A gelling polysaccharide was extracted from Ibaranori (Hypnea charoides LAMoRoux) and purified by gelation with potassium chloride. The total carbohydrate, ash and water content of the polysaccharide were 70.2, 20.4 and 3.2%, respectively. The content of total sulfate and sulfate in the ash was estimated to be 19.2 and 12.9%, respectively. Molecular mass of the polysaccharide was estimated to be about 2.3×10⁵. The polysaccharide was composed of D-galactose, 3, 6-anhydro-D-galactose and ester sulfate in a molar ratio of 1.2 : 0.9:1.2. The infrared spectrum and values of optical rotation of the polysac charide at different temperatures were in agreement with those of κ-carrageenan . Gelation occurred at a concentration of 0.2% aqueous solution upon addition of potassium chloride (0.2%) at room temperature. These results indicate that the gelling polysaccharide is a κ-carrageenan .

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The effects of hemicellulase on some rheological properties of wheat flour dough and on loaf volume in a home baker were studied. The addition of hemicellulase improved the specific volume of bread baked, and the volume was significant at the addition of more than 2.5×10³ U of the enzyme . The combined addition of calcium stealoyl-2-lactylate (CSL) or calcium gluconate (CG) with hemicellulase increased loaf volume distinctly, but the combination of those three additives did not obviously improve loaf volume. The arrival and development times of dough became short as compared to the control, and stability time decreased distinctly. Both the gelatinization temperature and enthalpy of starch in the dough tended to increase slightly, but the viscosity coefficient and modulus of elasticity decreased . The size of gas cells of crumb baked with hemicellulase did not change distinctly. The combined addition of hemicellulase with CSL or CG distinctly improved the baking property and slightly improved the storage property.

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Gelation occurred at a concentration of 0.5% of alginates (45M, 45G and 45) on addition of a mixed solution of NaCl and CaCl₂ at room temperature (25°C). The alginates showed plastic behavior at various concentrations even at 50°C. The dynamic modulus of 45M alginate rich in D-mannuronic acid was scarcely changeable with increasing temperature until 60°C, which was estimated to be a transition temperature, then it decreased gradually. Though the dynamic modulus of 45G alginate rich in L guluronic acid decreased gradually with increasing temperature until 60°C, which was also estimated to be a transition temperature, then it contrarily increased with further increases in the temperature. The dynamic modulus of alginate showed a large value in a wide range of pH values. The dynamic modulus of alginate stayed at low values on the addition of urea (4.0 M), even at low temperature (°C). An intermolecular Ca-bridge may play a dominant role in gel formation of alginate molecules in the presence of Ca²⁺ ions. Intramolecular hydrogen bonding an intermolecular hydrophobic interaction may also take part in the gel formation of D-mannuronic acid-rich and L-guluronic acid-rich alginate molecules, respectively.

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Pectin and pectic acid were used to improve the quality of sponge cake composed of flour, whole egg and sugar [3: 5 : 3 (w/w) ]. The added content of pectin or pectic acid was 0-4%, based on flour weight (0-1%, based on batter weight). A 300 g portion of basic batter was put into a pan with an 18 cm diameter and baked at 180°C for 30 min. The prepared sponge cakes were stored for 0-5 days at room temperature and the specific volume, moisture, firmness, elasticity, gelatinization degree, cross section and sensory evaluation were investigated. Pectin and pectic acid prevented a decrease in specific volume, elasticity and gelatinization degree, an increase in hardness during storage and improved the sensory properties of the sponge cake without morphological change. Pectin and pectic acid can thus be used to improve sponge cake quality as water-soluble dietary fiber sources.

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Pectin, a water-soluble dietary fiber, was used to improve the shelf life of bread. Bread was composed of flour, sugar, skim milk, shortening, sodium chloride, dry yeast and water. Dough making was prepared and baking was carried out using an automatic home bakery (National SD-BT100) . The amount of pectin added was 0-10%, based on flour weight. The prepared bread was stored at 25°C for 0-5 days and the specific volume, moisture, firmness, elasticity, pH, gelatinization degree, cross section and Hunter whiteness were investigated. The specific volume of bread increased with increases in pectin content from 1% to 6% as compared to the control. The firmness decreased with 1-7% addition of pectin, and elasticity somewhat decreased with 1-5% addition as compared to the control. The pH of the dough decreased with increases in pectin content (pH 4 at 10% pectin content) and showed the lowest value at fermentation, while recovering a little after baking. Cross sections of breads containing pectin indicated good sponge structure except for a 10% pectin content. Hunter whiteness was not substantially different from the control. Adding pectin to bread prevented decreases in specific volume, moisture, elasticity and gelatinization degree, and an increase in firmness during storage. Pectin can be effectively used to improve the shelf life of bread.

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Amylopectins of Koshihikari and Reiho were partially hydrolyzed with isoamylase immobilized on magnetic supports in order to obtain information on their sectional structures . After stopping the reactions at about 20% hydrolysis, the resulting hydrolysates were separated into three fractions (Fr1, Fr 2 and Fr 3) by gel chromatography with Toyopearl HW 65F. Chain-length distributions of the fractions were investigated by HPAEC-PAD after debranching perfectly with free isoamylase . From a comparison of these HPAEC-PAD data, it was suggested that longish chains are somewhat plentiful in the outer portion of Reiho Ap and that Koshihikari Ap is composed of shortish chains in all portions .

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Using methyl 3-O-acetyl-6-O- (p-tolylsulfonyl)-β-D-glucopyranoside (1), which was prepared in quantitative yield by lipase-catalyzed regioselective acetylation as the common intermediate, highly deoxidized monosaccharides, 2, 6-dideoxy-D-arabino-hexopyranose (6), 2, 4, 6-trideoxy-D- threo -hexopyranose(10) and 2, 4-dideoxy-D-threo-hexopyranose (12) were chemically synthesized. Compounds 6, 10, and 12 were obtained from 1 in about 37 (5 steps), 28 (4 steps) and 40% (4 steps) yields, respectively.

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A strain of Brevibacterium sp. No. 9605 was isolated from soil and produced cyclodextrin glucano transferase (CGTase, EC. 2.4.1.19). The CGTase produced mainly γ-cyclodextrin (γ-CD) from starch in the initial stage of reaction, but then the proportion of β-CD was increased and also produced a small amount of α-CD. CD formation was affected by the presence of calcium ions and ethanol. In the presence of ethanol, the yield of γ-CD from soluble starch was increased. It was thought to be due to the repression of γ-CD production from starch and the repression of γ-CD degradation by coupling reaction. Brevibacterium CGTase showed a broad acceptor specificity to various monosaccharides and phenolic compounds. When D-mannose, L-rhamnose, 1, 3-dihydroxybenzene, 1, 3, 5-trihydroxybenzene, 3-hydroxybenzyl alcohol and (+) -catechin were used as acceptors, Brevibacterium CGTase synthesized much greater amounts of transfer products than either Bacillus macerans and Bacillus stearothermophilus CGTases. The transfer products of D-mannose and kojic acid were 4-O-α-D-glucosyl-D-mannose and kojic acid 7-O-α-D-glucoside, respectively.

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Levan is a microbial polyfructan which is constructed from the β-2, 6-fructofuranosyl main chain and, β-2, 1-fructofuranosyl-linked side chains. The enzymatic synthesis of levan has been well-studied for several decades. However, there was no detailed report describing the degradation of levan and levan degrading enzymes, and most of the previous reports were on unpurified enzymes. This paper deals with the screening of levan-degrading enzymes, enzymatic properties and classification of the newly obtained enzymes.

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Zymomonas mobilis produces an exocellular fructosyltransferase (EC 2.4.1.10, sucrose: 2, 6-β-Dfructan6-β-D-fructosyltransferase, FTase) that is capable of synthesizing branched fructan (levan) of high molecular mass from sucrose. In addition, the enzyme catalyzes the fructosyl transfer from sucrose to numerous acceptors other than levan, such as water, short-chain acylalcohols, and various mono- and disaccharides. In the present study, molecular genetic approaches have been utilized to confirm the structure of the catalytic site of Z. mobilis FTase (sucZE2), as follows; 1) Cloning and characterization of the gene cording for FTase, 2) Presumption of function domains in FTase by random mutation, 3) Alteration of transfructosylating activity by site-directed mutagenesis, 4) Alteration of substrate specificities of FTase by site-directed mutagenesis, 5) Identification of catalycally important residues, Glu278, His295 and His296, in the FTase gene, and 6) Presumption of transfructosylating reaction of FTase via a general acid-base catalytic mechanism.

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We performed a chemo-enzymatic transformation of maltooligosaccharide into both end-modified oligosyl-lactone to be of potential utility as a substrate analogue inhibitor for mammalian α-amylases. Enzymatic modification at the nonreducing end glucosyl residue of maltooligosaccharide was first carried out using the transglycosylation of β-D-galactosidase from Bacillus circulars. When maltotriose and maltotetraose were acceptors, the enzyme regioselectively synthesized 4³-O-β-D-galactosyla-maltotriose (LG3) and 4⁴-O-β-D-galactosyl-α-maltotetraose (LG4) from lactose as a donor, respectively. LG4 was further selectively hydrolyzed with specific G2-forming amylase to afford 4²- 0-β-Dgalactosyl-α-maltose (LG2). The anomer hydroxyl groups of LG2 and LG3 were chemically oxidized to the corresponding lactones 4²-O-β-D-galactosyl-cr-maltobionolactone (LG20) and 4³-O- β-Dgalactosyl-α-maltotrionolactone (LG3O), respectively. LG2O and LG3O, which are competitive inhibitors for mammalian α-amylases, had K, values on the order of 1.87×10^-5-2.80×10^-6 M, using pnitrophenyl-α-maltopentaoside (G5P) as a substrate and 3.20×10^-4-1.38×10^-5 M using soluble starch. In 1H-NMR analysis, these oligonolactones were shown to exist in two forms of the lactone-ring and the open-chain form of aldonic acid in aqueous solution. In this case, the lactone form was essential for the occurrence of α-amylase inhibitor.

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Human α-amylases are produced as one of the major proteins in the salivary gland and pancreas, and exist in saliva, pancreatic juice, blood and urine. Changes in α-amylase activities are known to be in correlation with certain diseases. Therefore, they have been known as typical marker enzymes for diagnosis. The two α-amylases are isozymes as they are encoded on different genes. They show multiple forms with different isoelectric points. The multiple forms of salivary α-amylase are divided into two groups, one is named "human salivary a-amylase family A (HSA-A)" and has one mole of biantennary complex-type oligosaccharide; the other is named "human salivary α-amylase family B (HSA-B) " and has no oligosaccharide. The mechanisms of the formation of the two families and their multiple forms have been unclear. Recently, we found the conversion of HSA-A to HSA-B in human saliva, and then purified the enzyme which catalyzed the reaction, whith was identified as endo-β-Nacetylglucosaminidase. We named the enzyme "endo-β-N-acetylglucosaminidase HS (Endo HS) ". Endo HS showed a molecular weight of about 270, 000 and three multiple forms. Endo HS was specific for complex-type oligosaccharides and was able to release bi, tri and tetraantennary complex-type oligosaccharides from native glycoproteins, glycopeptides and asparaginyl oligosaccharides regardless of the existence of fucosyl residue attached to the proximal G1cNAc residue of oligosaccharides. So Endo HS shows a high specificity for oligosaccharide structure and low specificity for aglycon. Endo HS is originated from epithelial cell peeled from oral epithelium into saliva. It is a plasma membranebinding protein. HSA-A was converted to HSA-B by purified Endo HS or Endo HS on plasma membrane of the epithelial cell. The multiple form pattern of HSA-A was converted to the same as that of HSA-B. We speculated the formation mechanism of the carbohydrate-deficient human salivary a-amylase molecules by enzymatic deglycosylation with Endo HS of human saliva. On the other hand, the cDNA sequence of human salivary a-amylase indicated the presence of two potential glycosylation sites. A human α-amylase having two oligosaccharides has not been found. We tried to purify a salivary α-amylase having two oligosaccharides at the two potential glycosylation sites to confirm the existence in human saliva, and found it in a new α-amylase fraction, named "human salivary α-amylase family C (HSA-C) ". The ratio of HSA-C in total α-amylase was about 1%. It showed a molecular weight of about 66, 000 and antigenicity for anti-HSA-B antiserum. HSA-C was thought to have two biantennary complex-type oligosaccharides from the conversion pattern by Endo HS and reactivity with lectins. HSA-C was converted to HSA-C via HSA-A by the action of Endo HS.The multiple form pattern of HSA-C was also converted to the same pattern as that of HSA-B . The existence of HSA-C and Endo HS, and the conversion of HSA-C to HSA-B via HSA-A by the action of Endo HS in human saliva suggest that human salivary α-amylase was glycosylated at the two glycosylation sites, secreted into saliva as HSA-C and then deglycosylated by Endo HS to be converted to HSA-A and HSA-B.

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A crystalline α-glucosidase from Aspergillus niger (ANGase), a so-called "maltase"-type enzyme, was composed of two different subunits, P1(227 amino acids) and P2 (719 amino acids), which were not linked via S-S bridge. Their primary structures were determined by EDMAN degradation and genetic methods. The genomic nucleotide sequence revealed that the Pl and P2 polypeptides were encoded by a single reading frame in this order. Between two subunits, 14 amino acids were removed, suggesting a unique post-translational modification with limited proteolysis. The Asp (224) of P2 was identified to be one of the catalytic amino acids. Seven N-linked high-mannose type oligosaccharides were isolated and their structures were determined. Four sugar chains contained one galactofuranosyl residue bound via α- (1→2) -linkage to Man (A) . Three of them are novel types of N-linked oligosac charides. The O-linked sugars were also isolated from ANGase. A structural analysis of the two compounds indicated that mannose and α-mannosyl- (1→2) -mannose are bound directly to Ser/Thr of ANGase.

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Sugar beet α-glucosidase exhibits the highest hydrolytic activity, among α-glucosidases, towards soluble starch. α-Glucosidases including sugar beet enzyme produce α-anomeric compounds from maltotriose, soluble starch, phenyl α-maltoside, D-glucal, D-gluco-octenitol and α-glucosyl fruoride. These enzymes, therefore, are not glucoamylases. Conduritol B epoxide (CBE), an affinity labeling reagent, inactivated sugar beet α-glucosidase following pseudo-first-order kinetics. Analyzing an ³HCBE-labeled peptide indicated that the active site sequence was-DGIWIDMNE-. The cDNA (3056 bp), cloned from a library constructed from mRNA of suspension cultured cells, had an open reading frame encoding a polypeptide of 913 amino acid residues. The deduced amino acid sequence contained the active site region determined by CBE modification, and showed a high homology to those from spinach (68%) and barley (55%) α-lucosidases. The primary structure is discussed in relation to some α-amylases.

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New phosphoryl oligosaccharides (POS) were prepared from potato starch hydrolysate. The inhibitory effect of POS on the formation of a calcium phosphate precipitate were investigated. POS were fractionated by ion-exchange chromatography into two fractions, PO-1 and PO-2. Both fractions, PO-1 and PO-2, had the ability to form a soluble complex with calcium, and fraction PO-2 had a stronger inhibitory effect on the formation of calcium phosphate precipitate, and made a more stable and soluble complex with the calcium ion. Fraction PO-1 was the main component of POs, and was composed of maltotriose, maltotetraose and maltopentaose to which one phosphate group was attached. Fraction PO-2 was predominantly composed of maltopentaose and maltohexaose to which at least two phosphate groups were attached. According to the reaction specificities of glucoamylase (GA) (EC3.2.1.3) and bacterial saccharifying a-amylase (BSA) (EC 3.2.1.1), and spectrometrical as well as chemical analyses, we determined the precise structure of the components fraction PO-1. In addition, it was found that fraction PO-1 was a suitable compound in producing the conjugate with ovalubmin (OVA) through the Maillard reaction and the conjugate could exhibit an inhibitory effect on the formation of a calcium phosphate precipitate.

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