A new method for simultaneously analyzing positional isomers of α-linked glucooligosaccharides was described. Four α-glucobioses (kojibiose, nigerose, maltose and isomaltose) were analyzed by using the p-aminobenzoic acid ethyl ester (ABEE)-conversion method under the following conditions: mobile phase, 0.1 M ammonium acetate buffer (pH 4.0) containing 12% acetonitrile; column temperature, 25°C. Furthermore a mixture of four α-glucobioses, five α-glucotrioses, 22- ο -α -D-glucosyl-D-maltose (kojibiosyl glucose), 32-ο-α-D-glucosyl-D-maltose (nigerosyl glucose), 4-ο -α-D-glucosyl-D-maltose (maltotriose), 62-ο-α-D-glucosyl-D-maltose (panose), 6-ο-α-D-glucosyl-D-maltose (isomaltotriose) and glucose was able to be analyzed by using the ABEE-conversion method with 0.1 M ammonium acetate buffer (pH 4.0) containing 10.5% acetonitrile as an eluent and a column temperature of 25°C. This separation was further improved by connecting two col-umns in series. The relationship between the peak areas and quantities of ABEE-converted c-linked glucooligosaccharides was linear in the range of 30 pmol to 3 μmol.
Lactobionic acid (LA) is derived from lactose and expected to be a versatile material for grow-ing bifidobacterium and forming mineral salts with high solubility in water for supplements. We aimed to develop microbial or enzymatic production systems of LA. To this aim, we screened lactose-oxidizing microorganisms, and obtained a strain of Burkholderia cepacia. The lactose-oxidizing activity existed in the membrane fraction of disrupted cell preparation of the strain. Only oxygen was necessary for lactose-oxidizing activity as a proton acceptor. A crude cell-free enzyme preparation was prepared, and its oxidizing ability and other properties on several saccharides were examined. The cell-free preparation oxidized D-glucose, D-mannose, D-galactose, D-xylose, L-arabinose and D-ribose. It also reacted with lactose, cellobiose, maltose, maltotriose, maltotetaose and maltopentaose. The strain accumulated LA in the culture supernatant with no loss of lactose. The strain is advantageous to production of LA by both fermentation and enzymatic reaction.
Several types of starch hydrolysates that have dextrose equivalents of 12-16 were compared in terms of structural analysis, in vitro digestibility via the Prosky method, and digestibility as esti-mated by measuring the glycaemic index in humans, with their correlations noted. Results showed that digestion of starch hydrolysates decreased with the initial moisture content during their prepa-ration. Structural analyses of samples suggested that the decrease in digestion was due to glycosidic linkage conversion. Conceivably, this glycosidic linkage conversion translates to condensation, a re-verse reaction to hydrolysis. Furthermore, it was also shown that digestibility of maltodextrins clearly differed between enzymatic hydrolysis and acidic hydrolysis, and that the molecular weights of indigestible fractions differed according to the hydrolysis method. In vitro digestibility of starch hydrolysates was also compared with their digestibility in humans by measuring glycaemic index. The portions determined indigestible from the in vitro test coincided with findings in humans. Similar results were obtained from the in vitro and in vivo tests. The in vitro test method used in this study may provide a simple and reliable approach to evaluate the digestibility of starch hydro-lysates in humans.
A gene (TM1751, Swissprot Q9X273, GenBank AAD36816) of Thermotoga maritima encoding endo-β-1, 4-glucanase of family 5 was cloned and expressed in Escherichia coli. The recombinant protein was purified by combination of Ni-NTA and Q-Sepharose FF column chromatography. SDS-PAGE analysis of purified fractions showed a homogeneous protein band with an expected molecular mass of 38 kDa. The specific activity of the enzyme increased about 16-fold while the recovery was 21% after purification. The optimal temperature of the enzyme was found to be 90°C while its optimal pH was 6.6. The enzyme was active over a wide pH range (4-9.5) and stable up to 85°C. Kinetic studies showed that the PNP-β-D-cellotetaoside and PNP-β-D-cellopentaoside had similar Km values of 0.25 and 0.24 mM respectively. However, maximum catalytic efficiency (kcat/ Km) was observed with PNP-β-D-cellopentaoside. Further analysis of the enzyme hydrolyzate by HPLC suggests that the carbohydrate binding cleft of the enzyme may be composed of four sub-sites for glucopyranose units. At the initial stage, the hydrolysis of PNP-oligosaccharides (DP up to 5) yielded a range of products with cellobiose, cellotriose and cellotetraose being the major prod-ucts. CMC hydrolysis also predominantly produced cellobiose and cellotriose as end products. The enzyme hydrolyzed mixed linked β-1, 3/1, 4 soluble substrates such as barley glucan and lichenan more strongly than CMC. Transglycosylation activity was also found to be taking place with smaller soluble cellooligosaccharides.
An extracellular pectate lyase of alkaliphilic Nocardiopsis sp. TOA-l, designated NPLase, was purified to homogeneity. The molecular mass of NPLase was estimated to be 54 kDa. Highest ac-tivity of this enzyme was obtained at pH 10.0 and 40°C and the isoelectric point (p1) was 4.7. NPLase was an endo type pectate lyase which degrades polygalacturonic acid in a random manner.
The oligosaccharide units of xyloglucans from the fruit cell walls of strawberry, persimmon, prune and banana were analyzed by enzymatic digestion followed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The oligosaccharide units of the polysaccharides were XXXG, XXLG, XLXG, XXFG, XLLG and XLFG [where each (1-4)-β-linked D-glucosyl residue in the backbone is given a one-letter code according to its sub-stituents: G, β-D-Glc; X, a-D-XyI-(1→6)-β-D-Glc; L, -D-Gal-(1→2)-a-D-XyI-(1→6)-β-D-G1c; F, a-L-Fuc-(1→2)-β-D-Gal-(1→2)-a-D-XyI-(1→6)-β-D-Glc] in an approximate molar ratio of 31: 4:9:38:5:13 for strawberry, of 26:12:7:6:39:10 for persimmon, of 31:16:10:21:6:16 for prune and of 38:4:16:9:28:5 for banana. The ratios of the six oligosaccharide units are different among them. However, the xyloglucans of the cell walls of these fruits are considered to be similar to each other in basic structure.