Kanefumi Kitahara

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.

Inhibition of the expression of the starch synthase II gene leads to lower pasting temperature in sweetpotato starch

The sweetpotato cultivar Quick Sweet (QS) with a lower pasting temperature of starch is a unique breeding material, but the biochemical background of this property has been unknown. To assess the physiological impact of the reduced isoform II activity of starch synthase (SSII) on the starch properties in sweetpotato storage root, transgenic sweetpotato plants with reduced expressions of the SSII gene were generated and evaluated. All of the starches from transgenic plants showed lower pasting temperatures and breakdown measured by a Rapid Visco Analyzer. The pasting temperatures in transgenic plants were approximately 10–15°C lower than in wild-type plants. Distribution of the amylopectin chain length of the transgenic lines showed marked differences compared to that in wild-type plants: more chains with degree of polymerization (DP) 6–11 and fewer chains with DP 13–25. The starch granules from the storage root of transgenic plants showed cracking on the hilum, while those from wild-type plants appeared to be typical sweetpotato starch. In accordance with these observations, the expression of SSII in the storage roots of the sweetpotato cultivar with low pasting temperature starch (QS) was notably lower than in cultivars with normal starch. Moreover, nucleotide sequence analysis suggested that most of the SSII transcripts in the cultivar with low pasting temperature starch were inactive alleles. These results clearly indicate that the activity of SSII in sweetpotato storage roots, like those in other plants, affects the pasting properties of starch through alteration of the amylopectin structure.

Retrogradation of sweetpotato starch

Hardness and the percentage of leaked water of sweetpotato starch gels after storage were investigated as indexes of starch retrogradation. Starches of some varieties of sweetpotato were retrograded rapidly, but those of others were not retrograded during storage for one week. After one month of storage, starches of all varieties were highly retrograded, but the varietal order of hardness and the percentage of leaked water were almost the same as that after storage for one week. The study of chain length distribution by gel permeation chromatography after debranching by isoamylase showed that the hardness and the percentage of leaked water from the gel were positively correlated with amylose content and the proportion of Fr 1, the fraction of the highest molecular weight, containing amylose and extremely long chains of amylopectin, and negatively correlated with the proportion of Fr 3, the fraction with the lowest molecular weight, containing shorter chains of amylopectin. In addition, it was demonstrated by high performance anion exchange chromatography that the proportion of extra-short chains (around DP 10) of amylopectin after isoamylase treatment was negatively correlated with the retrogradation index of starch. These results suggest that retrogradation of sweetpotato starch was promoted by amylose and extremely long chains of amylopectin and was inhibited by extra-short chains (around DP 10) of amylopectin.

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.

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.

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.

Characterization of Molecular Structure of Starch Granules in Suspension-cultured Cells from Ipomoea cordatotriloba Denn.

The molecular structure of starch granules formed in suspension-cultured cells of Ipomoea cordatotriloba Denn. was characterized by its chain length distribution, which was compared to those of the starches from the root and leaf of the original plant. The cultured cell starches were spherical and had a very small granule size (about 2 μm). The debranched starches roughly separated into three fractions during gel-permeation chromatography, and the fractions were defined as Fr.1, 2, and 3, respectively. The chain length distribution of the debranched cultured cell starch showed that the high molecular weight fraction (Fr.1), referred to as an amylose fraction, was much less than those of the root and leaf starches. The ratio of the two lower fractions (Fr.3/Fr.2) of the cultured cell starch, which was mainly derived from unit chains of amylopectin, was greatest among the starches, suggesting that the amylopectin from the cultured cell starch has much shorter unit chains. By X-ray diffraction analysis, it was found that both cultured cell and leaf starch granules have low crystallinity.

Bifidobacterium longum subsp. longum Exo-β-1,3-Galactanase, an Enzyme for the Degradation of Type II Arabinogalactan

ABSTRACT Type II arabinogalactan (AG-II) is a suitable carbohydrate source for Bifidobacterium longum subsp. longum, but the degradative enzymes have never been characterized. In this study, we characterized an exo-β-1,3-galactanase, BLLJ_1840, belonging to glycoside hydrolase family 43 from B. longum subsp. longum JCM1217. The recombinant BLLJ_1840 expressed in Escherichia coli hydrolyzed β-1,3-linked galactooligosaccharides but not β-1,4- and β-1,6-linked galactooligosaccharides. The enzyme also hydrolyzed larch wood arabinogalactan (LWAG), which comprises a β-1,3-linked galactan backbone with β-1,6-linked galactan side chains. The k cat/Km ratio of dearabinosylated LWAG was 24-fold higher than that of β-1,3-galactan. BLLJ_1840 is a novel type of exo-β-1,3-galactanase with a higher affinity for the β-1,6-substituted β-1,3-galactan than for nonsubstituted β-1,3-galactan. BLLJ_1840 has 27% to 28% identities with other characterized exo-β-1,3-galactanases from bacteria and fungi. The homologous genes are conserved in several strains of B. longum subsp. longum and B. longum subsp. infantis but not in other bifidobacteria. Transcriptional analysis revealed that BLLJ_1840 is intensively induced with BLLJ_1841, an endo-β-1,6-galactanase candidate, in the presence of LWAG. This is the first report of exo-β-1,3-galactanase in bifidobacteria, which is an enzyme used for the acquisition of AG-II in B. longum subsp. longum.