Journal of applied glycoscience最新文献

Trehalose is known to protect enzymes from denaturation. In the present study, we observed promotion of apple polyphenol oxidase (PPO) inactivation in a trehalose solution with thermal treatment. Crude PPO from Fuji apple was mixed with either sucrose or trehalose solutions, then the samples treated at 25 or 65 °C. In the presence of trehalose, PPO activities were markedly decreased upon treatment at 65 °C with increasing trehalose concentration. Furthermore, the reduction in PPO activity in the presence of trehalose was proportional to storage time after thermal treatment and thermal treatment time. Comparing PPO activities between treatment time 0 and 90 min at 65 °C, activities decreased 89 % for trehalose concentration of 0.2 M. These results indicates that trehalose acts not only as inhibitor but as promoter of inactivation of PPO. The Lineweaver-Burk plot indicated that trehalose acts on PPO as a non-competitive inhibitor during the 65 °C treatment. Two mechanisms of PPO inactivation in the presence of trehalose were suggested; one is the suppression of PPO activation cause by a thermal treatment, and another is the conformational change to inactivation form of PPO in conjunction with trehalose and a thermal treatment. Additionally, apple juice including 0.2 or 0.5 M trehalose with 65 °C treatment indicated slow browning than the juice with 0.2 or 0.5 M sucrose or without sugars. This result demonstrates that the preventing of browning with trehalose is a viable industrial food process.

α-Mannosidase (ALMAN) extracted from onion (Allium cepa) was purified by column chromatography such as hydrophobic and gel filtration. ALMAN is an acidic α-mannosidase that exhibits maximum activity against pNP-α-Man at pH 4.0-5.0 at 50°C. Amino acid sequence analysis of ALMAN was consistent with α-mannosidase deduced from Allium cepa transcriptome analysis. The gene alman was amplified by PCR using mRNA extracted from onions, and a full-length gene of 3,054 bp encoding a protein of 1,018 amino acid residues was revealed. ALMAN is classified as Glycoside Hydrolase Family (GH) 38 and showed homology with other plant-derived α-mannosidases such as tomato and hot pepper.

This review discusses sugar isomerization with organogermanium compounds. Organogermanium compounds markedly increase the aldose-ketose (glucose-fructose or lactose-lactulose) isomerization ratio, double the initial reaction rate, and significantly reduce the base-catalyzed degradation of sugars. ¹H-nuclear magnetic resonance analysis reveals that the affinity of organogermanium compounds with a 3-(trihydroxygermyl)propanoic acid (THGP) structure toward ketoses is 20-40 times stronger than that toward aldoses; thus, such organogermanium compounds form complexes more readily with ketoses than with aldoses. Stable ketose complexes, which contain multiple cis-diol structures and high fractions of furanose structures, suppress the reverse ketose-aldose reaction, thereby shifting the equilibrium toward the ketose side. These complexes also protect sugar molecules from alkaline degradation owing to the repulsion between anionic charges. The increased rate of the initial reaction in the alkaline isomerization process results from stabilizing the transition state by forming a complex between THGP and a cis-enediol intermediate. The cyclic pentacoordinate or hexacoordinate THGP structures give rise to a conjugated system of germanium orbitals, which is extended through dπ-pπ interactions, thereby improving the stability of the complex. Based on these results, we have developed a bench-scale lactulose syrup manufacturing plant incorporating a system to separate, recover, and reuse organogermanium poly-trans-[(2-carboxyethyl)germasesquioxane]. This manufacturing plant can be used as a model of an alkaline isomerization accelerator for continuous industrial production.

Some probiotics including lactobacilli, colonize host animal cells by targeting glycosaminoglycans (GAGs), such as heparin, located in the extracellular matrix. Recent studies have shown that several lactic acid bacteria degrade GAGs. Here we show the structure/function relationship of Lacticaseibacillus rhamnosus 4-deoxy-L-threo-5-hexosulose-uronate ketol-isomerase (KduI) crucial for metabolism of unsaturated glucuronic acid produced through degradation of GAGs. Crystal structures of ligand-free and bound KduIs were determined by X-ray crystallography and the enzyme was found to consist of six identical subunits and adopt a β-helix as a basic scaffold. Ligands structurally similar to the substrate were bound to the cleft of each enzyme subunit. Several residues located in the cleft interacted with ligands through hydrogen bonds and/or C-C contacts. In addition to substrate analogs, a metal ion coordinated to four residues, His198, His200, Glu205, and His248, in the cleft, and the enzyme activity was significantly inhibited by a chelator, ethylenediaminetetraacetic acid. Site-directed mutants in Arg163, Ile165, Thr184, Thr194, His200, Arg203, Tyr207, Met262, and Tyr269 in the cleft exhibited little enzyme activity, indicating that these residues and the metal ion constituted an active site in the cleft. This is the first report on the active site structure of KduI based on the ligand-bound complex.

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.

Foreign amylase addition to honey in an effort to disguise diastase activity has become a widespread form of food fraud. However, since there is no report on the investigation in Japan, we investigated foreign amylases in 67 commercial honeys in Japan. First, the α-glucosidase and diastase activities of honeys were measured, which revealed that only α-glucosidase activity was significantly low in several samples. As both enzymes are secreted from honeybee glands, it is unlikely that only one enzyme was inactivated during processing. Therefore, we suspected the presence of foreign amylase. α-Amylase in honey were assigned using protein analysis software based on LC-QTOF-MS. As a result, α-amylases from Aspergillus and Geobacillus were detected in 13 and 6 out of 67 honeys, respectively. To detect foreign amylases easily, we developed a cost-effective method using native PAGE. Conventional native PAGE failed to separate the α-amylase derived from honeybee and Geobacillus. However, when native PAGE was performed using a gel containing 1 % maltodextrin, the α-amylase from honeybee did not migrated in the gel and the α-amylase could be separated from the other two α-amylases. The results from this method were consistent with those of LC-QTOF-MS method, suggesting that the novel native PAGE method can be used to detect foreign amylases.

The purpose of the present study was to clarify the browning behavior of calcium maltobionate (CaMb). CaMb samples with varying water content (0-50 %) and water activity (0-0.98) were prepared. Absorbance due to the browning of CaMb-water samples increased linearly with an increase in holding time, and the browning rate was evaluated as a pseudo-zero-order reaction at each temperature (353-413 K). The effect of temperature on the browning rate of CaMb samples was analyzed using the Arrhenius formula, and the pre-exponential factor and activation energy were determined. In addition, the browning rate at 298 K (typical ambient condition) was determined according to the Arrhenius behavior. The browning rate at 298 K slightly decreased, markedly increased, and then decreased with an increase in water content and water activity. This behavior can be explained by the monolayer effect, plasticizing effect, and dilution effect of water molecules. There was a linear relationship between the natural logarithm of the pre-exponential factor and activation energy according to the thermodynamic compensation rule. The effect of water content on the activation energy was fitted using the cubic function. From these results, an approach for prediction of the browning of CaMB during thermal condensation was established.

Pectin, a type of soluble fiber, promotes morphological changes in the small intestinal villi. Although its physiological significance is unknown, we hypothesized that changes in villus morphology enhance the efficiency of nutrient absorption in the small intestine and investigated the effect of pectin derived from persimmon on calcium absorption using polarized Caco-2 cells. In polarized Caco-2 cells, pectin altered the mRNA expression levels of substances involved in calcium absorption and the regulation of intracellular calcium concentration and significantly reduced calcium absorption. Although this was comparable to the results of absorption and permeability associated with the addition of active vitamin D, the simultaneous action of pectin and active vitamin D did not show any additive effects. Furthermore, as active vitamin D significantly increases the activity of intestinal alkaline phosphatase (ALP), which is known to be involved in the regulation of intestinal absorption of calcium and lipids, we also investigated the effect of pectin on intestinal ALP activity. As a result, it was found that, unlike the effect of active vitamin D, pectin significantly reduced intestinal ALP activity. These results suggest that pectin stimulates polarized Caco-2 cells through a mechanism distinct from the regulation of calcium absorption by vitamin D, modulating total calcium absorption from the elongated villi through morphological changes in the small intestine by suppressing it at the cellular level.

Lytic polysaccharide monooxygenases (LPMO) are key enzymes for the efficient degradation of lignocellulose biomass with cellulases. A lignocellulose-degradative strain, Paenibacillus xylaniclasticus TW1, has LPMO-encoding PxAA10A gene. Neither the C1/C4-oxidizing selectivity nor the enzyme activity of PxAA10A has ever been characterized. In this study, the C1/C4-oxidizing selectivity of PxAA10A and the boosting effect for cellulose degradation with a cellulase cocktail were investigated. The full-length PxAA10A (rPxAA10A) and the catalytic domain (rPxAA10A-CD) were heterologously expressed in Escherichia coli and purified. To identify the C1/C4-oxidizing selectivity of PxAA10A, cellohexaose was used as a substrate with the use of rPxAA10A-CD, and the products were analyzed by MALDI-TOF/MS. As a result, aldonic acid cellotetraose and cellotetraose, the products from C1-oxidization and C4-oxidization, respectively, were detected. These results indicate that PxAA10A is a C1/C4-oxidizing LPMO. It was also found that the addition of rPxAA10A into a cellulase cocktail enhanced the cellulose-degradation efficiency.