Properties and molecular structure of Furunori (a kind of paste gel for the design of Japanese painting, and the need to store it for over 10 years to have recommended quality for usage after preparation from wheat starch gel) was investigated. The results are as follows: 1) Granule structure was not observed in the SEM image of the gel. 2) Furunori is proved to be an extremely retrograded starch gel by X-ray diffraction pattern, degree of gelatinization measurement and DSC analysis. 3) GPC analysis showed that both components (amylase and amylopectin) were greatly decomposed and they formed small-sized fragments which showed a single peak an GPC (Toyopearl HW-75). But, extremely small oligosaccharide was not obtained with methanol extraction from Furunori. 4) The above observations suggest that disruption of amylopectin may mainly take place at the linkage of clusters and that cluster units may be liberated. 5) It is considered that decomposition of starch gel will mainly occur by physical stress such as the cycle of freezing and thawing during storage than by amylase of microbes in the gel.

View full abstract

A Fucoidan was isolated from Okinawamozuku (Cladosiphon okamuranus T0KmA) and its chemical and physicochemical properties were investigated . The total carbohydrates, uronic acid, ash, sulfuricacid, and moisture of the fucoidan was estimated to be 67.2, 13.5, 23.0, 11.9, and 3.2%, respectively. The L-fucose and n-xylose residues were identified by paper and liquid chromatography from the acid hydrolysate of the fucoidan. The molecular weight of the fucoidan was estimated to be about 500, 000. The optical rotation of the fucoidan showed a value of -0.351° at a temperature of 60°C, then it decreased a little with decreasing temperature. The infrared spectrum of the fucoidan was essentially in agreement with that of a fucoidan (Sigma), which was used as a standard sample, in a wide range of wave numbers.

View full abstract

The effects of fucoidan extracted from Okinawamozuku (Cladosiphon okamuranus TOKIDA) on serum cholesterol levels in cholesterol-fed rats were studied in comparison with those of cellulose. The liver weight of rats fed fucoidan and Okinawamozuku diets was lower than those in rats fed cellulose diet. The free and total cholesterol levels of serum for fucoidan diets showed lower values than those for cellulose diets. However, HDL-cholesterol levels of fucoidan diets in serum showed higher values than those of cellulose diets (not significant). On the other hand, the total cholesterol levels of liver for fucoidan diets also showed lower values than those of cellulose diets. The serum cholesterol normalizing effect was estimated to be greater for Okinawamozuku diets in both serum and liver than for fucoidan diets. These results suggest that fucoidan and Okinawamozuku have serum cholesterolnormalizing effects.

View full abstract

Bacillus stearothermophilus K-12481, which was isolated from soil, produced an intracellular cyclodextrinase. The strain seemed to produce at least three components of cyclodextrinase and one of them was purified to homogeneity. The enzyme was a dimer and the molecular weight of a subunit was 67, 000 and it showed the highest activity at 60°C and pH 6 .5. The enzyme degraded α-cyclodextrin (α-CD) two times faster than β-CD and γ-CD and had low activity toward high-molecular-weight substrates such as glycogen and amylopectin. The enzyme also had high activity toward malto oligosaccharides of DP 4-7. α-CD was the best substrate among those tested . All CDs were hydrolyzedinto glucose and maltose.

View full abstract

The reversible conversion between maltobionolactone and maltobionic acid was analyzed kinetically; the rate constants were determined at each pH, and the pKa of maltobionic acid was estimated to be 3.60±0.02. The inhibition by maltobionolactone for the hydrolysis of soluble starch was inves tigated at pH 5.4 and 25t, and it was found that maltobionolactone was a competitive inhibitor with a K₁ of 0.40 mNi. The unitary binding affinity (7.0 kcal?mol^-1) was the same as that of maltotetraose (a substrate). From the result, taking the subsite affinities of this enzyme into account, it was presumed that maltobionolactone consisting of a gluconolactone, with half-chair or sofa conformation, and a glucose (Cl form) is a transition state analogue and binds at subsites 1 and 2.

View full abstract

The actions of a-amylases (Taka-amylase A and porcine pancreatic α-amylase) and soybean β-amylase for four synthetic oligosaccharides, which are contained one or two D-xylose residues linked via α-1, 4 glycosidic bond, were investigated. A synthetic analogue of maltotetraose, Ο-α-Dglucopyranosyl-(14) -Ο-α-D-xylopyranosyl- (1→4) -Ο-α-D-xylopyranosyl- (1→4) -D-glucopyranose (GXXG) was not hydrolyzed by a- and β-amylases and was a competitive inhibitor only for both aamylases. GXXG had almost the same binding affinities as maltotetraose. An analogue of maltotriose, Ο-α-D-glucopyranosyl- (1→4) -Ο-α-D-glucopyranosyl- (1→4) -D-xylopyranose (GGX) was αcompetitive inhibitor (K, = 7.8 mM) only for soybean β-amylase. From the result, it was found that the CHZOH group at C-5 of the glucose residue of substrate plays a very important role in the catalytic action or in the productive binding of substrate for α-and β-amylases, and that GXXG and GGX arevery useful for the X-ray crystallographic study on the productive binding mode of substrate for α-and fl-amylases, respectively.

View full abstract

Transgalactosylated derivative (α-GALA) of L-ascorbic acid was synthesized by Candida guilliermondii H-404 α-galactosidase from the mixture of raffinose (as a donor substrate) and L-ascorbic acid (as an acceptor). The structure of α-GALA was identified as 6-Ο-α-D-galactopyranosyl i.-ascorbicacid by hydrolysis with α-galactosidase, UV_max, FAB-mass and ¹³C-NMR spectroscopies. α-GALA wasmore stable than L-ascorbic acid in aqueous solution at both pH 4.0 and 7.0, especially at pH 7.0.

View full abstract

Bamboo shoot starch granules were prepared from five samples of two species of bamboo shoot, Moso bamboo (A and B; produced in Shimada, Yasugi, Shimane and in Yamashiro, Kyoto, respectively) and Black bamboo [C-1 (the upper part), C-2 (the middle part), C-3 (the lower part) ; produced in Yamashiro, Kyoto] and the properties of the starch granules were examined. The results obtained were as follows: 1) The mean particle size of bamboo shoot starches were 2.4-5.0, μm. 2) Starch granules in the slices of bamboo shoot were found to be round in shape on scanning electron microscopy. 3) The susceptibility of the bamboo shoot starch granules to α-amylase was higher than that of normal maize starch. 4) The gelatinization temperature of Moso bamboo A starch determined by Differential Scanning Calorimetry was lower than that of other bamboo shoot starches. 5) The solubility and swelling power of Moso bamboo A starch was higher than those of other bamboo shoot starches. 6) The amylose contents of bamboo shoot starch, determined by amperometric titrimetry, and enzymatic and chromatographic method, were 21.6-26.6% and 21.3-25.0%, respectively. 7) By X-ray diffractography, the Moso bamboo A starch showed a C-type pattern and other bamboo shoot starches showed C_A-type patterns.

View full abstract

The effects of sodium (GNA) or potassium (GK) gluconate supplemented with monoglyceride (MG) on some viscoelastic properties of wheat flour dough, on the interaction between gluten and starch in dough, and on loaf volume were studied. The addition of either GNA, or GK singly or in combination with MG to the ingredients for breadmaking increased the specific volume of bread. When the physicochemical properties of the dough were measured in a farinograph, the development time of gluten formation was decreased by the addition of small quantities of GNA and the stability time of the dough increased. Scanning electron micrographs of dough mixed with GNA and MG showed fibrous elements in the gluten; the gluten seemed to cover the starch entirely. The addition of GNA singly and in combination with MG decreased the size of gas cells in the crumb grain. From the result of reflectivity of sliced bread crumb, the retrogradation was suppressed by the combined addition of GNA and MG 90.

View full abstract

The production of β-xylosidase by an imperfect fungus, Fusidium sp. BX-1 was remarkably enhanced in a medium containing 0.5% glycerol and 3.0% washed wheat bran. From this culture, the enzyme was purified as an electrophoretically homogeneous protein. Its molecular weight was estimated to be about 120, 000. The optimum pH for its activity was 3-4 and the optimum temperature was 65°C. The enzyme was stable within the rage of pH 5-10. About 80% of the initial activity remained after heating for 30 min at 60°C. The activity was enhanced 1.5-1.8 times by incubating with 50-60% ethanol.The K_m values for xylobiose, xylotriose and phenyl β-xyloside were 39.4 m_M, 48.3 m_M and 4.42 m_M, respectively. The enzyme could slightly degrade xylan to xylose.

View full abstract

Amylolytic activity that converts soluble starch to α, α-trehalose, was found in the cell homogenate of the hyperthermophilic archae, Sulfolobus. Purification of the activity using new reliable assay methods showed two enzymes to be essential for the activity. The reaction mechanisms of the enzymes were examined by analysis of the reaction products with the two enzymes on maltooligosaccharides using ¹H. and ¹³C-NMR spectra and HPLC. One of the enzymes was a new glycosyltransferase which catalyzed the conversion of maltooligosaccharides to the corresponding glycosyltrehaloses with atrehalose structure as an end unit. The other was a new a-amylase which hydrolyzed the glycosyltrehaloses to isolate trehalose specifically. The genes encoding the enzymes were cloned and expressedin E. coli. The yield of trehalose from starch amounted to 80% using the two enzymes and debranching enzyme.

View full abstract

Two novel enzymatic pathways for trehalose synthesis were discovered in bacteria. One involves synthesis from maltooligosaccharides catalyzed by two novel enzymes, and the other involves synthesis from maltose by a different novel enzyme. A bacterium, Arthrobacter sp. Q36, isolated from soil, showed an ability to produce trehalose from maltooligosaccharides. Two novel enzymes were found to be related to the trehalose synthesis. One enzyme catalyzed the conversion of maltooligosaccharide into 4-Ο-α-maltooligosyl trehalose by ntramolecular transglycosylation, but did not act on maltose. The other enzyme specifically catalyzed the hydrolysis of the α-1, 4-glucosidic linkage that bound the maltooligosyl and trehalose moieties of maltooligosyl trehalose. Through the actions of these enzymes on maltodextrin, the trehalose content in the reaction mixture reached more than 80%. These enzymes were also found to exist in the thermoacidophilic archaebacteria, Sulfolobus. A bacterium, Pimelobacter sp. R48, also isolated from soil, showed an ability to produce trehalose from maltose. This activity was found to be due to a novel enzyme which catalyzed the conversion of maltose into trehalose by intramolecular transglucosylation without requiring phosphate. This Pnzvme activity was also found to exist in the genus Thermus.

View full abstract

The short article is a comments on the two papers of new trehalose-forming enzymes in the Symposium for Amylases and Related Enzymes. Recently, the new enzymes for trehalose-producing pathways, which have been suggested by L_AMA et al. (1991), were made clear by two Japanese research groups. Trehalose was shown to be directly produced in high yield from maltooligosaccharides, amylose and soluble starch through the new pathways mainly consisting of the two enzymes. There are some disagreements in the analytical data for the enzyme reactions and the designations for the enzymes between the two groups. Especially, the names proposed for the new enzymes seems to be unreasonable. It is considered that more suitable names for the enzymes should be introduced on the reaction mechanisms.

View full abstract

To investigate the role of the C-terminal region of barley, Q-amylase, plasmid Δ54 was constructed with an expression vector of barley β-amylase by site-directed mutagenesis. Escherichia coli JM109 harboring plasmid Δ54 was expected to express Δ54 β-amylase in which 54 amino acid residues were deleted from the C-terminus. A comparison of the properties of the mutant enzyme with those of the original recombinant fl-amylase revealed that the isoelectrophoretic heterogeneity of the original recombinant β-amylase was apparently due to its C-terminal region and the C-terminal region was significantly involved in the thermostability of β-amylase. To increase the thermostability of fl-amylase, seven kinds of single-mutant plasmids were constructed. The remaining activity versus temperature curves were used to determine the temperatures (T₅₀) at which 50% of the initial activity was lost during a 30-min heating period. These mutations increased the T₅₀ values by amounts ranging from 0.8 to 3.2°C. To express the sevenfold-mutant β-amylase in E. coli, plasmid pB927 was constructed. E. coli harboring plasmid pB927 produced sevenfold-mutant β-amylase. The T₅₀ value of purified sevenfold-mutant fl-amylase (69.0°C) was higher than that of not only the original recombinant, Q-amylase (57.4°C) by 11.6°C but also soybean fl-amylase (63.2°C) by 5.8°C. The intragenic amino acid replacements were found to have simple additive effects on the thermostability of β-amylase. The data obtained from kinetics studies suggested that the sevenfold mutant β-amylase acquired enhanced thermostability, but its function as a fl-amylase remained unchanged.

View full abstract

Thermostable β-galactosidase of Saccharopolyspora rectivirgula, a thermophilic actinomycete, is a unique multimetal enzyme that requires multiple divalent metal ions per monomeric enzyme (Mr, 136977). We found that the enzyme had eight specific binding sites for divalent metal ions. These sites were classified as follows: a very tight (class I) site for Cat+, three tight (class II) sites consisting of two Cattspecific and one Mn²⁺-specific sites (Kd for Mn²⁺, 2 nM), and four loose (class III) sites for Mn²⁺ and Mg²⁺ (Kd for Mn²⁺, 1.2 jiM). Titration, kinetic, and thermostability studies showed that class II Mn²⁺ ions was essential for catalytic functioning of the enzyme whereas occupation of class III sites with Mn²⁺ or Mg²⁺ ion was important to attain sufficient thermostability by which this extracellular enzyme remained active for a prolonged time at elevated temperatures, as was observed during growth of S. rectivirgula. We cloned the β-galactosidase gene to determine its primary strucutre. The β-galactosidase consisted of 1251 amino acid residues including a 27-residue signal peptide for secretion, and N-terminus of the mature form of the enzyme was pyroglutamic acid 28. Although the enzyme exhibited the highest sequence similarity to Escherichia coli lacZ, Q-galactosidase; it also contained the following unique sequences: (i) a sequence of 200 amino acid residues with no sequence similarity to any known proteins, located as if inserted into the sequence corresponding to the sequence constituting the active-site domain of E. coli lacZ enzyme; (ii) a sequence which seemingly satisfies a rule for the loop in the Ca²⁺-binding helix-loop-helix 'EF-hand' motif.

View full abstract

The reaction mechanism of chitosanase from Streptomyces sp. N174 was investigated by analysis of the products from substrate, 25-35% acetylated chitosan or glucosamine (G1cN) oligosaccharide, using the wild-type enzyme and its site-directed mutants. The chitosanase was found to hydrolyze G1cNAc-G1cN linkages as well as G1cN-G1cN linkages in the chitinous polysaccharide chain. When the chitosanase digestion of G1cN hexamer was followed by ₁H-NMR spectroscopy, only α-form was produced in the early stage of the reaction, indicating that the chitosanase is an inverting enzyme. The chitosanase hydrolyzed G1cN hexamer in an endo-splitting manner, producing predominantly trimer, and dimer and tetramer in lesser amounts. From site-directed mutagenesis studies, G1u22 and Asp40 were found to be essential for catalysis, and Asp37 was found to play an important role in the stabilization of active site structure.

View full abstract

A new cyclization catalyzed by branching enzyme (BE, EC 2.4.1.18) was described. The BE from Bacillus stearothermophilus decreased the molecular size of synthetic amylose (weight-average degree of polymerization, 1860) without increasing reducing power. Structural analyses of the product indicated that the BE catalyzed intramolecular transglycosylation to form a cyclic structure with a side chain. The cyclic part was isolated from the product by removing the side chain with glucoamylase. The evidence of its cyclic nature was provided by mass spectrometry. The product from amylopectin by BE was indicated to have a cyclic structure with several non-cyclic side-chains. Based on these findings, a new mode of action of BE is proposed. It is also suggested that BE from plants catalyzes the cyclization of amylose and amylopectin. The possible role of BE on glucan metabolism and the potential applications for the cyclic glucan are discussed.

View full abstract

The aim of our study is to synthesize various kinds of oligosaccharide units in glycoproteins or glycolipids by organic chemical methods and enzymatic methods for the elucidation of the function of carbohydrate chains. This paper focuses on the enzymatic synthesis of oligosaccharide components by the two types of reactions using glycosidases: reverse hydrolysis reactions and transglycosylation reactions. In reverse hydrolysis reactions, various linkages are formed so that the regioselectivity is usually low. On the other hand, in transglycosylation reactions using glycosidases, the regioselectivity is relatively high if the enzyme is properly selected. We have synthesized Manal-2Man, Manal-2Manal-2Man, G1cNAcβ1-2Man and GIcNAcf 1-6Man using the reverse hydrolysis activities of α-mannosidase and β-N-acetylglucosaminidase. We also synthesized regioselectively Galfl-4G1cNAcβ1-2Man and Galβ1-4G1cNAc/I1-6Man using the transglycosylation activities of β-galactosidases, Neu5Acα2-3Galβ1-4G1cNAc, Neu5Acα2-6Galβ1-4G1cNAc using sialidases, Fucal-2Gal, Fucal-3GIuNAc, Fucal-6GluNAc using α-fucosidases and Gal/il-3Glc and Galβ1-4G1c using the transglycosylation activities of β-galactanase. The proper use of reverse hydrolysis reaction and transglycosylation reaction turned out to be quite effective for the synthesis of oligosaccharides. These oligosaccharides can be used for the chemical synthesis of glycoconjugates.

View full abstract

The enzymatic glycosylation and phosphatidylation of biologically active compounds are described. Aromatic, monoterpene, and indole alcohols (benzyl alcohol and its related alcohols, geraniol, citronellol, f arnesol, geranyl-geraniol, and tryptophol) were glycosylated when these alcohols and cellobiose (or lactose) were incubated with Aspergillus niger R-glucosidase (or A, oryzae f-galactosidase), and all β-glycosylated compounds of these alcohols were odorless. A purified cyclo maltodextrin glucanotransferase (CGTase) from Bacillus stearothermophilus was found to catalyze the transfer reaction of the glucosyl residue from dextrin not only to CH2OH groups of the acceptors such as benzyl alcohol and its related alcohols, riboflavin (B₂), pyridoxine (PN), thiamin (B₁), and n-butyl alcohols with high efficiency, but also to the OH group at the inositol moiety of kasugamycin, at the C-4 positions of glucose moieties of ginsenosides Rc and. Rg1, and of n-octyl-a (Q) -glucoside at the C-3 position of fructose, and also to the OH group of sec- and tert-butyl alcohols, quercetin, and aromatic hydroxy compounds (vanillin, ethyl vanillin, phenol, pyrocatechol, pyrogallol, gallic acid, protocatechuicacid, sesamol) at a considerable efficiency, resulting all α-glucosides of these compounds except quercetin were isolated in crystalline and powder forms and characterized. B. stearothermophilus CGTase had relatively broad acceptor specificity. α-Glucosylated compounds of aromatic alcohols, vanillin, ethyl vanillin, and B₁ were odorless. All glucosylated antioxidants were much more stable than aglycones against the oxidation by peroxidase in the presence of hydrogen peroxide. Phospholipase D from Streptomyces sp. (PLD) was observed to be highly active in transferring he dipalmitoylphosphatidyl (DPP-) residue from 1, 2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) to the CH₂OH group in the acceptors such as vitamins (B₁, B₂, PN, pantothenic acid, B₁ disulfide-related compounds), arbutin, kojic acid, genipin, and dihydroxyacetone in a biphasic system. DPP-arbutin and DPP-kojic acid showed the same inhibitory activity to tyrosinase as arbutin and kojic acid, respectively. DPP-genipin showed 6-52 times stronger cytotoxity than genipin to HeLa, HEL, and MT-4 cells. DPP-genipin was found to react with L-phenylalanine in organic solvents to give a clear blue solution having a similar color to an aqueous solution of the natural blue pigment "gardenia blue." This is the first example for the preparation of hydrophobic pigment from a phosphatidyl derivative of a water soluble compound. Moreover, the PLD was first found to bring about the transfer of DPP-residue from DPPC to aromatic hydroxy group of acceptors such as various phenols, naphthol, and 5-hydroxyindole in a biphasic system of an organic solvent with low water solubility and buffer. Immobilized PLD with Amberlite IRC-50 retained 74% of its initial activity after 10 times-repeated batch reaction for DPPcompound synthesis.

View full abstract

Measurement of sialic acid in various structural fractions of hen's egg showed that it is distributed in all parts of the egg. Quantitatively, sialic acid was mainly found in the yolk fraction. A suspension of delipidated egg yolk was treated with protease and purified by column chromatography. The obtained sialyloligosaccharide fraction was subjected to structural characterization, and there were three major types of biantennary sialyloligosaccharides. The sialyloligosaccharides were absorbed and distributed to various tissues, when orally administrated to rats. The sialyloligosaccharide fraction was shown to inhibit infection of rotavirus in vitro and was effective in lowering the incidence of diarrhea in rats infected with rotavirus. Also, the sialyloligosaccharide fraction was found to be effective in promotion of learning performance in infant rats when examined by the maze test.

View full abstract