A cellodextrin phosphorylase (CDP) gene was cloned from Clostridium thermocellum YM4 strain by using a PCR cloning method. The nucleotide sequence of the gene contained an open reading frame of 2952 by encoding a polypeptide of 984 amino acid residues. The deduced protein sequence was very close to that of CDP from C. thermocellum ATCC 27405 (92% identity) but not so similar to that of CDP from C. stercorarium (20%). The recombinant CDP phosphorolyzed cellooligosaccharides larger than cellotriose and utilized cellooligosaccharides larger than cellobiose as the acceptor molecule. The enzyme was stable up to 60°C and showed highest activity at pH 7.5.
Synergistic effects in the degradation of bacterial cellulose (BC), Avicel and H3PO4-swollen Avicel (HA) by cellobiose dehydrogenase (CDH) and ?A-1, 4-glucanases from Irpex lacteus including exo-cellulase (Ex-1), endo-cellulase (En-1) and ?A-glucosidase were investigated. CDH effectively promoted both the degradation of celluloses by Ex-1 and synergism of Ex-1 and En-1 but moderately the degradation by En-1. Kinetic studies indicated that CDH enhances cellulose degradation by Ex-1 more effectively than ?A-glucosidase does by preventing product inhibition. The enhancement in the cellulose-degrading capability of cellulases by synergistically acting with CDH was significant and independent of the crystallinity of substrate while the synergistic effect of Ex-1 and En-1 by themselves was limited when substrates were at low crystallinity. Despite the large amount of cellobiose consumed by CDH in the presence of electron acceptors, the cellulolytic enzyme system from 1. lacteus effectively degraded different cellulose sources and continued producing sufficient glucose for the cell.
Water sorption isotherms of powdered crude drugs and their starches were measured by a gravimetric method. The powder and the starch were prepared from the roots of Panax ginseng C.A. Meyer (PG) and Panax notoginseng (Burk.) F.H. Chen (PN), the rhizomes of Pinellia ternata (Thunb.) Breitenbach (PT) and Alisma orientale Juzepczuk (AU), and the seed of Coix lacrymajobi Linne var. ma-yuen Stapf (CL). The Guggenheim-Anderson-de Boer equation was well applicable to the water sorption isotherms. The optimum amount of water added to knead and to granulate the powders was approximately 25-28 (w/w)%. The water contents of granules or globules have to be determined on the basis of the isotherms at storage temperature and depend on the species of powders. The water sorption capacity of starch is high at water activity less than approximately 0.5 and it is higher at lower temperatures. The results of Gibbs free energy indicate the change in free energy of a water-starch solution formed by the water sorption was larger than that of the water-powder solution; that is, starch is more hydrophilic than powder. The enthalpy suggests water was sorbed endothermically on powders and exothermically on starches. The entropy suggests freedom of water was decreased in the solution and that those of powder and starch were reversed.
In this study we compared methods of evaluating early stage (within 24 h storage at 4 °C) of retrogradation process in cooked rice. The degree of retrogradation in cooked rice could be estimated by the value of L* measured by a chroma meter; however, the data showed low precision . A small peak according to the retrogradation of cooked rice appeared on the DSC curve and the Xray diffraction pattern after the rice had been refrigerated for 6 and 9 h, respectively. The degree of gelatinization measured using the BAP method showed a difference among the three samples with different water contents refrigerated for over 12 h, whereas a difference among the samples after 9h refrigeration was noted in the degree of retrogradation by the DSC measurement. In the cooked rice samples, a difference in water content of 8% did not influence the enthalpy calculated from the DSC curve. From the advantage of being able to detect the difference of the sample refrigerated for a shorter period and to do direct measurement, we considered that the DSC measurement was a suitable method for evaluating the retrogradation of cooked rice at the early stage .
Amylases of Streptomyces griseus, Pseudomonas stutzeri and Klebsiella pneumoniae produced mainly G3, G4 and G6 at the initial stage of the reaction. The amylase of Bacillus licheniformis had a dual product-specificity for the formation of G5 and G3. Amylases of Pseudomonas stutzeri and Bacillus licheniformis catalyzed the degradation of water-insoluble, cross-linked blue starch . All four amylases also hydrolyzed partially-oxidized potato amylose and the degree of hydrolysis increased gradually. The action patterns of four amylases were investigated by two-dimensional paper chromatography by using 14C-reducing-end-labeled maltooligosaccharides. Three amylases of S. griseus, P. stutzeri, and K pneumoniae were characterized as exo-amylases, and that of B. licheniformis was an endo-amylase. Three such exo-amylases, namely maltotriohydrolase, maltotetraohydrolase, and maltohexaohydrolase formed products having α-configuration . I propose to classify this new group of amylases as “exo-α-amylase” with high product-specificity . Maltohexa ose was also formed from maltotetraose by a transfer reaction of the exo-maltohexaohydrolase, with an action pattern dependent on the substrate concentration. In addition, continuous production of maltotetraose using a dual immobilized enzyme system of maltotetraohydrolase and pullulanase was studied. The effects of operating conditions on the maltotetraose production were examined to confirm that the maltotetraose content of the products could be analyzed using the specific space velocity, SSV. The effectiveness of using immobilized pullulanase along with the maltotetraohydro lase was confirmed from constant-conversion operations in which the maltotetraose content in the product was kept at 50% (w/w) for 60 days in laboratory and bench scale experiments. Furthermore, industrial production and utilization of brand-new starch-related functional oligosaccharides will be described in this paper.
Using possible monodeoxy derivatives of p-nitrophenyl (pNP) α-D-glucopyranoside, -mannopyranoside, and -galactopyranoside as probe substrate, glycon specificities of α-glucosidases, -mannosidases, and -galactosidases from various sources were investigated, through hydrolysis of them. α-Glucosidases of Saccharomyces cerevisiae, Bacillus stearothermophilus, and honeybee hydrolyzed no deoxy derivatives, while the enzymes of rice, sugar beet, flint corn, and Aspergillus (A.) niger hydrolyzed the 2-deoxy derivative with substantially high activities. Moreover, flint corn and A. niger enzymes showed, although low, activities against the 3-deoxy derivative. Jack bean and almond α-mannosidases both showed sufficient activities toward 6-deoxy derivative. A. niger α-galactosidase acted on only 2-deoxy derivative with substantially high activity, while the enzymes of green coffee bean and Mortierella vinacea hydrolyzed not only the 2-deoxy derivative but also 6-deoxy one with low activities. Oligosaccharides that contain 2- or 3-deoxygenated glucose were synthesized by the transesterification reaction of A. niger α-glucosidase. α- and β-Anomers of methyl 6-ο-(p-tolylsulfonyl)-D-glucopyranoside, -mannopyranoside, and -galactopyranoside were acetylated partially by lipase-catalyzed transesterification with vinyl acetate. A lipase from Pseudomonas cepacia (lipase PS) reveaed high activity and regioselectivity for the esterification of them.α-Glycopyranosides were acetylated preferentially at the C-2 hydroxyl group, while corresponding β-anomers were acetylated preferentially at the C-3 hydroxyl group. The best selectivity was observed in the reaction of the glucopyranosides. Using methyl 3-ο -acetyl-6-ο -(p-tolylsulfonyl)-β-Dglucopyranoside, which was prepared in quantitative yield by the lipase PS-catalyzed regioselective acetylation, as a common starting material, highly deoxygenated monosaccharides, namely 2, 6-dideoxy-D-arabino-hexopyranose, 2, 4-dideoxy-D-threo-hexopyranose, and 2, 4, 6-trideoxy-D-threo-hexopyranose, respectively, were chemically synthesized in good yields.
Isomalto-dextranase (EC 184.108.40.206) was purified from the culture of a soil bacterium, Arthrobacter globiformis T 6 by successive chromatographies on CM-cellulose and CM-sepharose to a homoge neous state as confirmed by PAGE. The molecular weight of the enzyme was estimated to be about 69 kDa by SDS-PAGE. The enzyme hydrolyzed α-1, 6-glucosidic linkages of dextran or isomalto oligosaccharides to release exolytically α-isomaltose from the non-reducing ends . The optimum pH and temperature of the enzyme were pH 5.3 and 65°C, respectively . The enzyme showed a weak isopullulanase activity, an endo-type attack on pullulan to produce isopanose. The isomalto dextranase expressed by the recombinant E. coli cells also produced isopanose from pullulan . The enzyme hydrolyzed α-1, 4-glucosidic linkage of panose as well as α-1, 6-glucosidic linkage of isomaltotriose. The kinetic features of the experiments with the mixed substrates of isomaltotriose and panose were in good agreement with those expected for a single catalytic site mechanism. The ionization constants, pKel and pKe2, of the essential ionizable groups 1 and 2 of the enzyme were 3.3 and 6.3 for dextran T2000 and 3.5 and 6.1 for isomaltotriose. The heats of ionization for groups 1 and 2 were 0 kcal/mol or less with both the substrates. These kinetic results suggested that the ionizable groups essential for the enzyme activity were carboxyl and carboxylate . Modification experiments with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), modifying carboxyl residues specifically, also indicated that the carboxyl groups were essential to the enzyme activity . The subsite affinities of the enzyme were culculated to be >7.3, <-7.2, 6.7, 0.74 and 0.18 kcal/mol for subsites 1, 2, 3, 4 and 5, respectively, from the rate parameters (Km and 10) for the hydrolysis of isomaltooligosaccharides. Subsites 1 and 3, showing large affinity values, were thought to attract the substrates and form the productive bindings. A new method for preparation of isomaltose was developed by using the enzyme and an acid-treated dextran. The branch points of dextran were selectively hydrolyzed by a mild acid pretreatment. When the acid-treated dextran was acted on by the enzyme, the maximal degree of hydrolysis went up to over 90%.
4G-Galactosylsucrose (β-D-fructofuranosyl 4-ο -β-D-galactopyranosyl-α-D-glucopyranoside or lactosucrose; LS) is selectively utilized by bifidobacteria in the human intestinal canal. This saccharide is found in the fermentation of yogurt containing sucrose as a sweetener. In 1957, Avigad and co-workers reported that LS was synthesized from sucrose and lactose by transfructosylation of levan sucrase. Production of LS was then proposed, using transfructosylation of levan sucrase or transgalactosylation of β-galactosidase from sucrose and lactose. However, industrial production of LS was not undertaken at that time. We have therefore attempted to establish production of LS, and to develop utilization of LS as an ingredient in health foods. Arthrobacter sp. K-1 isolated from soil produces β-fructofuranosidase. The enzyme catalyzes both transfructosylation and hydrolysis when incubated with only sucrose. However, in the presence of a suitable acceptor such as lactose, the enzyme predominantly catalyzes transfructosylation and transfers the fructosyl residue preferentially to an acceptor molecule rather than to a sucrose molecule. LS is commercially produced as follows. The mixture of sucrose and lactose (45-55: 55-45, w/w) is solubilized in water at 40% (w/w), and incubated with Arthrobacter sp. β-fructofuranosidase and invertase-deficient yeast at a temperature of 30-35°C for 24 h. The yeast is added to remove residual products of glucose derived from sucrose by assimilation, as these materials inhibit LS production. Utilizing this method, LS production is increased more than 65%. After heating is used to terminate the reaction, the reaction mixture is purified by decoloration, carbonation, filtration, desalination, ultra filtration and concentration. LS syrup containing over 55% LS can then be obtained. Three kinds of products, Nyuka-oligo LS-40 L, LS-55 L, and LS-55 P are commercially available, with LS contents of 40, 55, and 55%, respectively. The sweetness of LS, LS-40 L, LS-55 L, and LS-55 P is about 30%, 79%, 50-55% that of sucrose, respectively. These products have a high quality taste similar to sucrose. LS is not digestible in the human small intestine, but human intestinal microorganisms, particularly bifidobacteria, ferment it. The minimum effective dose of LS to improve intestinal microflora fecal conditions and defecation is 2 g/day. The saccharide is less likely to result in watery stool compared to other low-caloric sweetening agents. LS is used in soda, soft drinks, frozen yogurt, candy, biscuits, cookies, powdered soft drinks, sweet pastries (e.g., croissants), and table
It is well known that the carbohydrate moieties of glycoproteins or glycolipids, which exist on mammalian cell surfaces, are modified by sulfate or phosphate, etc, in a few cases. These modifiers are recognised as possible receptors in intercellular communications. On the other hand, mammalian milk or colostrum contains many kinds of free oligosaccharides other than lactose in common. These oligosaccharides are also suggested to have biological roles such as inhibition of the adhesion of pathogenic microorganisms to the intestinal tract of infant or brain development - stimulation in infants. Recently, a few milk oligosaccharides have also been shown to be modified and the biological significance of these saccharides are assumed as follows: 1) Sialyllactose lactone: Some proportion of Neu5Gc(α2-3)Gal(β1-4)Glc was found to occur as free lactones between the carboxyl group of Neu5Gc and OH of Gal or Neu5Gc itself (sialyllactose lactone) in ovine colostrum. The sialyl oligosaccharides in milk or colostrum may attach to bacterial toxin and viruses, such as influenza virus in the intestine, thus protecting the infant. The lactone can be assumed to be resistant to virus sialidase. When the sialic acid is liberated from sialyl oligosaccharides by virus sialidase, they would lose their protective effect but the lactone should retain this effect because of its resistance to the action of virus sialidase. 2) 4-O-acetylated sialyllactose: Neu5Ac(α2-3)Gal(β1-4) Glc, whose Neu5Ac was O -acetylated at OH-4, was identified in the milk of echidna, one of the monotremes. Although this saccharide was assumed to be a possible inhibitor for the adhesion of viruses or bacteria whose receptors are O -acetylated Neu5Ac, the exact function of this saccharide is still unknown. 3) sulfated oligosaccharides: Gal(β1-4)Glc-6'-O-sulphate (lactose 6'-O -sulfate) and Neu5Ac(α2-3)Gal(β1-4)Glc-6'-O-sulfate (N-acetylneuraminyllactose 6'-O-sulfate) were identified in rat milk, whereas Gal(β1-4)Glc-3'-O-sulfate (lactose 3'-O-sulfate) was found in dog milk. The presence of N-acetylneuraminyllactose 6'-O -sulfate is suggested in human milk, too, and, in addition, this milk contained oligosaccharides whose GlcNAc residues were replaced by sulfate at OH-6 position. These compounds may permit the simultaneous delivery of two essential nutrients, sulfate and calcium, in early life, avoiding the precipitation of insoluble calcium sulfate in milk. 4) phosphorylated oligosaccharides: Bovine or mare colostrum contained phosphorylated oligosaccha-rides including Neu5Ac(α2-6)Gal(β1-4)GlcNAc-a1-phosphate (cow), Neu5Ac(α2-6)Gal(β1-4)GlcNAc-6-phosphate (cow) and Gal(β1-4)GlcNAc-α1-phophate (horse). These oligosaccharides may also permit the simultaneous delivery of phosphate and calcium, avoiding the precipitation of calcium phosphate. The existence of these modified oligosaccharides in milk may offer valuable in-formation for manufacturing bio-functional materials in the food industry.