β-Fructofuranosidase from Arthrobacter sp . K-1 was immobilized onto a carrier resin (FE4611) with glutaraldehyde to effectively produce 4G-β-D-galactosylsucrose, which increased Bifidobacterium in the human intestine. Maximum immobilized enzyme was approximately 30 U/g·wet on carrier resin and its immobilized enzyme yield was 76.6%. Optimal pH broadened and optimum temperature increased in response to immobilization. The pH stable range for immobilized enzyme was wider than that for the native enzyme. Both immobilized and native enzymes were stable at temperatures lower than 45°C, whereas the immobilized enzyme was less stable than the native enzyme at temperatures higher than 65°C. The column reactor was successfully operated for 35 days to produce more than 30% LS using a constant conversion flow system on a pilot plant scale. The present results suggest the possibility of LS production on an industrial scale using a column reactor.
Isopullulanase (IPU) from Aspergillus niger ATCC 9642 is a cell-bound glycoprotein that hydrolyzes pullulan into isopanose. The sugar content of the recombinant enzyme expressed in Aspergillus oryzae M-2-3 decreased from 33.8 to 2.1% by 13 h-treatment with endoglycosidase H (Endo H), and deglycosylated rec-IPU had 65% of the original activity. 3 Deg-IPU (prepared by 3 h-treatment of Endo H; 6.8% sugar) showed the same substrate specificities, optimum pH and optimum temperature as native IPU and rec-IPU, while its kinetic parameters, ko and ko/Km values for pullulan, and Km, ko and ko/Km values for panose decreased with deglycosylation, except Km for pullulan. The oligosaccharide chains of rec-IPU were typed using lectin-peroxidase reagents and classified as hybrid- and/or high-mannose types.
Two distinct endo-cellulase components derived from Acremonium cellulase, a commercial cellulase preparation from Acremonium cellulolyticus, were extensively purified by consecutive column chromatography and designated as cellulase III-A and cellulase III-B. Cellulases III-A and III-B were each homogeneous on both Native- and SDS-PAGE, and were completely free from β-glucosidase. The molecular mass (SDS-PAGE) and pI Tvalues of cellulases IIIV-A and III-B were 58 kDa and 4.6, and 49 kDa and 4.2, respectively. Both enzymes contained 14-16% carbohydrates (as glucose). The N-terminal amino acid sequences from the 2nd up to the 20th residue of bothenzymes were determined by Edman degradation. Some enzymatic properties of the purified cellulases were investigated. The optimum pH and temperature for cellulases III-A and III-B were pH 5.5 and 55°C, and pH 5.5 and 65°C, respectively. Cellulases III-A and III-B were completely stable over the range of pH 4.2-8.0 at 4°C for 24 h and at temperatures below 55°C, and pH 3.3-7.8 and below 60°C, respectively. Cellulases III-A and III-B retained 25 and 88% of the original CMC-saccharification activities, respectively, after heating at 70°C for 10 min. The hydrolysis of CMC by cellulase III-B was more endo-lytic than that by cellulase III-A. Both enzymes splitvarious soluble and insoluble substrates to produce predominant cellobiose and a small amount of glucose as the final hydrolysis products.
A gelling polysaccharide was extracted from Togekirinsai (Eucheuma serra), which was collected from Miyako Island, Okinawa Prefecture, Japan, and purified by gelation with calcium chloride. The purified polysaccharide obtained from Togekirinsai was colorles fibrous powder; yield, 38.3% (w/w) based on dried seaweed and 4.6% (wet seaweed). The total carbohydrate, ash and moisture contents of the polysaccharide were 71.4, 21.2 and 7.1%, respectively. The content of total sulfate was estimated to be 23.8%. The polysaccharide was composed of D-galactose, 3, 6-anhydro-D-galactose and ester sulfate at a molar ratio of 1.2: 1.0: 1.5. Molecular mass of the polysaccharide was estimated to be about 2.8×105. The infrared spectrum and values of optical rotation of the polysaccharide at different temperatures were in agreement with those of standard ι-carrageenan. The 13C- and 1H-NMR spectra showed the polysaccharide isolated from Togekirinsai was composed of D-galactopyranosyl-4-sulfate and 3, 6-anhydro-D-galactopyranosyl-2-sulfate . These results indicate that the gelling polysaccharide is a ι-carrageenan.
White-rot fungus Irpex lacteus produces two cellobiose dehydrogenases (CDH I and CDH II) which have different electron acceptor specificity. CDH II which is a major CDH produced by I. lacteus was purified about 262 times to homogeneity and characterized. CDH II was a hemoflavo protein with the molecular mass of 97 kDa on SDS-PAGE, and it was a glycoprotein with carbohydrate content of 7.2% as glucose. Optimum temperature, heat stability as well as substrate specificity of this enzyme were similar to CDH from Phanerochaete chrysosporium. However, the stable pH range of CDH II was pH 3 to 5, which was different from other CDHs reported (pH 4 to 10). Cellobiose and cytochrome c were the best substrate and electron acceptor with kcat/Km values of 429.4- and 4405 mM-1s-1, respectively. From the studies on adsorption isotherm of this enzyme on cellulose, the one-binding site mechanism was suggested. Moreover, CDH II had higher adsorption affinity but lower adsorption capacity on crystalline cellulose than on amorphous one.
For the purpose of determining physicochemical properties of low-amylose rice starch, amylose content and amylopectin unit-chain distribution were studied by amperometric titration and gel filtration methods. Further, also examined were photopastography, viscosity and gel texture to determine characteristic gelatinization and retrogradation. Four kinds of low-amylose rice, an aromatic rice, a large-grain rice and a high-yield rice were used as the rice samples for comparison with two kinds of high-amylose rice, Hoshiyutaka and Yumetoiro, with Nipponbare as the control. The amylose content of low-amylose rice ranged from 4.3 to 9.6%. Snow pearl was the lowest, Ou 354 at 4.4%, Milky queen at 7.5%, Soft 158 at 9.6%, and Nipponbare at 17.1%. Fr. II, the longer chain length fraction in amylopectin, was noted at 24.0-26.3%, which was large, similar to the 4 kinds of low-amylose rice. Fr. III, the shorter chain length fraction in amylopectin, was noted at 55.8-69.8%. Fr. III/Fr. II was noted at 2.6-2.8, which was smaller, similar to the 4 kinds of low-amyloserice, than Nipponbare by 3.0. With respect to the rice starch, the amylose content showed a negative correlation for both maximum viscosity and breakdown, and there was a positive correlation with the viscosity when cooled at 50°C. The firmness of rice flour paste negatively correlated with the highest viscosity of the rice starch, while the firmness of rice flour paste and starch paste positively correlated with the setback.
The high hydrophobicity of Levoglucosan was verified by a newly developed assessment method for monosaccharide hydrophobicity. This method is a simple one, in which the hydrophobicity of a monosaccharide is estimated by the retention time of HPLC (partition chromatography) concerning monosaccharides. By this method, the hydrophobicity of Levoglucosan was estimated to be 1.36 times as large as that of D-Deoxyribose: this sugar was known as the monosaccharide with the highest hydrophobicity. We also assumed the hydrophobicities of L-Arabinose and L-Rhamnose. Based on these estimated hydrophobicities, we analyzed the data on the hardening phenomena of "Shiratama-ko" gels which were supplemented with monosaccharides. We came to the conclusionthat hardnesses of the gels has an obvious tendency to be decreased with increasing hydrophobicity.
The physicochemical properties of starches obtained from various parts of sago palm trunks at different growth stages were reported in a previous paper. Relative crystallinity by X-ray diffraction with calcium fluoride as the internal standard, amylose contents and amylopectin unit-chain distri-butions, photopastegraphy, and differential scanning calorimetry (DSC) of starches were determined. Results obtained were: (1) Relative crystallinity of starch was higher in 14.5-year-old palm than 9-year-old palm, and this also held true for upper part of the trunk as compared to the lower part of the trunk. (2) Amylose content, the total amount of Fr. I, of the starch from the lower part of the trunk was higher than that of the starch from the upper part of the trunk, and increased as the growth period increased. (3) Starch from the 14.5-year-old palm as well as the upper part of the trunk showed lower transmittance curves by photopastegraphy. (4) Gelatinization onset temperature of the starch from the upper part of the trunk ranged from 65.3 to 68.2°C, and gelatinization con-clusion temperature ranged from 75.0 to 76.0°C. Starch from the lower part of the trunk showed lower gelatinization onset temperature and higher gelatinization conclusion temperature compared with that of the starch from the upper part of the trunk.
A new method to evaluate the texture and sensory attributes of cooked rice was studied in order to characterize the properties of cooked rice in which amylose content was low, under 15%. Measurement using a new model of tensipresser was carried out under two different conditions of low and high compression, using either the one-grain or mass-grain method. The properties of cooked rice in respect to strength, flexibility and fracturability were estimated from measurements at low and high compressions. A new cooking procedure which allows the measurement of these items after cooking without transferring to a measuring cup was applied to the mass-grain method. Four kinds of low-amylose rice, an aromatic rice, a large-grain rice and a high-yield rice were used as samples for comparison with Nipponbare as the control. Information as to firmness and adhesiveness of both the surface part and the whole grain was obtained from the physicochemical data of cooked rice measured by the one-grain method at low and high compressibilities. Thus, it was found that the physicochemical properties were significantly different between even the two kinds in which the amylose contents were only slightly different. Further, there were also significant differences in strength, flexibility and fracturability between the samples. The characteristics of each cooked rice in which the amylose content was lower than 15% could be clarified by the one-grain method. Moreover, similar results to those from the mass-grain method were obtained by the onegrain method. It was found that the new cooking procedures applied to the mass-grain method were useful for making uniform rice samples, as well as for convenient and rapid measurement. Thus, this new method is effective for measuring the texture of cooked rice, and there is a definite conelation between its sensory attributes and amylose contents and the structure of amylopectin.
The physiological effects of L-arabinose on rats (male Wistar, 5-week-old) fed a diet containing 1 or 2.5% L-arabinose together with 20% sucrose were investigated up to 30 days. First, markedly strong inhibition of L-arabinose against the rat intestinal sucrase was confirmed in vitro. The body weights of rats fed the 2.5% L-arabinose diet for 30 days were significantly decreased, but there was no significant difference in the feed conversion rate. The concentration of plasma neutral lipid and the weights of adipose tissues (epididymal and retroperitoneal depots) were significantly decreased dose-dependently with L-arabinose. The concentration of total serum cholesterol and lipid content in liver also showed a slight decrease. The weight of the cecum with contents and the amount of total organic acids, such as acetate, lactate, propionate and succinate in the cecum, were markedly increased by L-arabinose ingestion with sucrose. However, the physiological effects of Larabinose were not observed without sucrose. In conclusion, L-arabinose ingestion with sucrose appears to be effective for decreasing the serum triacylglycerol content and weight of adipose tissues, and for increasing the production of organic acids in the cecum.