Toshihiko Suganuma

Inhibition of the gene expression for granule-bound starch synthase I by RNA interference in sweet potato plants

Granule-bound starch synthase I (GBSSI) is one of the key enzymes catalyzing the formation of amylose, a linear α(1,4)D-glucan polymer, from ADP-glucose. Amylose-free transgenic sweet potato plants were produced by inhibiting sweet potato GBSSI gene expression through RNA interference. The gene construct consisting of an inverted repeat of the first exon separated by intron 1 of GBSSI driven by the CaMV 35S promoter was integrated into the sweet potato genome by Agrobacterium tumefaciens-mediated transformation. In over 70% of the regenerated transgenic plants, the expression of GBSSI was inactivated giving rise to storage roots containing amylopectin but not amylose. Electrophoresis analysis failed to detect the GBSSI protein, suggesting that gene silencing of the GBSSI gene had occurred. These results clearly demonstrate that amylose synthesis is completely inhibited in storage roots of sweet potato plants by the constitutive production of the double-stranded RNA of GBSSI fragments. We conclude that RNA interference is an effective method for inhibiting gene expression in the starch metabolic pathway.

Antioxidant and α-Amylase Inhibitory Compounds from Aerial Parts of Varthemia iphionoides Boiss

Various extracts of aerial parts of Varthemia (Varthemia iphionoides Boiss) were investigated for radical-scavenging activity, antioxidative activity, and porcine pancreas α-amylase inhibitory activity. The ethanol and water extracts showed a pronounced 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity, with inhibition of about 90% at a concentration of 100 μg/ml, and α-amylase inhibitory activity of about 70% at a concentration of 200 μg/ml by the 2-chloro-4-nitrophenyl α-maltotrioside (CNP-G3) degradation method. The ethanol extract was purified by column chromatography to give seven 3-methoxyflavones (1–7) and eudesmane sesquiterpene, selina-4,11(13)-dien-3-on-12-oic acid (8). The structures of these compounds were established by NMR, MS, and UV spectroscopy. Of 3-methoxyflavones, 5,7,4′-trihydroxy-3,6-dimethoxyflavone (1), 5,7,4′-trihydroxy-3,3′-dimethoxyflavone (2), and 5,4′-dihydroxy-3,7,3′-trimethoxyflavone (3,7,3′-tri-O-methyl-quercetin) (7) exhibited pronounced radical-scavenging activity. The antioxidative activity in the linoleic acid system was considerable in compounds 1, 2, and 5,4′-dihydroxy-3,6,7-trimethoxyflavone (4). Compounds 1, 2, 4, 5 (5,7,4′-trihydroxy-3-methoxyflavone), and 6 (5,4′-dihydroxy-3,7-dimethoxyflavone) showed markedly high inhibitory activity against porcine pancreas α-amylase. Eudesmane sesquiterpene did not show any activity.

Release of bound lipids in cereal starches upon hydrolysis by glucoamylase

ABSTRACT The raw starch granules from corn, rice, and wheat were hydrolyzed by practically pure glucoamylase (Rhizopus niveus). The bound lipids remaining in the residual starches were investigated, of which the major components of the lipids, free fatty acids (FFA) in corn starch, FFA and phospholipids (PL) in rice starch, and PL in wheat starch were determined. In each case, the bound FFA and PL were decreased to some extent during the initial stage of hydrolysis. During the later stages, the FFA continued to gradually decrease, while the level of PL stabilized. It was interesting that some of the bound lipids were released from the granules upon glucoamylase hydrolysis, differing from the model amylose-lipid complexes. Furthermore, the structures of the residual starches were investigated. The blue value and λmax of the starches were increased by partial hydrolysis of the starch granules using practically pure glucoamylase. Two gel-permeation chromatography analyses revealed that the relative amount of...

Molecular cloning and characterization of a β-L-arabinobiosidase in Bifidobacterium longum that belongs to a novel glycoside hydrolase family

Extensin is a glycoprotein that is rich in hydroxyprolines linked to β-l-arabinofuranosides. In this study, we cloned a hypBA2 gene that encodes a novel β-l-arabinobiosidase from Bifidobacterium longum JCM 1217. This enzyme does not have any sequence similarity with other glycoside hydrolase families but has 38–98% identity to hypothetical proteins in Bifidobacterium and Xanthomonas strains. The recombinant enzyme liberated l-arabinofuranose (Araf)-β1,2-Araf disaccharide from carrot extensin, potato lectin, and Araf-β1,2-Araf-β1,2-Araf-β-Hyp (Ara3-Hyp) but not Araf-α1,3-Araf-β1,2-Araf-β1,2-Araf-β-Hyp (Ara4-Hyp) or Araf-β1,2-Araf-β-Hyp (Ara2-Hyp), which indicated that it was specific for unmodified Ara3-Hyp substrate. The enzyme also transglycosylated 1-alkanols with retention of the anomeric configuration. This is the first report of an enzyme that hydrolyzes Hyp-linked β-l-arabinofuranosides, which defines a new family of glycoside hydrolases, glycoside hydrolase family 121.

Elucidation of the subsite structure of bacterial saccharifying alpha-amylase and its mode of degradation of maltose

The subsite structure of bacterial saccharifying alpha-amylase (BSAm) was elucidated by two methods using a series of maltooligosaccharides labeled with [14C]D-glucose at the reducing end. The rate parameter k0/Km and the cleavage frequency were obtained using the labeled substrates at sufficiently low concentrations to eliminate transglycosylation and condensation. This evaluation showed that the active center is composed of five subsites, with the catalytic site located between the 3rd and the 4th subsites from the nonreducing end. The evaluated affinity values of a subsite varied with the set of data used, which suggests some stimulation factor resulting from the chain length effect. The appearance of a time lag during the digestion of the poor substrate, maltose, was studied using radioactively labeled maltose (81.6 mM). Radioactive oligosaccharides larger than maltose were found at a significant level of more than 2% of the initial substrate in the digests, including a product peculiar to condensation, G-G*-G, as 8-10% of the maltotriose in the digests. This indicates that transglycosylation is a main side reaction (ca. 90%). A degradation pathway for maltose via maltosyl transfer was proposed, in which G3 behaves as a kind of catalyst.

Study of the action of human salivary alpha-amylase on 2-chloro-4-nitrophenyl α-maltotrioside in the presence of potassium thiocyanate

The degradation mechanism of a synthetic substrate, 2-chloro-4-nitrophenyl alpha-maltotrioside (CNP-G3), by human salivary alpha-amylase (HSA) was investigated by kinetic and product analyses. It was observed that the enzyme attacked the various CNP-maltooligosaccharides (CNP-G3 to CNP-G6) releasing free CNP. Addition of 500 mM potassium thiocyanate (KSCN) was also found to greatly increase the rates of CNP-release. It was the fastest with CNP-G3, and, in the presence of KSCN, was almost comparable to that of degradation of maltopentaose (G5). On the other hand, addition of KSCN decreased the rate of cleavage between glucan-glucan bonds in maltopentaose. Product analysis showed that KSCN addition altered the cleavage distribution which occurred 100% at the bond between CNP and G3, and that product distribution of free CNP was largely dependent on substrate concentration. Formation of CNP-G6, a larger product than the original substrate CNP-G3, was found to be present in the digest at high concentrations of substrate and in the presence of KSCN. Based on these results, a degradation pathway for CNP-G3 involving transglycosylation besides direct hydrolysis is proposed. The increase of the CNP-release by the addition of KSCN would result from a corresponding increase in the interaction between the CNP moiety and the corresponding subsite near the catalytic site, as well as the enhancement of the catalytic efficiency.

Analysis of the characteristic action of D-enzyme from sweet potato in terms of subsite theory

Abstract Disproportionating enzyme (D-enzyme, EC 2.4.1.25) was purified chromatographically from a β-amylase-deficient variety of sweet potatoes, and obtained free of amylases and glucosidases. Some heavy metal ions, particularly Hg2+ and Ag+, inactivated the enzyme. The Km value for maltrotriose was found to be 7.3m m . Analysis by h.p.l.c. of digests of malto-oligosaccharides (G3–G7) showed that, as with other D-enzymes, maltose is not formed in any case. The products from all substrates except G4 are those resulting from maltosyl transfer as the predominant reaction, wherein Gn−2 and Gn+2 are produced from Gn. The various malto-oligosaccharides undergo reaction at almost the same rates. The frequency of cleavage of the various bonds in malto-oligosaccharides was estimated from the observed molar ratios of products smaller than the substrates. A possible subsite structure for the D-enzyme is proposed on the basis of computer modelling of data describing the cleavage pattern etc.

Characterization of a Novel β-l-Arabinofuranosidase in Bifidobacterium longum FUNCTIONAL ELUCIDATION OF A DUF1680 FAMILY MEMBER

Background: β-l-Arabinofuranosyl linkages are found in many plant biopolymers, but the degradation enzyme has never been found. Results: A novel β-l-arabinofuranosidase was found in Bifidobacterium longum. Conclusion: β-l-Arabinofuranosidase plays a key role in Bifidobacterium longum for β-l-arabinooligosaccharides usage. Significance: The members of DUF1680 family might be used for the degradation of plant biopolymers. Pfam DUF1680 (PF07944) is an uncharacterized protein family conserved in many species of bacteria, actinomycetes, fungi, and plants. In a previous article, we cloned and characterized the hypBA2 gene as a β-l-arabinobiosidase in Bifidobacterium longum JCM 1217. In this study, we cloned a DUF1680 family member, the hypBA1 gene, which constitutes a gene cluster with hypBA2. HypBA1 is a novel β-l-arabinofuranosidase that liberates l-arabinose from the l-arabinofuranose (Araf)-β1,2-Araf disaccharide. HypBA1 also transglycosylates 1-alkanols with retention of the anomeric configuration. Mutagenesis and azide rescue experiments indicated that Glu-366 is a critical residue for catalytic activity. This report provides the first characterization of a DUF1680 family member, which defines a new family of glycoside hydrolases, the GH family 127.

Degradation of cell wall materials from sweetpotato, cassava, and potato by a bacterial protopectinase and terminal sugar analysis of the resulting solubilized products

Cell wall materials (CWMs) from sweetpotato, cassava, and potato starch residues were degraded using a crude enzyme solution from the culture filtrate of a Bacillus sp. isolated from soil, Bacillus sp. M4. This organism has been found to secrete polygalacturonic acid lyase (PGL) and glycan depolymerase activities, especially arabinanase, but cellulase activity was nearly absent. Sugar analysis of the solubilized product after enzyme treatment at pH 7.0 revealed that it is mainly composed of galacturonic acid, galactose, and arabinose, the sugars found commonly in the pectin fraction. This suggested the presence of a protopectinase (PPase) activity in the culture filtrate. The presence of EDTA completely inhibited PGL but PPase activity was almost retained, suggesting that the PGL is not the primary activity responsible for pectin solubilization. The mode of action of the crude enzyme was determined by terminal sugar analysis using HPAEC-PAD after hydrolysis of the reduced products. Results revealed that galactose is the main neutral sugar at the reducing terminal of the products, although rhamnose was also present in the higher molecular weight component. This suggested that at neutral pH, the primary activity in the culture filtrate of Bacillus sp. M4 is a B-type PPase, which attacked the galactan as well as rhamnogalacturonan moieties of the protopectin, resulting in the release of a soluble pectin fraction.

Determination of kinetic parameters for maltotriose and higher malto-oligosaccharides in the reactions catalyzed by α-d-glucan phosphorylase from potato

Abstract For kinetic studies on its synthetic and phosphorolytic reactions, α- d -glucan phosphorylase from potatoes was purified chromatographically until free of D-enzyme. Purified maltotriose (G 3 ) is a poor primer in the phosphorylase-catalyzed synthetic reaction, showing an anomalous time course and making previous attempts to determine its kinetic parameters unsuccessful. In the present work the true rate of the G 3 -primed reaction was obtained from linear plots obtained by incorporating a sufficient quantity of β-amylase in the digest to eliminate the more rapidly reacting G 4 formed from the G 3 . A K m value of 9.4 ± 0.8 m m for G 3 was calculated from the data by a nonlinear least-squares method. Kinetic parameters for a series of higher malto-oligosaccharides (G 4 –G 8 ) were also determined in both the synthetic and the phosphorolytic directions. A large change in the values of K m and V/e was seen on going from G 3 to G 4 for the synthetic reaction, and from G 4 to G 5 for the phosphorolytic. For the higher saccharides the V/e values do not vary strongly with increasing d.p., while the K m values tend to decrease, as has seen in the reactions of other plant phosphorylases.