We investigated the effects of the major short chain fructooligosaccharides, 1-kestose and nystose, on the intestinal microorganisms and on the intestinal and systemic immune responses of mice. Both 1-kestose and nystose promoted intestinal Lactobacillus number. However, the balance of Lb. reuteri and Lb. intestinalis, the major Lactobacillus species in the mice was not altered. The IgA content in the feces of mice treated with both 1-kestose and nystose increased from day 4 to day 7 after starting the administration and returned to the same level of control mice on day 14. Splenocyte responses to Con A, anti-CD3 plus anti-CD28 antibodies and LPS were reduced by 1-kestose and nystose. Nystose lowered IL-2, IFN-γ, IL-12 and IL-4 secretion from the splenocytes more than 1-kestose. These results suggested that both 1-kestose and nystose can influence the microorganisms as well as the intestinal and systemic immune responses, but to different degrees.
α-Glucuronidase from Aspergillus niger specific for O-α-D-glucosyluronic acid α-D-glucosiduronic acid (trehalose dicarboxylate, TreDC) was hydrolyzed TreDC to produce D-glucuronic acid. For the application of the α-glucuronidase to D-glucuronic acid production, the enzyme was immobilized on a baked diatomite. The immobilized enzyme treated with glutaraldehyde retained about 60% of the initial activity after repeated uses of 20 cycles. Although the immobilized enzyme completely hydrolyzed 100 mM TreDC, the reaction was significantly inhibited by D-glucuronic acid over 200 mM TreDC. To hydrolyze TreDC completely, the reaction mixture was diluted with buffer to keep the D-glucuronic acid concentration below 300-400 mM. The immobilized enzyme in the diluted reaction mixture hydrolyzed 500 mM TreDC (initial concentration) almost completely to produce D-glucuronic acid. The immobilized enzyme packed in a column continuously produced D-glucuronic acid and the activity of the enzyme remained about 80% of the initial activity after 20 days of operation.
α-Amylase isoforms I-1 and II-4 were found in rice grains during ripening, α-amylase II-4 being the most predominant isoform. To determine their functions in ripening seeds, we generated a series of transgenic rice plants transformed with α-amylase I-1 and α-amylase II-4 cDNA under the control of Cauliflower mosaic virus 35S promoter. These isoforms were increased in young shoot and mature leaf tissues of the transgenic plants at both mRNA and protein levels. The starch accumulation in leaves was reduced to 42-82% of that in the wild-type. The transgenic lines A3-1 and D1-4, which overexpressed α-amylase I-1 and α-amylase II-4, respectively, were examined further. The enzyme activity was increased in both seeds, and the increase was greater in D1-4. The dry weight of A3-1 and D1-4 seeds was decreased approximately 4 and 11%, respectively. White immature grains frequently appeared in both lines, with severer abnormalities seen in D1-4. These results strongly suggest that the increase of α-amylase activities inhibits the accumulation of reserve starch and lowers the grain quality of rice.
Resistance to chelators, as well as thermostability, of an alkaline α-amylase (AmyK, formerly named LAMY) from an alkaliphilic Bacillus sp. strain was significantly improved by deletion of Arg181-Gly182. To clarify the mechanism of thermostability and chelator resistance conferred by the deletion mutation, we constructed models of AmyK with the 3D structure of α-amylase from Bacillus amyloliquefaciens as the template. In the structural models, Ala186 and Asp188 on a loop were both coordinated with a structural calcium ion. Molecular dynamics simulations suggested that the affinity of the Ala186 carbonyl oxygen to the calcium ion in the mutant enzyme was strengthened, thereby causing enhanced thermostability and the chelator resistance.
Malto-oligosyltrehalose synthase (EC 188.8.131.52, MTSase) catalyzes the conversion of α-1,4-glucan to glycosyltrehalose by forming an α,α-1,1-glucosidic linkage on the reducing side of the α-1,4-glucan. This enzyme also slightly hydrolyses the glucan and releases glucose from the reducing end of the glucan. We mutated the gene of MTSase from Sulfolobus acidocaldarius ATCC 33909 and expressed the mutated gene in E. coli. The mutants of Asp228, Glu255 or Asp443 corresponding to the catalytic residues of the α-amylase family enzymes showed no enzymatic activity. The transglycosylation activity of the mutants of Lys390 or Lys445 decreased, but the hydrolytic activity of the mutants increased in comparison to the wild-type enzyme. The substitution of Lys390 or Lys445 for a bulky residue, tryptophan, caused the loss of the transglycosylation activity of the enzyme, and provided a novel hydrolase reacting on the reducing side of the α-1,4-glucan.
Starch is synthesized in semi-crystalline granular structures. Starches of different botanical origins possess different granular sizes, morphology, polymorphism and enzyme digestibility. These characteristics are related to the chemical structures of the amylopectin and amylose and how they are arranged in the starch granule. In this paper, structures and locations of amylose and amylopectin molecules in the granule are reviewed. The branch structures of amylopectin molecules and their relationship with the polymorphism, structures, and morphology of the starch granules are discussed. Internal structures of starch granules revealed by confocal laser-scattering microscopy and by using a surface-gelatinization method are compared and their effects on surface pinholes and serpentine channels of the starch granules are discussed.
An improved method for the quantitative analysis of isomaltooligosaccharide (IMO) products by HPLC with a polymer-based amino column was developed. The column was much higher in durability than a silica-based amino column used for the conventional method. The column durability enabled us to determine each IMO using the calibration curve of RI-detector response against a concentration of standard IMO and maltosaccharide reagents. The linear relationship between peak height of RI response and concentration of saccharide was found for glucose, maltose, kojibiose, nigerose, isomaltose, maltotriose, panose, isomatotriose, maltotetraose and isomaltotetraose. The linearity was obtained at concentrations of up to 17 mg/mL, and correlation coefficients were ≥0.999. The slope of peak height versus concentration differed from saccharides, of which glucose was the highest while isomaltotetraose was the lowest. The relative slope of each saccharide to glucose, (slope for saccharide)/(slope for glucose), referred as a conversion factor, was calculated, and the concentration of each saccharide in commercial IMO products was determined from peak height on a HPLC chromatogram by the following equation: (concentration of saccharide A, mg/mL)=(concentration of standard glucose, mg/mL)×(peak height of A)/(conversion factor of A)/(peak height of standard glucose). A commercial IMO product was analyzed and the result obtained was as follows: isomaltose (19.2 g), isomaltotriose (10.3 g), panose (4.9 g), nigerose (2.0 g), kojibiose (3.5 g) and isomaltotetraose (2.8 g), respectively. The total amount of the sugars identified by the improved method from IMO was higher than those determined by the conventional method, which may have resulted from the higher resolution of each saccharide. The method showed clearly the presence of nigerose and kojibiose together with four unknown components. A major unknown component was identified to be isomaltotriosylglucose by 1H- and 13C-NMR analyses.