The action pattern and mechanism of the Taka-amylase A-catalyzed reaction were studied quantitatively and kinetically by product analysis, using a series of maltooligosaccharides from maltotriose (G3) to maltoheptaose (G7) labeled at the reducing end with 14C-glucose. A marked concentration dependency of the product distribution from the end-labeled oligosaccharides was found, Especially with G3 and G4 as substrates. The relative cleavage frequency at the first glycosidic bond counting from the nonreducing end of the substrate increases with increasing substrate concentration. Further product analyses with unlabeled and end-labeled G3 as substrates yielded the following findings: 1) Maltose is produced in much greater yield than glucose from unlabeled G3 at high concentration (73 mM). 2) Maltooligosaccharides higher than the starting substrate were found in the hydrolysate of labeled G3. 3) Nonreducing end-labeled maltose (G-G), which is a specific product of condensation, was found to amount to only about 4% of the total labeled maltose. Based on these findings, it was concluded that transglycosylation plays a significant role in the reaction at high concentrations of G3, although the contribution of condensation cannot be ignored. A new method for evaluating subsite affinities is proposed; it is based on the combination of the kinetic parameter (ko/Km) and the bond-cleavage distribution at a sufficiently low substrate concentration, where transglycosylation and condensation can be ignored. This method was applied to evaluate the subsite affinities of Taka-amylase A. Based on a reaction scheme which involves hydrolysis, transglycosylation and condensation, the time courses of the formation of various products were simulated, using the Runge-Kutta-Gill method. Good agreement with the experimental results was obtained.

Background: ␤-L-Arabinofuranosyl linkages are found in many plant biopolymers, but the degradation enzyme has never been found. Results: A novel ␤-L-arabinofuranosidase was found in Bifidobacterium longum. Conclusion: ␤-L-Arabinofuranosidase plays a key role in Bifidobacterium longum for ␤-L-arabinooligosaccharide usage. Significance: The members of the DUF1680 family might be used for the degradation of plant biopolymers.

Extensin is a glycoprotein that is rich in hydroxyprolines linked to ␤-L-arabinofuranosides. In this study, we cloned a hypBA2 gene that encodes a novel ␤-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)-␤1,2-Araf disaccharide from carrot extensin, potato lectin, and Araf-␤1,2-Araf-␤1,2-Araf-␤-Hyp (Ara 3 -Hyp) but not Araf-␣1,3-Araf-␤1,2-Araf-␤1,2-Araf-␤-Hyp (Ara 4 -Hyp) or Araf-␤1,2-Araf-␤-Hyp (Ara 2 -Hyp), which indicated that it was specific for unmodified Ara 3 -Hyp substrate. The enzyme also transglycosylated 1-alkanols with retention of the anomeric configuration. This is the first report of an enzyme that hydrolyzes Hyplinked ␤-L-arabinofuranosides, which defines a new family of glycoside hydrolases, glycoside hydrolase family 121.

The cell wall materials (CWMs) from sweetpotato (Ipomoea batatas cv. Kokei 14), cassava (Manihot esculenta), and potato (Solanum tuberosum cv. Danshaku) and commercial sweetpotato fiber as well as their polysaccharide fractions were analyzed for sugar composition by the high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) method. The separation of arabinose and rhamnose, and xylose and mannose, by this method has been improved using a CarboPac PA 10 column. Pretreatment of the CWMs and cellulose fractions with 12 M H(2)SO(4) was required for complete hydrolysis to occur. Commercial sweetpotato fiber was found to be mainly composed of glucose (88.4%), but small amounts of other sugars were also detected. Among the root crops, sweetpotato CWM had the highest amount of pectin and galacturonic acid. Fucose was detected only in cassava CWM and its hemicellulose fraction, while galactose was present in the highest amount in potato CWM. Among the polysaccharide fractions, it was only in the hemicellulose fraction where significant differences in the sugar composition, especially in the galactose content, were observed among the root crops.

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Various extracts of aerial parts of Varthemia (Varthemia iphionoides Boiss) were investigated for radical-scavenging activity, antioxidative activity, and porcine pancreas alpha-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 microg/ml, and alpha-amylase inhibitory activity of about 70% at a concentration of 200 microg/ml by the 2-chloro-4-nitrophenyl alpha-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 alpha-amylase. Eudesmane sesquiterpene did not show any activity.

Cereal Chem. 74(1):1-6The 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 λ max 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 amylose fraction was increased by glucoamylase hydrolysis, and also that the increments were reduced by the defatting of bound lipids. The results suggest that the increase in amylose fraction is attributable to the existence of bound lipids in the granules.