SSI accounts for 60% of the total SS activity in the soluble fraction in the developing rice endosperm. Rice SSI-deficient mutants were identified by using reverse genetics, and the chain-length analysis of the endosperm starch showed that SSI distinctly synthesizes DP 8-12 chains from short DP 6-7 chains emerging from the A chains and the branch points in the B1 chains of amylopectin.1) In this study, to evaluate by in vitro study the function of recombinant SSI of rice (rSSI), the change in the chain-length distribution of glycogen or amylopectin in the activity band after rSSI enzymatic reactions in native-PAGE gel was examined. When glycogen was used as the substrate, the α-glucan produced in the rSSI activity band on the native-PAGE gel had specifically fewer DP 6 chains and more DP 8 chains than unmodified glycogen did. When rice amylopectin was used as the substrate, the α-glucan produced in the rSSI activity band showed the oscillation of a short turn in the range of DP ≤ 20 compared with the unmodified amylopectin; the chains with DP 6, 7, 10, 11, 13 and 17 decreased, while the extent of decrease was reduced in the chains with DP 8, 12 and 15. These results suggest that rSSI preferentially elongates the chains up to DP 8 from the short and long B1 and B2 chains with DP 6 or 7 from branch points to the non-reduced end as well as the A chains with DP 6 or 7 by adding one or two glucose moieties.
The absorbance of 500 nm of various concentration of cyclodextrin (CD) containing 0.02 mM congo red (CR) solution were obtained. The absorbance was plotted toward the concentration of CDs to obtain curves showing the relation of the variation of the absorbance to the concentration of CDs. In the case of the α-CD series (α-CD, G1-α-CD and G2-α-CD), the absorbance increased in accordance with the CDs’ concentration, and the increasing ratio of G2-α-CD was considerably higher than those of α- and G1-α-CD. On the other hand, in the case of the β-CD and γ-CD series, the absorbance curves showed that they reached a plateau at a certain concentration. It was almost same among the β-CD series. A certain interaction, which was maximum at a ratio less than CR: β-CD/1:250, might occur between CR and β-CD, except inclusion complex formation. The absorbance curves for the γ-CD series showed that it reached a plateau at a considerably lower concentration than that of β-CD. The formation ratio of CR: γ-CD complex was calculated as 1:2 at pH 5.0. We inferred that cavity size and the character of CDs affected accessing style to CR molecules and that the CR molecule was unable to access α-CD because the cavity size was too small to incorporate a CR molecule. The cavity of β-CD may be able to attract a CR molecule, but is unable to incorporate it. The cavity of γ-CD may be able to incorporate a CR molecule. As a conclusion, the α- and β-CD series were able to interact with CR molecules except in inclusion complexes, whereas, the γ-CD series were able to form inclusion complexes. In the NMR spectroscopy of CR in CDs containing D2O, the 1H NMR profile of CR was greatly changed by the addition of the γ-CD series, slightly changed by the addition of the β-CD series and not changed by the addition of the α-CD series. The chemical shift of the γ-CD series was also greatly changed by the addition of CR, but not the α-CD or β-CD series. By the addition of the same molar concentration of γ-CD to CR in D2O, 1:1 complex formation was achieved, and the pH of this solution was around 8. We inferred that the complex formation ratios’ difference between NMR of spectroscopy and spectrophotometry depends on the solutions’ pH.
Fermented beverage of plant extract was prepared from 55 kinds of fruits and vegetables. Natural fermentation was conducted by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp. and Pichia spp.). We have previously found that the fermented beverage contained the novel saccharide, O-β-D-fructopyranosyl-(2→6)-D-glucopyranose, which is produced by the fermentation process. The characteristics of O-β-D-fructopyranosyl-(2→6)-D-glucopyranose were investigated. The saccharide showed 0.2 times the sweetness of sucrose, non-cariogenicity and low digestibility. Furthermore, the unfavorable bacteria, Clostridium perfringens, Escherichia coli and Enterococcus faecalis that produce mutagenic substances did not use the saccharide. Therefore, it was thought that saccharide 1 could be a new material for foods.
In this study, we isolated alkaliphilic Bacillus sp. strain HM-127 as the source of α-glucosidase. An analysis of the purified enzyme for molecular mass was carried out by SDS-PAGE, which revealed a single band (63 kDa). Maximum activity of the enzyme against maltose was at pH 6.4. When p-nitrophenyl-α-glucopyranoside (pNPG) was used as substrate, two pH optima, 6.4 and 8.3, were observed. The enzymatic activity was strongly inhibited by Fe2+, Zn2+ and ethylenediaminetetraacetic acid, when using both substrates. Phenylmethylsulfonyl fluoride and dithiothreitol almost completely eliminated the pNPG-hydrolyzing activity and maltose-hydrolyzing activity, respectively. At high concentrations of sucrose and turanose, the activity of the enzyme against pNPG was markedly inhibited at pH 8.3 but no inhibitory effect occurred at pH 6.4. The present results suggest that the Bacillus sp. strain HM-127 α-glucosidase hydrolyzes pNPG and maltose by different catalytic mechanisms at pH 6.4 and 8.3.
Amylose and amylopectin unit-chain distributions of starches from the roots of Panax ginseng C.A. Meyer (PG) and Panax notoginseng (Burk.) F.H. Chen (PN), tuber of Pinellia ternata (Thunb.) Breitenbach (PT), rhizome of Alisma orientale Juzepczuk (AO) and seed of Coix lacryma-jobi Linné var. ma-yuen Stapf (CL) were investigated by means of a gel filtration method. The amylose contents of the PG, PN, PT, AO and CL starches were 15.4-28.2, 25.9-35.7, 20.1-34.9, 26.6-35.2 and 17.2-26.4%, respectively. The ratios of subfractions (Fr. III/Fr. II) of amylopectin in PG, PN, PT and AO starches were 0.82±0.19, 0.93±0.13, 1.22±0.25 and 1.23±0.05, respectively. Those of CL starches were 0.97 and 3.49. The weight-average chain-lengths (CLw) of amylopectin in PG, PN, PT, AO and CL starches were 33-47, 28-35, 22-32, 25-28 and 23-38, respectively. It was confirmed that the ratio of subfractions (Fr. III/Fr. II) was negatively correlated with CLw. The weight-average degrees of polymerization (DPw) of amylose from PG, PN, PT and CL starches were 2720-3590, 3240-3730, 2390-2710 and 3090-3880, respectively.
The influence of the grain-filling temperature of rice on the super-long chain (LC) content of amylopectin and its chain-length distribution was examined by the fluorescent labeling/HPSEC method. The LC content of Kirara 397, which is the main cultivar in Hokkaido, was 2.58, 1.30 and 0.48% for the grain-filling temperature (°C, day/night) of L21/17, M25/21 and H29/25, respectively. The lower temperature increased the LC content and also the molar and weight ratios (A+B1)/(B2+B3) of amylopectin unit-chains. These findings were confirmed by the experiment for five varieties and four lines of rice recently bred in Hokkaido and Milky Queen, cultivated at the grain-filling temperature of L22/16, M26/20, H30/24 and HH34/28. From the LC contents at 19, 23, 27 and 31°C (average temperature of day and night) the increase of LC content per 1°C was determined to be 0.542 0.152 and 0.037%/°C for the temperature ranges of 19-23, 23-27 and 27-31°C, respectively. This clarified that the lower temperature increased the LC content more, as in the case of the apparent amylose content (Amylose+Amylopectin LC). The proportion of the LC in the apparent amylose content was larger for low-amylose varieties than for non-glutinous varieties. Less varietal difference in the LC content among the varieties and lines was observed under the grain-filling temperature. Therefore, the target of rice breeding for cooked rice with good texture appeared to be the low LC content of amylopectin with stable structure against grain-filling temperature. This might be achieved by the gene analysis of enzyme(s) involved in the biosynthesis of amylopectin LC.
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