We found that various kinds of hydrophobic food stuffs such as polyphenols and fatty acids were dispersed into water phase by the action of CGTase (cyclodextrin producing enzyme) on the mixture of hydrophobic food stuffs and starch, and supposed that some complex of the stuffs might be formed with cyclodextrins and maltooligosaccharides resultantly produced from starch by the action of CGTase. In the case of using α-amylase instead of CGTase, some hydrophobic food stuffs such as green tea powder was also dispersed into water phase in the same way. We named these complexes “CD wrap” and “Sugar wrap” . To make these phenomena clear by using spectrophotometry and the NMR method, we showed that maltooligosaccharides having some degree of polymerization changed the color of congo red and that the NMR profile of such maltooligosaccharides was changed by the addition of congo red. From these results, we inferred that congo red interacted with maltoheptaose mildly to form some complex.
High activity of alginate lyase was produced by a marine bacterium (strain No. 1786), which was isolated from the intestinal contents of an arthropod. Strain No. 1786 was classified in the genus Pseudoalteromonas by morphological, biochemical and physiological characterization and 16S rDNA sequencing. The extracellular enzyme obtained from the culture supernatant was partially purified by diethylaminoethyl (DEAE) high-performance liquid chromatography (HPLC). The molecular mass of this enzyme was approximately 32.0 kDa on SDS-PAGE. The optimum temperature and pH of the enzyme reaction were 50°C and 7.1-7.7, respectively. The enzyme was stable up to 40°C and in the pH range 6.3-8.9. Furthermore, the enzyme activity was enhanced over 300% compared with the control level by the addition of 10 mM MgSO4, MgCl2, or CaCl2. In the enzyme reaction products, we detected peculiar peaks of unsaturated oligosaccharides on DEAE-HPLC derived from poly-β-D-mannuronate (PM) and poly-α-L-guluronate (PG) rich substrates, respectively. These results indicate that we have isolated a new extracellular alginate lyase in the genus Pseudoalteromonas, possessing high optimum temperature and enhancing the enzyme activity remarkably by adding Mg2+ and Ca2+.
The characteristic effect of such amino acid couples as aspartic acid (Asp) and glutamic acid (Glu), Glu and lysine (Lys), and asparagine (Asn) and glutamine (Gln) selected by the difference in net charge on the gelatinization temperature (GT) and viscosity of potato starch granules was analyzed from the results evaluated by differential scanning calorimeter and rapid viscoanalyzer, respectively. These results were compared with those of the individual amino acids. Asp, Glu and Lys had a greater effect than Asn and Gln on increasing GT, and markedly reduced the peak viscosity (PV). The effect of the Asp+Glu couple, which both have a negative net charge, on GT was according to the sum of the increasing effect of the individual amino acids on GT, and that of the Glu+Lys couple, which respectively have a negative and positive net charge, was synergistically multiplied by those of the two. However, the effect of the Asn+Gln couple, which both have a zero net charge, on GT was hard to estimate due to little change in GT. The effect of the Asp+Glu, and Glu+Lys couples on PV both approached the sum of the decreasing effect of the respective two.
Physicochemical properties of common buckwheat starches prepared from seven cultivars, Miyamaminamimiyajizairai, Onozairai, Imajozairai, Ikedazairai, Hitachiakisoba, Sinano 1 go and Kitawasesoba, were investigated. The gelatinization temperature for RVA ranged from 66.5 to 70.8°C. The range of amylose contents for the gel filtration method was 27.3-31.8%, and the ratio of short chains to long chains of amylopectin was 3.0-3.3. Swelling power of buckwheat starch at 93°C correlated negatively with amylose content. The swelling power and solubility of buckwheat starch at 93°C were about 30 and 12%, respectively. The digestibility by pancreatin was 77-81% for 3 h. Buckwheat starch granules were oval and polygonal in shape, and were 3-10 μm in size. The X-ray diffraction pattern is shown in Figure A.
The genes for 1,2-α-L-fucosidase and endo-α-N-acetylgalactosaminidase have been cloned from Bifidobacterium bifidum JCM1254 and Bifidobacterium longum JCM1217, respectively. The catalytic domain of 1,2-α-L-fucosidase (AfcA) specifically hydrolyzed the terminal α-(1→2)-fucosidic linkages of human milk oligosaccharides and sugar chains of glycoproteins. It exhibited high sequence similarity to several hypothetical proteins in a database, and thus, novel glycoside hydrolase (GH) family 95 has been created. Catalytically important residues of the domain, which were revealed by X-ray crystallographic analysis, were well conserved within the family except for one residue located at the +1 subsite. Endo-α-N-acetylgalactosaminidase (EngBF) that was expressed as C-terminally histidine-tagged protein was found to liberate Galβ1,3GalNAc disaccharide from Core 1-type O-glycans. Its homologues were found in the genomes of several bacteria, and thus a novel GH family (GH101) has been established. Considering that both AfcA and EngBF were able to degrade natural substrates present in intestine, it was envisaged that the enzymes play important roles for the organisms in making their habitats in the colon. The recent finding of the GNB/LNB pathway in bifidobacterial cells by Kitaoka’s group further supports this notion.
Glycoside hydrolase family 31 (GH 31) is one of the most intriguing glycoside hydrolase families. This family contains α-glucosidase, α-xylosidase, α-glucan lyase and isomaltosyltransferase. Escherichia coli YicI (α-xylosidase) is a representative enzyme of GH 31 because its biochemical and structural studies have been thoroughly carried out. YicI is a strict α-xylosidase, which rigidly recognizes α-xyloside at the non-reducing terminal end, even though its amino acid sequence apparently displays similarity with α-glucosidases. Phe277, Cys307, Trp345 and Lys414 at the subsite-1 are important for α-xylosidase activity. The mutant YicI enzymes, which possesses Ile307/Asp308 instead of Cys307/Phe308 and which has a shorter β→α loop 1 of (β/α)8 barrel in place of the original longer loop, respectively, possess α-glucosidase activity. In the transxylosylation of YicI, glucose, mannose and allose are able to act as acceptors, but galactose, talose and gulose never do, implying that equatorial OH-4 of the aldopyranose is crucial for acting as an acceptor. YicI transfers α-xylosyl moieties to a specific hydroxy group in the acceptor sugar (except fructopyranose) showing 1,6 regioselectivity, which is in agreement with the structural feature of the aglycone-biding site. Among the transxylosylation products of YicI, α-D-xylopyranosyl-(1→6)-D-mannopyranose, α-D-xylopyranosyl-(1→6)-D-fructofuranose, and α-D-xylopyranosyl-(1→3)-D-fructopyranose are novel sugars. α-D-Xylopyranosyl-(1→6)-D-mannopyranose and α-D-xylopyranosyl-(1→6)-D-fructofuranose have the ability to inhibit rat intestinal α-glucosidases.
We screened for the carbohydrate-active enzymes that catalyze transglycosylation reactions on carboxylic compounds. Sucrose phosphorylase from Streptococcus mutans showed remarkable transglucosylating activity on benzoic acid, especially under acidic conditions. Sucrose phosphorylase from Leuconostoc mesenteroides also showed the activity, although it was very weak. Three main products were detected from the reaction mixture with sucrose, benzoic acid and S. mutans sucrose phosphorylase. These compounds were identified as 1-O-benzoyl α-D-glucopyranose, 2-O-benzoyl α-D-glucopyranose and 2-O-benzoyl β-D-glucopyranose on the basis of their isolation and the isolation of their acetylated products and subsequent spectroscopic analyses. Time-course analyses of the enzyme reaction and the degradation of 1-O-benzoyl α-D-glucopyranose proved that 1-O-benzoyl α-D-glucopyranose was initially produced by the transglucosylation reaction of the enzyme, and 2-O-benzoyl α-D-glucopyranose and 2-O-benzoyl β-D-glucopyranose were produced from 1-O-benzoyl α-D-glucopyranose by intramolecular acyl migration reaction. The acceptor specificity in the transglucosylation reaction of S. mutans sucrose phosphorylase was also examined. The enzyme could transglucosylate toward various carboxylic compounds. Comparison of the pH-dependence of transglucosylation activities of the enzyme on phosphate, hydroquinone and acetic acid suggest that an undissociated carboxylic group is essential as the acceptor molecule for the transglucosylation reaction on carboxylic compounds. We also obtained 1-O-acetyl α-D-glucopyranose using the transglucosylation reaction of the enzyme. The sensory test of acetic acid and the glucosides revealed that the sour taste of acetic acid was markedly reduced by glucosylation.
A lactose-oxidizing enzyme was obtained from culture supernatant of a fungal strain of Paraconiothyrium sp. KD-3. The enzyme was a flavoprotein with a molecular mass of 54 kDa. The purified enzyme oxidized various monosaccharides and oligosaccharides such as D-glucose, D-galactose, D-xylose, cellooligosaccharides and maltooligosaccharides in addition to lactose, using molecular oxygen as a good electron acceptor, to accumulate the corresponding aldonic acids and hydrogen peroxide. The Paraconiothyrium enzyme was suitable for the production of calcium lactobionate compared with a commercial hexose oxidase owing to the stability during the conversion. The enzyme converted 10-20% (w/v) lactose completely to calcium lactobionate in a 500-mL reactor under aeration, agitation and pH regulation at 5.5. The neutralization was successfully performed by the addition of 10% (w/w) slurry of calcium carbonate, while neutralization with 25% (w/v) sodium hydroxide solution inactivated the enzyme gradually. The supplement of catalase to the mixture promoted the conversion by degrading hydrogen peroxide. The immobilized enzyme, which was prepared by the adsorption to a cation exchange resin followed by the condensation with carbodiimide, oxidized 18.5% (w/v) lactose to produce calcium lactobionate in a batch reaction. Calcium lactobionate and aldonates derived from D-galactose, D-xylose and L-arabinose showed high aqueous solubility and low calcium binding capability.
Efficient enzymatic digestion for soybeans, soybean milk residue (okara), and coffee beans was carried out by a selected food-processing cellulase or pectinase. Enzymatic digestion based on a consideration of cells and cell-wall structures brought about efficient digestion. Cell-walls, components and organs of plant are very complicated. Starch, protein and oil are stocked in the particles of plant cells, and the residues after their extraction are certainly indigestible fibers. These enzymatic digestions are considered to be difficult; however, if the reaction order, pre-treatment conditions, and selection of the enzymes and the combinations are investigated, the digestions will achieve a high yield. The efficient digestion of the cells of soybeans and coffee beans was carried out as model cases. The solution approaches and the components of the cells are shown and discussed.
Fermented beverage of plant extract was prepared from about fifty kinds of fruits and vegetables. Natural fermentation was conducted by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp. and Pichia spp.). Fourteen kinds of oligosaccharides have been isolated from this beverage; their structures were confirmed by methylation analysis, MALDI-TOF-MS and NMR measurements. In these saccharides, five novel oligosaccharides have been found to be constructed by fructose residue of pyranose form, and fructosyl residues of sucrose bond with the β-D-galactose and β-D-glucose. The characteristics of one of the novel saccharides, O-β-D-fructopyranosyl-(2→6)-D-glucopyranose (Fp2-6G) were investigated, and included non-cariogenicity and low digestibility. Furthermore, the unfavorable bacteria, Clostridum perfringens, Escherichia coli and Enterococcus faecalis, that produce mutagenic substances did not use the saccharide. The Fp2-6G synthesis activity of crude enzyme of the 5 yeast strains isolated from plant extract was examined using the ABEE-converting method. Fp2-6G synthetic activity was observed only in a reaction mixture using crude enzyme from the Y-1 strain. The Y-1 strain was identified as Pichia spp.
Arabinogalactan-proteins (AGPs) are a family of complex proteoglycans found throughout the plant kingdom. AGPs are massively glycosylated, and are implicated in diverse plant growth and development. Gum arabic and larch arabinogalactan, which are a kind of AGP, were used for food materials and additives. Carbohydrate moieties of AGPs consist of β-1,3-galactan backbone having β-1,6-galactan side-chains. Galactanases that hydrolyze β-1,3- or β-1,6-galactans are important in degrading AGPs. We succeeded in, for the first time, cloning an exo-β-1,3-galactanase and an endo-β-1,6-galactanase genes from Phanerochaete chrysosporium (Pc1,3Gal43A) and Trichoderma viride (Tv6GAL), respectively. Pc1,3Gal43A consisted of two modules resembling glycoside hydrolase family 43 (GH43) and carbohydrate binding module family 35 (CBM35). It specifically hydrolyzed only the β-1,3-linkage of two galactosyl residues in an exo-acting manner. However, it produced oligosaccharides together with galactose from AGPs, suggesting that Pc1,3Gal43A is able to accommodate β-1,6-linked galactosyl side-chains. The C-terminal CBM35 specifically bound to β-1,3-galactan. Using the Pc1,3Gal43A sequence as a query in BLAST search, we newly obtained the enzymes from Clostridium thermocellum and Streptomyces avermitilis. Both enzymes contained GH43 and CBM13. We found similar sequences not only in bacteria but also in plants. The two kinds of gene products from Arabidopsis thaliana demonstrated exo-β-1,3-galactanase activity when they were expressed in yeast. Thus, exo-β-1,3-galactanases are distributed in the fungal, bacterial and plant kingdoms. The putative endo-β-1,6-galactanase gene from S. avermitilis which was found by using Tv6GAL sequence was cloned. The recombinant enzyme catalyzed the hydrolysis of β-1,6-linked galactosyl linkages of oligosaccharides and polysaccharides in an endo-acting manner.
Chitinase (Pa-Chi) and chitin oligosaccharide deacetylase (Pa-COD) are involved in the production of a heterodisaccharide, β-D-N-acetylglucosaminyl-(1,4)-D-glucosamine (GlcNAc-GlcN). These enzymes were recovered from the supernatant of Vibrio parahaemolyticus KN1699 cell culture and purified and characterized. For each enzyme, an ORF encoding gene and its signal sequence were cloned from genomic DNA of strain KN1699. In addition, the expression plasmid was constructed for each enzyme gene and inserted into Escherichia coli cells, and recombinant Pa-Chi and Pa-COD (Pa-rChi and Pa-rCOD) were secreted into the culture medium with the aid of signal peptides. Di-N-acetylchitobiose [(GlcNAc)2] was produced in 60% (w/w) yield by cultivating the Pa-rChi-secreting E. coli cells in 2% (w/v) β-chitin-containing medium. Moreover, GlcNAc-GlcN was produced in high yield by treating (GlcNAc)2 with the culture supernatant of Pa-rCOD-secreting E. coli cells.