α-Linked galactooligosaccharide (α-GUS) B was produced from lactose hydrolyzates by reverse reaction of hydrolysis of α-galactosidase from Candida guilliermondii H-404. The reaction mixture (2300 ml), containing 1 .93 kg lactose hydrolyzates and 400 g wet cell (62 U/g-galactose) was incubated at pH 4.5 at 50°C for 87 hr. From the reaction mixture, 350 g of α-GUS B was obtained by active carbon chromatography. α-GOS B contained 84% disaccharides and 15% oligosaccharides (mainly trisaccharides). The solubility of α-GOS B was 82% (w/w) or above at 25°C. The sweetness of α-GUS B was about 30% of that of sucrose. It was stable both at neutral pH (pH 7. 0, 80°C, 2 hr), and at acidic pH (pH 3. 0, 80t, 2 hr), and at temperatures up to 180°C (pH 4. 5, 10 min), and on long-time storage (pH 3 .0 and 7. 0, 25°C and 60°C, 3 months). The powder preparation was highly hygroscopic, and its solution showed higher activity of retaining moisture than sucrose. The water activity of α-GUS B was 0.83 (25°C, 70% (w/w)), α-GUS B was not hydrolyzed in an in vitro digestion method, and had activity to inhibit the synthesis of insoluble glucan from sucrose . α-GUS B had also strong selective growth activity for Bifidobacterium. α-GUS A was also produced from only galactose, and was compared with α-GUS B. But there were no large differences with respect to all properties tested.

View full abstract

Studies have been made of the effects of surfactants on the apparent viscosity of waxy corn starch (WCS) pastes, and the intrinsic viscosity and transparency of WCS solutions. The viscosities of the pastes and solutions, and the transparency of the solutions were increased by the addition of all anionic surfactants used, with the exception of sodium caprylate, in order of sodium caprate < sodium laurate < sodium dodecyl sulfate < sodium dodecylbenzene sulfonate. They were markedly increased by the addition of cationic surfactants, stearyltrimethlylammonium chloride and cetyldimethylbenzylammonium chloride. The surfactants which increased the intrinsic viscosities of the solution increased also the viscosities of the paste and the transparency of the solutions. The amphoteric (lysolecithin) and nonionic (Tween 60 and sucrose fatty acid ester) surfactants had little effects on the viscosities of the paste and solution. The increase in the apparent viscosity of the paste by sodium dodecyl sulfate was depressed by adding nonionic surfactants, Tween 60 and sucrose fatty acid ester. It was concluded from these results that the surfactants with the appropriate lengths of hydrocarbon chains interact with the outer branches of WCS and the interaction of WCS with anionic and cationic surfactants leads to a further expansion of the swollen granules and dispersed starches due to an electrostatic repulsion between adjacent branches of amylopectin molecules, which may result in the increases in the viscosities of the pastes and solutions, and the transparency of the solutions.

View full abstract

Two kinds of acidic arabinogalactan (F2A and F2B) were extracted from the cotyledon of Azuki bean (Phaseolus angularis) with 0.5 M NaCI, and purified by column chromatographies of DEAE-Sephadex A-50 (Cl-form) and Sepharose CL-2B. Both polysaccharides, as constitutional monosaccharides, contained L-arabinose, D-galactose and D-galacturonic acid in a ratio of 13. 15.9 : 1.0 (F2A) and 3.0 : 2.2 : 2.0 (F2B). From the results of methylation and ¹³C-NMR studies, it could be concluded that F2A and F2B respectively have a backbone consisting of β-(1→3)-linked D-galactose and D-galacturonic acid residues, with α-(1→5)-linked arabinofuranosyl side-chains attached at C6 of D-galactose residue. The molecular weight of F2A and F2B determined by gel permeation chromatography was 50, 000 (F2A) and 94, 000 (F2B), respectively. And on gel permeation chromatographic analysis, elution volume of the polysaccharides was easily influenced by the ion strength of the eluant. This suggests that F2A and F2B could assemble to form a macromolecule through ionic bonds between polysaccharide molecules.

View full abstract

A sucrose phosphorylase was purified from Leuconostoc mesenteroides ATCC 12291 cells to an electrophoretically homogeneous state with hydrophobic, ion-exchange and gel filtration column chromatographies. The enzyme had a molecular weight of 55, 000 and pI of 4.0. It showed the maximum activity at pH 6.5 in a transfer reaction of glucose residue from a-D-glucose -l-phosphate (G-1-P), at pH 7.0 in a transfer reaction from sucrose and is fully stable at pH between 5.5 and 7.5. It showed the maximum activity at 35°C and is stable below 40°C. D-Fructose, D-xylulose, L-arabinose, Lsorbose and L-ribulose can serve as an acceptor in the transfer reaction from G-1-P, and the last four can serve as an acceptor in the reaction from sucrose, too. It was found that the reactions with sucrose gave a higher yield than those with G-1-P . Among the reactions tested, glucosylxyluloside synthesis from sucrose and xylulose gave the highest yield.

View full abstract

A levanase from Arthrobacter sp. No. 51A was purified to a homogeneous state. The molecular weight of the levanase was estimated to be 60, 000 by SDS-polyacrylamide gel electrophoresis, and 64, 000 by gel filtration. The pl of the enzyme was 6.37. The optimum pH and temperature were pH 5.8 and 65°C. The enzyme was stable between pH 7.0 and 9.0, and below 45°C. The levanase hydrolyzed levan to produce a series of levanoligosaccharides at an early stage of reaction. It was a typical endo-type enzyme. The limit of hydrolysis of levan was 96.3%. The enzyme slightly acted on nystose, 1-kestose, and inulin. The Km values against levan, nystose, and 1-kestose were 0.23%, 0.15 M, and 0.14 M respectively.

View full abstract

Retrogradation of starches, potato and sweet potato as a root (or tuber) starch and corn and wheat as a cereal one, were examined in two ways of gelatinization at various starch concentrations (5, 10, 15%). One is the gelatinization by heating at 100°C with stirring for 10 min (SH). The other gelatinization consisted of two steps: 2% (or 3%) starch suspension was heated at 100°C with stirring for 10 min, and then starches were added up to each final concentration, suspended well and heated at 100°C for 30 min without stirring (NH). The gelatinized starches were stored at 5°C, and retrogradation of the starches measured at the storage periods. The degree of gelatinization of the hot pastes was higher in SH than in NH. Lower retrogradation of the starch was observed in those gelatinized by NH than in those gelatinized by SH. These differences between the gelatinization methods may be related to the conditions, stirring, pre-heating, heating time, included in the methods. The suppression of the retrogradation was noticeable in the cereal starches compared to the root (or tuber) starches.

View full abstract

The pore size distribution of sobium Carboxymethyl starch (Na-CMS) was obtained from the nitrogen adsorption isotherm to elucidate the relation between the degree of substitution (DS) and the microstructure of Na-CMSs and to evaluate the importance of the micropores in determining their greater moisture sorption capacity at lower water activity (a_w). Although the total pore volume increased through carboxymethylation, the larger micropores (17.5<r≤19.5Å) disappeared in Na-CMSs with a lower DS (0.13-0.64) and larger micropores and smaller micropores (13.5≤r≤17.5Å) appeared in Na-CMSs with a higher DS (0.81-1.01). The smaller micropores of Na-CMSs, rather than their larger micropores or mosopores (r>20Å), increased the moisture contents at lower aw than 0.4.

View full abstract

The cooked rice preparations with low palatabilities were inferior to those with high palatabilities in hardness, stickiness and luster in sensory test. Instrumental analyses showed that the rice preparations were characterized by their low values of degree of gelatinization, stickiness, luster and hot-water extractable starch content and by high values of hardness. These results indicate that the common characteristics of unfavorable cooked rice are a hard and unsticky texture, and a dull appearance. A major factor contributing to the hardness is a low degree of starch gelatinization in whole cooked grains, and the main factor contributing to low degrees of stickiness and luster is a low level of hot-water extractable starch from the cooked rice.

View full abstract

The cyclodextrins (CD) including flavor essences of c, citron, c, sudachi, c. iyo, c. orange, c. lemon, menthol, essences of horseradish and ethanol were prepared . These prepared CD complexes could effectively be used as food ingredients because of their higher thermostabilities.

View full abstract

The acid hydrolysis rate and iodine-binding power of the acid-treated (with 2.2 N HCl at 35°C) starch, as hydrolysis time increased, showed three stages. The amylose and amylopectin were hydrolyzed simultaneously in the first phase, but decrease of absorbance and λ_max of iodine-starch complexes indicate that amylose is more affected than amylopectin. The second phase corresponds to amorphous parts which were hydrolyzed α-1, 6 glucosidic bonds or removal of blocks containing the bonds of amylopectin. The third phase corresponds to crystalline parts which were not attacked so much by acid. These results suggest that acid hydrolysis pattern of rice starch showed three stages and the amylose and amylopectin are hydrolyzed partly in the first phase.

View full abstract

Modification at the nonreducing end residue of p-nitrophenyl a-maltopentaoside (G5P) was carried out by using the transglycosylation of lysozyme or β-D-galactosidase. p-Nitrophenyl 35-O-β-IV acetylglucosaminyl-a-maltopentaoside was regioselectively formed through hen egg-white lysozyme-mediated reaction from di-1V acetylchitobiose as a donor and G5P as an acceptor, p-Nitrophenyl 4⁵-O-β-D-galactosyl-β-maltopentaoside (LG5PI) with 6⁵-O-β-D-galactosyl-β-maltopentaoside were similarly synthesized by β-D-galactosidase from Bacillus circulans from lactose and G5P. LG5PI was very useful as a novel synthetic substrate for the colorimetric assay of human amylase in serum. A series of modified maltooligosaccharides were shown to be effective for examining the interaction between the substrates and the active site of human salivary β-amylase.

View full abstract

Two distinct groups of a-amylase isozymes (designated Amylase-I and -III) in suspensioncultured rice (Oryzae sativa L. cv. Sasanishiki) were characterized. These two enzymes differed from the a-amylase found in germinating rice seeds in molecular weight, isoelectricpoint, and Km for soluble starch. The Amylase-III showed a unique property with a low-temperature optimum at 25°C unlike other plant, animal and microbial amylases that generally had optimum at around 35-55°C. The Arrhenius plot for the enzyme also gave an unusual curve, that is, the plot deviated very much from a straight line above 25°C. This suggests the occurrence of a conformational change in the structure of Amylase-III around 25°C in the presence of substrate. By treatment of the Amylase-III with raw starch or maltodextrin (G17) at various temperature (4°C, 26°C and 37°C), the residual amylolytic activity was almost lost.

View full abstract

The cDNA clones coding for a form of soluble starch synthase from developing rice seeds have been isolated using a synthetic oligonucleotide as a probe. The deduced amino acid sequence of this enzyme includes a Lys-Xaa-Gly-Gly consensus sequence for the substrate-binding site of glycogen synthase. The precursor of the enzyme contains 626 amino acids, including a 113-residue transit peptide at the amino-terminus. The mature form of soluble starch synthase shares a significant, but low sequence identity with rice granule-bound starch synthase and Escherichia coli glycogen synthase. However, several regions, including the substrate-binding site, are highly conserved among these three enzymes. Northern blot analysis demonstrates that the gene encoding soluble starch synthase is expressed in both leaves and developing seeds. The cDNA cloning of two major forms of rice starch branching enzyme, RBE1 and RBE3, has been also carried out. The nucleotide sequences indicate that RBE1 and RBE3 are initially synthesized as 820- and 825-residue precursor proteins, including putative 64- and 65-residue transit peptides at the amino-termini, respectively. The consensus sequences of the four regions that form the catalytic sites of amylolytic enzymes are conserved in the central region of the RBE1 and RBE3 sequences. Thus, branching enzyme belongs to a family of amylolytic enzymes. RBE1 shares a noticeable degree of sequence identity with RBE3, especially at the central portion of the protein molecule. However, as compared with RBE1, RBE3 possesses an approximately 70-residue extra sequence at the amino-terminus, and lacks a carboxyl-terminal sequence of almost 50 residues. On the basis of the primary structures of the enzymes participating in starch biosynthesis, the possible functions of these enzymes are discussed.

View full abstract

Three kinds of novel cyclic isomaltooligosaccharides were isolated from the culture broth of a strain of Bacillus sp. T-3040, which had been isolated from soil. They were identified as cycloisomalto-heptaose, -octaose, and -nonaose, respectively, on the basis of their mass spectra, proton nuclear magnetic resonance spectra, carbon nuclear magnetic resonance spectra, infrared spectra, reducing power, and results of enzymatic analysis using endo-dextranase and exodextranase. They also showed hydrophobic features similar to cyclodextrins on high-pressure liquid chromatography analysis using ODS column, Furthermore, a novel enzyme, which catalyzes the conversion of dextran to cyclic isomaltooligosaccharides by intramolecular transglucosylation (cyclization reaction), was purified from the culture of Bacillus sp. T-3040 . The homogeneous enzyme was obtained by DEAE-Sepharose CL-6B colum chromatography, hydrophobic, anion exchange and molecular-sieve HPLC. The Mr of the enzyme was estimated to be 98 kDa by sodium dodecyl sulf ate-polyacrylamide gel electrophoresis. The enzyme had an optimum pH at pH 5.5, and was stable from pH 4.5 to 8.5 at 25°C for 24 hr. It was almost inactivated at 50°C for 15 min. The enzyme mainly catalyzed cyclization reaction and gave three cycloisomaltooligosaccharides in an about 20% total yield. Coupling and disproportionation reactions were also observed. These results showed that this enzyme is a multi-functional enzyme wShich catalyzes intramolecular and intermolecular transglucosylation.

View full abstract

The three-dimensional structure of cyclodextrin glucanotransferase (CGTase) has been determined at 2.5 Å resolution x-ray crystallography. The molecule folds into four globular domains, A, B, C and D. The structure of N-terminal domains, A and B, are similar to those of α-amylase. In addition, the active site has been studied using crystals containing maltose as the substrate in order to understand the enzymatic reaction mechanism. The substrate binding in the active cleft supports the putative catalytic residues (Asp-225, Glu-253 and Asp-324) inferred from sequence alignments and site-specific mutagenesis studies. It is considered that two hydrophobic residues (Phe-179 and Phe-225) in close to the catalytic center form the acceptor-binding site for transglycosylation of the enzyme reaction. Moreover, Phe-191 in the cleft is suggested to be associated with cyclization of the substrate for cyclodextrin formation through hydrophobic interaction between the substrate and the residue. Based on the information of the molecular structure and the substrate binding, we propose a model of reaction mechanism for cyclodextrin formation by CGTase.

View full abstract

Based on a series of experimental results using neopullulanase and the structural similarities, we previously reported a general idea for an enzyme super family, α-amylase family, including α-amylase, pullulanase/isoamylase, cyclomaltodextrin glucanotransferase, 1, 4-α-D-glucan branching enzyme, neopullulanase, and related enzymes. We also demonstrated a hypothesis that the enzymes belonging to α-amylase family can be altered each other by manipulating substratebinding specificity or by changing the environment of the active center. A unique enzyme, neopullulanase, was used to explore further into the hypothesis. The three-dimensional structure of neopullulanase was predicted by a homology modeling procedure. A computer simulated docking-study in three-dimension was also employed to investigate the enzyme-substrate structures. Based on the simulated docking-study, we introduced some replacement of the amino acid residues which are most likely to affect the substrate preference of neopullulanase by site-directed mutagenesis. By manipulating the amino acid residues, the enzyme specificities toward α- (1→6) -branched oligosaccharides and pullulan were reduced or increased. The characteristics of the mutated neopullulanase which was reduced in its specificity toward α- (1→6) -branched oligosaccharides and pullulan was quite different from those of wild-type enzyme and rather similar to those of α-amylase. Some experimental keys to explore our hypotheses are also discussed.

View full abstract

Polymerase chain reaction (PCR) amplification of mRNA from developing barley (cultivar Haruna two-rows) endosperm was used to clone and sequence full-length cDNA encoding β-amylase. The β-amylase cDNA was 1775 by in length . The β-amylase was deduced to be composed of 535 amino acid residues and its molecular weight was calculated to be 59, 610. To express the cloned -amylase cDNA in Escherichia coli under control of the tac promoter, a plasmid pBETA92 was constructed. The plasmid consisted of 6312 bp. The emzyme production started in the logarithmic phase, increased linearly, and reached a maximum after 12 hr. The recombinant barley β-amylase gave two major (pI 5.43 and 5.63) and four minor (pI 5.20, 5.36, 5.80, and 6.13) activity bands on isoelectric focusing and their pIs didn't change throughout the cultivation. But Western blot analysis found that one β-amylase having a molecular weight of about 56, 000 was synthesized. The purified enzyme gave a single band of protein on SDS-PAGE but showed heterogeneity on isoelectric focusing . The N-terminal amino acid sequence showed that the recombinant β-amylase lacked four amino acids at positions 2-5 (Glu-Val-Asn-Val) when compared with the presumed amino acid sequence of barley β-amylase. Therefore, the recombinant β-amylase consisted of 531 amino acids, and its molecular weight was calculated to be 59, 169. The N-terminal amino acid sequence of the recombinant β-amylase and the nucleotide sequence of the junction position in plasmid pBETA92 indicated that GTG (Val-5 in the case of barley β-amylase) at positions 27-29 from the SD sequence (AGGA) was the translation initiation codon. The properties of the recombinant β-amylase were almost the same as those of barley β-amylase except for the pI and the K_m values for maltohexaose and maltoheptaose.

View full abstract

In cellulolytic organisms, there are at least three cellulase components which together constitute a multienzyme system, and cellulosic substrates are converted to soluble sugars by their synergistic action. These components are an exo-cellulase, an endo-cellulase and a βglucosidase. However, the modes of degradation of cellulose have not been fully established. The modes of hydrolysis of highly purified cellulases, exo- and endo-cellulases, from fungi (Irpex lacteus, Trichoderma reesei, and Aspergillus niger) were investigated by using pure cellulosic materials with different crystallinities (CrI) and modified cellulose as substrates . In productivity of reducing sugar, exo-cellulases hydrolyzed effectively celluloses as compared with endo-type cellulases, and saccharifying activities of both cellulases, especially of endo-cellulases, drastically increased in parallel with decreasing CrI. Endo-cellulases lowered the degree of polymerization (DP) of crystalline celluloses much intensively as compared with exo-type cellulases, and DP lowering activity of endo-cellulase decreased with decreasing CrI. In the hydrolysis products, endo-cellulases produced several oligosaccharides from H₃PO₄-treated celluloses, but not from native celluloses, while exo-cellulases produced only cellobiose from insoluble celluloses. It was found that each cellulase component showed clearly the action of exo- and endo-fashion on the hydrolysis of insoluble cellulose, especially native cellulose as substrate . However, the strictness of their fashion was not obscure on the modified substrate such as p-nitrophenylβ-glycosides. Electron microscopy photographs showed clearly different morphologic changes of substrates treated with exo- and endo-cellulases. In the hydrolysis by exo-cellulase, the transverse deep cracks were observed on the surface of native cellulose and these cracks extended from fiber surface to lumen structure located in the inside of fiber . In endo-cellulase, these cracks seemed to be shallow and erosion areas were observed on the outside surface of fiber and lumen region. On the basis of these observations, it may be concluded that the hydrolysis by exo- and endocellulases produces quite different morphologic patterns with native cellulose, but little differences with H₃PO₄-treated celluloses. Basing on the results from these fungi, it may be concluded that the mode of action of exoand endo-cellulase is dependent on the structure of cellulose used as substrate .

View full abstract

A strain of Zoogloea ramigera isolated from soil produced an extracellular α-amylase with unusual characteristics. The extracellular α-amylase from Z. ramigera was purified to apparent homogeneity by starch adsorption and CM-Toyopearl ion-exchange chromatography. The enzyme had an isoelectric point of 8.8 and apparent molecular mass of 63, 000 Da, as estimated by SDSPAGE. The 15-amino acid NH₂ terminal sequence was different from other raw starch-digesting enzyme, α-Amylase activity on raw corn starch was optimal at pH 6.0 to 6.5. The enzyme was relatively stable between pH 5.0 and 8.0 and at temperatures below 50°C. When acted on raw corn starch, the enzyme produced maltopentaose alone in the early stage of hydrolysis. On further incubation, maltose and maltotriose were produced with concomitant decrease of maltopentaose. The COOH-terminal portion of the amylase from Z, ramigera contained several sequences significantly homologous, to those of other raw starch-digesting enzyme. In the synergism of the amylase from Z. ramigera and glucoamylase from Rhizopus niveus on hydrolysis of raw corn starch, role of the amylase was supplying of new substrate molecules for glucoamylase by endowise splitting of large molecules.

View full abstract