enzyme assay was carried out against wild, cultivate commercial stocks of strain and their variation stocks for analyzing their genetic differences and relations with the expression character. (1) Discrimination of various stocks of strain Flammulina velutipes : Zymograms of Esterase from F. velutipes showed many bands on the gel and variations among the different strains, and this method was very effective to distinguish the strains. The strains of F. velutipes in this experiment was largely classified to three groups (tentatively named A, B and C) according to the cluster analysis of esterase zymograms. All cultivate commercial strains belong to A group. The differ ences of physiological and morphological characteristic among the each groups were observed with mycelial growth, cap color and the size of basidiospore, and in addition these groups were different biological species each other from the crossbreeding test. (2) Screening of undesirable change in budding: The activity of peroxidase from F. velutipes was measured against normal stocks and variation stocks of strain for screening undesirable changes in budding. Also zymograms analysis of peroxidase was carried out concurrently. A specific band which is related with the germination abilities was observed on zymograms. Furthermore, a strong correlation (γ= 0.8590) between the peroxidase activities and the degree of germination abilities was found by statistical consideration. From this results it is suggested that peroxidase is one of the key enzyme in the process of germination of F. velutipes.

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Chitooligosaccharide and maltooligosaccharide were initially converted into useful oligosaccha rides, utilizing glycosidase-mediated transglycosylation. Hexa-N-acetylchitohexaose as a biologically active substance and p-nitrophenyl penta-N-acetyl β-chitopentaoside as a novel substrate of lysozyme assay were synthesized from chitooligosaccharides by hen egg-white lysozyme. p- Nitrophenyl α-maltopentaoside was synthesized from maltopentaose by maltotetraose-forming amylase. It was further converted into end-blocked p-nitropheny 4⁵-O-β-D-galactosyl-amaltopentaoside, which was designed as a substrate for diagnosis of human amylase in serum. Galactosylmaltooligonolactone was also designed and synthesized as substrate analogue inhibitor for mammalian α-amylase. Next an efficient method for synthesizing important di-, tri-, and tetrasaccharide units involved in glycoconjugate was developed. Beginning with β-galactosyl-disaccharide units library, a series of β-N-acetylglucosaminyl-, a-L-fucosyl- and β-mannnosyltrisaccharides were regioselectively synthesized by using some glycosidase-mediated transglycosylations. Consecutive synthesis of human milk tetrasaccharide and mucin type tisaccharide was carried out by enzymatic addition of two sugar residues. Such synthetic oligosaccharides were used to synthesize artificial glycopolymer. The polymers were shown to be useful as probes of carbohydrate recognition and modeling in the analysis of glycopolymer-lectin interaction.

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1. Starches were prepared from many kinds of sweet potatoes, which are mainly used for starch production, differing in variety, stage of development and tissue zone. Average granule size, amy lose content, gelatinization properties by differential scanning calorimetry and digestibility of raw starch by glucoamylase were compared. Digestibility by glucoamylase tended to be higher in the earlier stage of development and the cambium zone. Further, correlations among these starch properties were calculated to determine the factors relating to digestibility of raw starch by glucoamy lase. Negative correlations were observed between gelatinization heat and digestibility as well as average granule size and digestibility. 2. Starches were also prepared from many kinds of purple-and orange-fleshed sweet potatoes to investigate the effect of the cultivation conditions on starch properties. Earlier planting and harvesting dates apparently enhanced gelatinization temperature and tended to reduce the content of short chains of amylopectin. A major difference in starch properties, especially gelatinization temperature and amylopectin structure, occurs according to soil tem perature during the development of sweet potato tuberous roots. To elucidate the extensive variation in starch gelatinization properties within the same botanical origin for sweet potato, correlations were determined between gelatinization parameters and starch molecular characteristics. Starch with a lower gelatinization temperature had relatively abundant extremely shorter unit-chains of amylopectin.

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D-enzyme (EC 2.4.1.25) is believed to be involved in starch metabolism, but the function in vivo is not known. In order to investigate the role of D-enzyme, several approaches have been under taken. Transgenic potato plants with dramatically reduced D-enzyme activity were obtained by introducing sense and antisense D-enzyme cDNA sequences with the appropriate promoter sequence. The tubers from these plants sprouted later and the growth of sprouts was slower than the wild type. However, no significant difference was found in starch produced in tubers, either in its quantity or quality. These results seems to suggest the role of D-enzyme in starch degradation rather than starch synthesis. The analysis of the action of D-enzyme on amylose and amylopectin revealed that the enzyme can catalyze the cyclization reaction and produce cycloamylose with DP larger than 17. Cycloamylose produced are highly soluble in water and can incorporate various compounds in their helical cavities, thus expected to find exploitation in food and pharmaceutical industries.

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