To examine the applicability of microwave irradiation for the extraction of polysaccharides in the fruiting body of Hericium erinaceum, structures of polysaccharides obtained by microwave irradiation in water were compared to those obtained by hot water extraction using conventional external heating. A major polysaccharide obtained by microwave irradiation in water (140°C, 5 min) was (1→3;1→6)-β-D-glucan rich in (1→3) linkages, whereas polysaccharides obtained by hot water extraction using conventional external heating (100°C, 6 h) were fucogalactan and (1→3;1→6)-β-D-glucan rich in (1→6) linkages. In the case of microwave irradiation in water, fucogalactan was suggested to be depolymerized during heating. Microwave heating in water has an advantage over the conventional external heating for extraction of β-glucan rich in (1→3) linkages from the fruiting body of H. erinaceum.
Fresh edible burdock roots were stored in soil of 1 m depth underground from November to May. Three fructooligosaccharide derivatives without a terminal glucose residue, designated saccharides 1, 2 and 3, were generated in the stored burdock roots. They were purified from the sugar extract using carbon-Celite column chromatography. Saccharides 1, 2 and 3 have R-sucrose values (retention time of sucrose = 1) of 1.55, 2.15 and 2.73 by HPAEC, reducing terminal, molar ratios (reducing sugar to D-fructose) of 0.50, 0.33 and 0.25 and degrees of polymerization of 2, 3 and 4 by TOF-MS, respectively. Analyses by GLC and NMR confirmed the three different following structures: first was inulobiose [β-D-fructofuranosyl-(2→1)-β-D-fructopyranose], and the two others were inulotriose [β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl-(2→1)-β-D-fructopyranose] and inulotetraose [β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl-(2→1)-β-D-fructopyranose]. The NMR spectra showed that 70 to 80% of the terminal fructose residue of the three saccharides is pyranosyl form, while 20 to 30% is furanosyl form. The 13C- and 1H-signals were also assigned by 2D-NMR including COSY, HSQC, HSQC-TOCSY and HMBC. These saccharides could be synthesized by purified burdock fructan:fructan 1-fructosyltransferase from 1-kestose to free D-fructopyranose giving inulobiose and sucrose, while elongation of fructofuranosyl units occurred at this transferred fructofuranosyl residue to produce inulooligosaccharides having one, two and more additional units of fructofuranose.
The intact crystal structure of family 10 xylanase (SoXyn10A) from Streptomyces olivaceoviridis indicates that the catalytic domain of SoXyn10A consists of nine α-helices (α0-8) and eight β-sheets (β1-8). Interaction in the α-helices of N-terminal (α0) and C-terminal (α8) of catalytic domain of SoXyn10A by 3 hydrogen bonds and 8 hydrophobic interactions is observed and predicted to playing an important role for the stability of the molecule. Therefore, the importance for the stability and folding of SoXyn10A were examined by using C-terminal truncated mutants of SoXyn10A. The thermostability was gradually decreased when the C-terminal was shortened; however, the enzyme activities were not influenced by the length of the C-terminal. The investigation of the stability using guanidine hydrochloride agreed with the expected results; namely the hydrophobic core was completed at Leu-300 and the resulting stability was not changed if the C-terminal was longer than it. The thermostabilty slightly decreased when Asn-252 was replaced with Ala, suggesting hydrogen bonding of Gly-303 with Asn-252 is also important for the stability of the molecule. When the effect of the interaction was observed in chimeric xylanases, which have a slight distortion in the structure, the C-terminal 4 amino acids certainly increased the thermostability of the chimeric enzymes. However, the N- and C-terminal of CfXyn10A from Cellulomonas fimi displayed by that of SoXyn10A decreased thermostability at the same degree as SoXyn10A, suggesting that the limit temperature of the interaction agrees with that of SoXyn10A and the distortion of both terminals make denaturating of the protein easy.
A family 10 xylanase of Streptomyces olivaceoviridis E-86 (SoXyn10A) is known to have a modular structure where an N-terminal catalytic module and a C-terminal xylan-binding module (XBM) are connected by a Gly/Pro-rich linker. The crystal structure of native SoXyn10A indicates that the XBM is located beyond -3 subsite from the substrate binding cleft. To investigate the mechanism of xylan binding by SoXyn10A, several kinds of cleft mutants of SoXyn10A were constructed. The mutants had an hindrance in the outer side of the (-) subsite or (+) subsite in the substrate binding cleft. Circular dichroism spectral analysis indicated that the mutant enzymes fold as a native enzyme. Kinetic studies for the mutants were performed by using p-nitrophenyl-β-D-xylobioside. The kcat/Km value did not change for the mutants which had a hindrance in the outer side of the (+) subsite while the value significantly decreased for the mutants which had a hindrance in the outer side of the (-) subsite, suggesting that xylan come into the substrate binding cleft of SoXyn10A from the direction of (-) subsites. The activities of the mutants against insoluble xylan were tested with or without XBM. All the mutants possessing XBM showed higher activity than those possessing the catalytic modules without XBM, indicating that the linker sequence connecting the catalytic module and XBM is flexible so that XBM binds to insoluble xylan to increase the concentration around the enzyme.
The degradation process of D-galacturonic acid in subcritical water was measured from 160 to 220°C. The process at any temperature obeyed first-order kinetics. The temperature dependence of the degradation rate constant could be expressed by the Arrhenius equation, and the activation energy and the frequency factor were estimated to be 131 kJ/mol and 4.81 × 1012 s-1, respectively. The degradation process of sodium D-galacturonate in subcritical water was also measured from 160 to 190°C. Sodium galacturonate was more easily degraded than galacturonic acid. The degradation process of sodium galacturonate did not obey the first-order kinetics, but could be expressed by the Weibull equation. According to the Arrhenius equation, the activation energy and the frequency factor were estimated to be 147 kJ/mol and 1.26 × 1015 s-1, respectively, for the degradation of sodium galacturonate. The pH of the reaction mixture at room temperature was also measured for the degradation of both the galacturonic acid and sodium galacturonate.
Retrogradation of starch in steamed rice grains under sake-making conditions was examined. The enthalpy change derived from retrogradation of amylopectin increased during the koji and sake mash processes. Ethanol in the sake mash accelerated retrogradation of amylopectin. Enzyme digestibility and properties of endosperm starches of 136 Japanese rice samples used for sake production were examined. A digestion test of steamed rice grains was conducted after 24 h storage at 15°C to estimate more accurately digestibility during the sake-making process. The ratio of short-to-long chain amylopectin exhibited a significant correlation with enzyme digestibility, suggesting that the structure of amylopectin may be used as a predictor of digestibility. The gelatinization temperature of brown/milled rice and purified starch measured by DSC (differential scanning calorimetry) correlated well with the ratio of short-to-long chain amylopectin and with enzyme digestibility. Furthermore, the pasting temperature of milled rice as measured by RVA (Rapid Visco Analyser) correlated well with the ratio of short-to-long chain amylopectin and with enzyme digestibility. Therefore, DSC and RVA appear to be useful tools for estimating enzyme digestibility of steamed rice grains under sake-processing conditions because the measurements are done rapidly and require only small weights of brown/milled rice flour samples.
A special hydroxyl protecting group, the uni-chemo hydroxyl protection (UCHP), was applied to automated oligosaccharide synthesis. The UCHP is a newly developed hydroxyl protecting group, which is comprised of oligomeric amino acid derivatives. The UCHP method is able to deprotect one hydroxyl after another, selectively, by repeating a simple deprotection protocol. The use of the UCHP group facilitated the automated synthesis of a tri-branched pentasaccharide.
A yeast strain I-8 was isolated as an α-amylase producer from digestive juice of Nepenthes bicalarate. The yeast was identified as Pseudozyma aphidis by the morphological test and comparative 26S rDNA-D1/D2 and ITS-5.8S rDNA gene sequence analysis. The α-amylase was purified from the culture filtrate by (NH4)2SO4 precipitation, DEAE-TOYOPEARL 650M, Butyl-TOYOPEARL 650M, Hydroxylapatite and TOYOPEARL HW-55 chromatography. The purified enzyme was shown as a single band and the molecular mass was 55 kDa by SDS-PAGE. The specific activity was 1679 U/mg protein. The optimum temperature and pH were around 60°C and 5.0, respectively. The enzyme was stable in a pH range from 5.0 to 9.0 and at below 60°C. The enzyme hydrolyzed soluble starch and released glucose, maltose and oligosaccharides. Maltooligosaccharides (G3-G5) were also favorable substrate but it showed no activity toward maltose, isomaltose or pullulan. On the hydrolysis of soluble starch, the iodine color of the reaction mixture disappeared at almost 10% of reducing sugar formation and the hydrolysis limit was about 70% of soluble starch. From these results, the α-amylase was recognized as a unique α-amylase. The α-amylase was applied to the bread making process. Addition of the α-amylase to the bread making process presented no effect toward the crumb softness or color but the improved taste of the bread by sensory evaluation.
A reliable quantitative assay method for starch branching enzyme (BE) remains to be established whereas it has been required to characterize BE towards understanding the regulatory mechanism for the synthesis of starch in plant tissues. We describe a new quantitative assay method for BE activities with malto-oligosaccharides, amylose and amylopectin as substrates by using bicinchoninic acid (BCA) for measuring directly the reducing terminals of linear glucans formed after debranching the reaction products. The BCA method not only can be performed by simple procedures and easy handling with the use of cheap toxic-free reagents, but also shows highly color-stable properties after the treatment of the reaction mixture with the color-yielding reagents compared with the modified Park-Johnson method (Carbohydr. Res., 94, 205-213(1981)). The intensity and the spectrum of the purple color generated in the treated assay mixture are maintained in sugars and glucans in the range of glucose and amylose with degree of polymerization up to at least 1658. The BCA method can be also applied for characterization of BE when amylopectin is used as glucan substrate. In this paper we report this efficient, reproducible and quantitative BCA method by showing some experimental results obtained in an attempt to determine the kinetic parameters of BEIIb from rice endosperm.
The effects of sugars on biofilm formation by Escherichia coli O157:H7 were investigated by microtiter plate assay. In the cultivation using the medium containing a monosaccharide such as D-glucose, D-galactose, D-mannose, D-xylose, L-arabinose, L-fucose and L-rhamnose for 7 days, active biofilm formation was observed at 1 day, and then the level of biofilm formation decreased gradually after 3 day. When the bacterium was cultivated in medium containing 0.4, 0.04 or 0.004% of monosaccharides for 1 day, a low level of biofilm formation was observed in the medium containing 0.4% D-mannose or L-rhamnose. However, only D-mannose suppressed biofilm formation in the presence of other sugars such as D-glucose.
Physicochemical properties of acetylated fractionated potato starches, prepared using acetic anhydride, were investigated. 1) As levels of acetic anhydride increased, physicochemical properties of acetylated fractionated potato starches changed as follows: (1) Content of acetyl group increased. (2) In distilled water, RVA (Rapid Visco Analyser) characteristics parameters (pasting temperature, peak viscosity and breakdown) and DSC (differential scanning calorimetry) characteristic parameters (onset, peak and conclusion temperatures of gelatinization, and enthalpy change of gelatinization) decreased, but solubility and swelling power increased. (3) Viscoelastic parameters (storage modulus and loss modulus) of starch pastes prepared in distilled water decreased, and their frequency-dependences were observed. Shear stresses of starch pastes at each shear rate decreased, and the extent of thixotropy decreased. (4) Syneresis of 4% starch pastes prepared in 0.1 M NaCl solution decreased. 2) The following differences in physicochemical properties were found among acetylated fractionated potato starches (large, middle and small granules). (1) Acetyl group contents of small granule starches were higher than those of middle and large granule starches. (2) Extent of decrease in RVA peak viscosity measured in distilled water increased with decrease in the starch granule size. Solubility and swelling power in distilled water increased with increase in the starch granule size. (3) In 0.1 M NaCl solution, RVA peak viscosity of starch pastes of small and middle granules increased with the increase in levels of acetic anhydride, and the extent of increase in the viscosity was markedly large for the small granule. (4) Solubility and swelling power in distilled water increased with the increase in the starch granule size. In 0.1 M NaCl solution, with the increase in the starch granule size, solubility increased, but the swelling power decreased. 3) Concerning the effect of 0.1 M NaCl solution on physicochemical properties of acetylated fractionated potato starches, RVA peak viscosity, solubility and swelling power decreased greatly compared with those in distilled water.
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