Molecular Weight Distribution of Whole Starch in Rice Endosperm by Gel-permeation Chromatography.

Starch is comprised of very large α-glucan molecules composed primarily of linear amylose and highly branched amylopectin. Most methods for analyses of starch structure use hydrolytic enzymes to cleave starch. When undegraded, whole starch structures can be analyzed by gel-permeation chromatography (GPC), but this typically yields a single peak each for amylopectin and amylose. The objective of this study was to stably separate amylopectins in whole starch based on their molecular weight using GPC, and to determine the structure of each peak. When alkali-gelatinized whole starch was applied to GPC columns (Toyopearl HW75S × 2, HW65S, and HW55S), it was separated into three peaks. Iodine staining and chain length distribution analyses of debranched samples showed that peaks were mainly composed of high-molecular weight (MW) amylopectin consisting of many clusters, low-MW amylopectin consisting of a small number of clusters, and amylose.

Shochu Koji Microstructure and Starch Structure during Preparation.

In this study, we investigated the changes in composition, microstructure, and starch molecular structure of shochu koji during preparation. We observed that the gelatinized and outer part of starch was decomposed in priority during the early and middle preparation stages. The gap between the starch granules increased with the delayed time. Finally, the koji microstructure became spongy. Shochu koji mold produced two α-amylases in different expression manners. Acid-labile α-amylase was produced in the early and middle preparation stages. Acid-stable α-amylase and saccharification power were produced in the middle and late stages. Throughout the koji preparation, reducing sugars content reached approximately 13-20 % of the total sugar content, with glucose representing over 70 % of the reducing sugars. α-Glucan fragments with C chains of degree of polymerization (DP) 4-73 were observed in the early and middle stages (<23 h), indicating the degradation of amylopectin at long B chains. In the latter stage, the amount of C chains of DP 6-30 decreased, while the longer C chains (DP 30<) did not change. These results showed that acid-labile α-amylase, acid-stable α-amylase, and saccharification enzymes including glucoamylase and α-glucosidase work preferentially on the amorphous regions of starch granules, and cooperative action of these enzymes during koji preparation contributes to the formation of the observed microstructure. Our study is the first report on the decomposition schemes of starch and the microstructure forming process in shochu koji.

Elongated phytoglycogen chain length in transgenic rice endosperm expressing active starch synthase IIa affects the altered solubility and crystallinity of the storage α-glucan.

The relationship between the solubility, crystallinity, and length of the unit chains of plant storage α-glucan was investigated by manipulating the chain length of α-glucans accumulated in a rice mutant. Transgenic lines were produced by introducing a cDNA for starch synthase IIa (SSIIa) from an indica cultivar (SSIIa (I), coding for active SSIIa) into an isoamylase1 (ISA1)-deficient mutant (isa1) that was derived from a japonica cultivar (bearing inactive SSIIa proteins). The water-soluble fraction accounted for >95% of the total α-glucan in the isa1 mutant, whereas it was only 35-70% in the transgenic SSIIa (I)/isa1 lines. Thus, the α-glucans from the SSIIa (I)/isa1 lines were fractionated into soluble and insoluble fractions prior to the following characterizations. X-ray diffraction analysis revealed a weak B-type crystallinity for the α-glucans of the insoluble fraction, while no crystallinity was confirmed for α-glucans in isa1. Concerning the degree of polymerization (DP) ≤30, the chain lengths of these α-glucans differed significantly in the order of SSIIa (I)/isa1 insoluble > SSIIa (I)/isa1 soluble > α-glucans in isa1. The amount of long chains with DP ≥33 was higher in the insoluble fraction α-glucans than in the other two α-glucans. No difference was observed in the chain length distributions of the ß-amylase limit dextrins among these α-glucans. These results suggest that in the SSIIa (I)/isa1 transgenic lines, the unit chains of α-glucans were elongated by SSIIa(I), whereas the expression of SSIIa(I) did not affect the branch positions. Thus, the observed insolubility and crystallinity of the insoluble fraction can be attributed to the elongated length of the outer chains due to SSIIa(I).

Structures of starches from rice mutants deficient in the starch synthase isozyme SSI or SSIIIa.

Amylose and amylopectin of rice mutants deficient in a starch synthase (SS) isozyme in the endosperm, either SSI (ΔSSI) or SSIIIa (ΔSSIIIa), were structurally altered from those of their parent (cv. Nipponbare, Np). The amylose content was higher in the mutants (Np, 15.5%; ΔSSI, 18.2%; ΔSSIIIa, 23.6%), and the molar ratio of branched amylose and its side chains was increased. The chain-length distribution of the ß-amylase limit dextrins of amylopectin showed regularity, which appeared consistent with the generally accepted cluster structure, and the degrees of polymerization found at the intersections were taken as the boundaries of the B-chain fractions. The mole % of the B(1)-B(3) fractions was changed slightly in ΔSSI, which is consistent with the proposed role of SSI in elongating the external part of clusters. In ΔSSIIIa, a significant increase in the B(1) fraction and a decrease in the B(2) and B(3) fractions were observed. The internal chain length of the B(2) and B(3) fractions appeared to be slightly altered, suggesting that the deficiency in SS affected the actions of branching enzyme(s).

Characterization of pullulanase (PUL)-deficient mutants of rice (Oryza sativa L.) and the function of PUL on starch biosynthesis in the developing rice endosperm.

Rice (Oryza sativa) allelic sugary1 (sug1) mutants defective in isoamylase 1 (ISA1) accumulate varying levels of starch and phytoglycogen in their endosperm, and the activity of a pullulanase-type of a debranching enzyme (PUL) was found to correlate closely with the severity of the sug1 phenotype. Thus, three PUL-deficient mutants were generated to investigate the function of PUL in starch biosynthesis. The reduction of PUL activity had no pleiotropic effects on the other enzymes involved in starch biosynthesis. The short chains (DP < or = 13) of amylopectin in PUL mutants were increased compared with that of the wild type, but the extent of the changes was much smaller than that of sug1 mutants. The alpha-glucan composition [amylose, amylopectin, water-soluble polysaccharide (WSP)] and the structure of the starch components (amylose and amylopectin) of the PUL mutants were essentially the same, although the average chain length of the B(2-3) chains of amylopectin in the PUL mutant was approximately 3 residues longer than that of the wild type. The double mutants between the PUL-null and mild sug1 mutants still retained starch in the outer layer of endosperm tissue, while the amounts of WSP and short chains (DP < or = 7) of amylopectin were higher than those of the sug1 mutant; this indicates that the PUL function partially overlaps with that of ISA1 and its deficiency has a much smaller effect on the synthesis of amylopectin than ISA1 deficiency and the variation of the sug1 phenotype is not significantly dependent on the PUL activities.

Granule-bound starch synthase I is responsible for biosynthesis of extra-long unit chains of amylopectin in rice.

A rice Wx gene encoding a granule-bound starch synthase I (GBSSI) was introduced into the null-mutant waxy (wx) rice, and its effect on endosperm starches was examined. The apparent amylose content was increased from undetectable amounts for the non-transgenic wx cultivars to 21.6-22.2% of starch weight for the transgenic lines. The increase was in part due to a significant amount of extra-long unit chains (ELCs) of amylopectin (7.5-8.4% of amylopectin weight), that were absent in the non-transgenic wx cultivars. Thus, actual amylose content was calculated to be 14.9-16.0% for the transgenic lines. Only slight differences were found in chain-length distribution for the chains other than ELCs, indicating that the major effect of the Wx transgene on amylopectin structure was ELC formation. ELCs isolated from debranched amylopectin exhibited structures distinct from amylose. Structures of amylose from the transgenic lines were slightly different from those of cv. Labelle (Wx(a)) in terms of a higher degree of branching and size distribution. The amylose and ELC content of starches of the transgenic lines resulted in the elevation of pasting temperature, a 50% decrease in peak viscosity, a large decrease in breakdown and an increase in setback. As yet undetermined factors other than the GBSSI activity are thought to be involved in the control of formation and/or the amount of ELCs. Structural analysis of the Wx gene suggested that the presence of a tyrosine residue at position 224 of GBSSI correlates with the formation of large amounts of ELCs in cultivars carrying Wx(a).

Antioxidant flavonoid glycosides from the leaves of Ficus pumila L.

The Okinawan folks in Japan use Ficus pumila L. as a beverage or herbal medicine to treat diabetes and high blood pressure. Four flavonoid glycosides were isolated and identified as rutin (1 and 3), apigenin 6-neohesperidose (2), kaempferol 3-robinobioside (4) and kaempferol 3-rutinoside (5). Among these compounds, rutin exhibited the strongest antioxidant activity in DPPH radical scavenging assay and superoxide radical inhibition assay. The preparation of Ooitabi leaves in water provide sufficient amount of flavonoid glycosides to the Okinawan although 50% of aqueous ethanol extracted these flavonoid glycosides more effectively. These results show the potential of Ooitabi leaves as a natural source of antioxidant for health management.

Structure and physicochemical properties of starches from kidney bean seeds at immature, premature and mature stages of development.

Starches from kidney bean (Phaseolus vulgaris L. cv. Toramame) seeds at the immature, premature, mature stages of development were examined. The starch content increased from 94, 219 to 265 mg per seed. Starches showed the C(a)-crystalline type composed of small (<5 micrometer) and large (10-35 micrometer) granules, with the large granules largely increasing with maturity. The amylose content increased from 21, 26 to 27%, and rapid viscograms and DSC thermograms suggested that the mature-stage starch was gelatinized with ease. The amylose increased in size from DPn 820, 1000 to 1080 and a number of chains per molecule (NC) from 3.3, 4.2 to 4.5. The branched amylose was a minor component (11-18% by mole) with NC 20-22. The amylopectin was similar in CL (23), beta-amylolysis limit (59%), and chain-length distribution, but reduced in size (DPn 17,100-5270) and increased in content of phosphorus (114-174 ppm) with an increase in the amount of phosphorus linked to C-6 of the glucose residue (8-66%).

Examination of the structure of amylopectin molecules by fluorescent labeling.

Amylopectin molecules from rice, maize, sweet potato and potato were examined by fluorescent labeling followed by gel-permeation HPLC. The number-average degree of polymerization (dp(n)) was determined to be in range of 9600-15,900. The molar-based distribution revealed the presence of three molecular species, large (dp(n) 13,400-26,500), medium (4400-8400) and small (700-2100). Their molar proportions differed by plant origin. The large species was a major component (43-63% by mole). A relatively large amount of the medium (16-28% by mole) and small (19-38%) species was found although their weight proportion was small (8-15, 1-4%, respectively). The three species from waxy rice amylopectin had a similar chain-length distribution and also a similar size-distribution of C chains. These results suggested that the three species were basically similar in cluster structure but different in number of clusters per molecule.

Examination of molar-based distribution of A, B and C chains of amylopectin by fluorescent labeling with 2-aminopyridine.

A method for determination of a molar-based distribution of A, B and C chains of amylopectin was developed. Labeling with fluorescent 2-aminopyridine was proportional to the number-average degree of polymerization (dp(n)) of the chains in the range of 6-440. Number-average chain lengths (cl(n)) of amylopectins from six different plant sources (rice, maize, wheat, potato, sweet potato and yam) determined by the labeling method were in good agreement with values obtained by determination of non-reducing residues. The molar-based distributions were polymodal (A, B(1) and B(2)+B(3) fractions) and characteristic to botanical sources. Amylopectins from starches with A-crystalline type had higher amount of A+B(1) chains (90-93% by mole) than starches with B-type (68-87%). Molar ratios of (A+B(1))/(B(2)+B(3)) were 8.9-12.9 for the A-type starches and 2.1-6.5 for the B-type starches, suggesting that amylopectins of A-type starches had 1.5-2 times more branches per cluster than B-type. The distributions of C chains, except for amylomaize, showed a broad, asymmetrical profile from dp approximately 10 to approximately 130 with a peak at dp approximately 40 and were very similar among botanical sources, suggesting that the biosynthetic process for C chains is similar in different plant species.

Mutations of muscle glycogen synthase that disable activation by glucose 6-phosphate.

Glycogen synthase, an enzyme of historical importance in the field of reversible protein modification, is inactivated by phosphorylation and allosterically activated by glucose 6-phosphate (glucose-6-P). Previous analysis of yeast glycogen synthase had identified a conserved and highly basic 13-amino-acid segment in which mutation of Arg residues resulted in loss of activation by glucose-6-P. The equivalent mutations R578R579R581A (all three of the indicated Arg residues mutated to Ala) and R585R587R590A were introduced into rabbit muscle glycogen synthase. Whether expressed transiently in COS-1 cells or produced in and purified from Escherichia coli, both mutant enzymes were insensitive to activation by glucose-6-P. The effect of phosphorylation was studied in two ways. Purified, recombinant glycogen synthase was directly phosphorylated by casein kinase 2 and glycogen synthase kinase 3, under conditions that inactivate the wild-type enzyme. In addition, phosphorylation sites were converted to Ala by mutagenesis in wild-type and in the glucose-6-P desensitized mutants expressed in COS-1 cells. Phosphorylation inactivated the R578R579R581A mutant but had little effect on the R585R587R590A. This result was surprising since phosphorylation had the opposite effects on the corresponding yeast enzyme mutants. The results confirm that the region of glycogen synthase, Arg-578-Arg-590, is required for activation by glucose-6-P and suggest that it is part of a sensitive and critical switch involved in transitions between different conformational states. However, the role must differ subtly between the mammalian and the yeast enzymes.