Preparation of a Molecular Library of Branched β-Glucan Oligosaccharides Derived from Laminarin

To study the structure of β-glucans, we developed a separation method and molecular library of β-glucan oligosaccharides. The oligosaccharides were prepared by partial acid hydrolysis from laminarin, which is a β-glucan of Laminaria digitata. They were labeled with the 2-aminopyridine fluorophore and separated to homogeneity by size-fractionation and reversed phase high-performance liquid chromatography (HPLC). Branching structures of all isomeric oligosaccharides from trimers to pentamers were determined, and a two-dimensional (2D)-HPLC map of the β-glucan oligosaccharides was made based on the data. Next, structural analysis of the longer β-glucan oligosaccharide was performed using the 2D-HPLC map. A branched decamer oligosaccharide was isolated from the β-glucan and cleaved to smaller oligosaccharides by partial acid hydrolysis. The structure of the longer oligosaccharide was successfully elucidated from the fragment structures determined by the 2D-HPLC map. The molecular library and the 2D-HPLC map described in this study will be useful for the structural analysis of β-glucans.     

Production of Gentiobiose from Hydrothermally Treated Aureobasidium pullulans β-1,3-1,6-Glucan

We report production of the functional disaccharide gentiobiose β-D-Glcp-(1→6)-D-Glc by a hydrolysis reaction of hydrothermally treated Aureobasidium pullulans β-1,3-1,6-glucan as the substrate and Kitalase as the enzyme. Gentiobiose was produced over the pH range 4−6 and the concentration of gentiobiose produced decreased above pH 7. The maximum value of gentiobiose production was unaffected by the enzyme concentration. The maximum concentration of gentiobiose produced was dependent on the substrate concentration whereas the maximum ratio of gentiobiose to glucose was not. The production of gentiobiose from yeast β-1,3-1,6-glucan was lower than that from A. pullulans β-1,3-1,6-glucan.     

Characterization of Cell Wall α-1,3-Glucan–Deficient Mutants in Aspergillus oryzae Isolated by a Screening Method Based on Their Sensitivities to Congo Red or Lysing Enzymes

We previously reported that sensitivity to Congo Red (CR) or Lysing Enzymes (LE) is affected by the loss of cell-wall α-1,3-glucan (AG) in Aspergillus nidulans. We found that the amount of CR adsorbed to AG was significantly less than the amount adsorbed to β-1,3-glucan (BG) or chitin, suggesting that loss of cell-wall AG would increase exposure of BG on the cell surface, and thereby increase the sensitivity to CR. Generally, fungal BGs are known as biological response modifiers because of their recognition by Dectin-1 receptors in human immune systems. Therefore, isolation of AG-deficient mutants in Aspergillus oryzae has been used in the Japanese fermentation industry to create strains with increased ability to promote immune responses. Here, we aimed to isolate AG-deficient strains by mutagenizing A. oryzae conidia with chemical mutagens. Based on the increased sensitivity to CR in AG-deficient strains of A. nidulans and A. oryzae, we established a screening method for isolation of AG-deficient strains. Several candidate AG-deficient mutants of A. oryzae were isolated using the screening method; these strains showed increased sensitivity to CR and/or LE. Cytokine production was increased in the dendritic cells co-incubated with germinated conidia of the AG-deficient mutants. Furthermore, according to a Dectin-1 NFAT (nuclear factor of activator T cells)-GFP (green fluorescent protein) reporter assay, Dectin-1 response levels in the AG-deficient mutants were higher than those in wild-type A. oryzae. These results suggest that we successfully isolated AG-deficient mutants of A. oryzae with immunostimulatory effects.     

Enzymatic Synthesis of 1,5-Anhydro-4-O-β-D-glucopyranosyl-D-fructose Using Cellobiose Phosphorylase and Its Spontaneous Decomposition via β-Elimination

Cellobiose phosphorylase from Cellvibrio gilvus was used to prepare 1,5-anhydro-4-O-β-D-glucopyranosyl-D-fructose [βGlc(1→4)AF] from 1,5-anhydro-D-fructose and α-D-glucose 1-phosphate. βGlc(1→4)AF decomposed into D-glucose and ascopyrone T via β-elimination. Higher pH and temperature caused faster decomposition. However, decomposition proceeded significantly even under mild conditions. For instance, the half-life of βGlc(1→4)AF was 17 h at 30 °C and pH 7.0. Because βGlc(1→4)AF is a mimic of cellulose, in which the C2 hydroxyl group is oxidized, such decomposition may occur in oxidized cellulose in nature. Here we propose a possible oxidizing pathway by which this occurs.     

Characterization of a GH36 β-L-Arabinopyranosidase in Bifidobacterium adolescentis

β-L-Arabinopyranosidases are classified into the glycoside hydrolase family 27 (GH27) and GH97, but not into GH36. In this study, we first characterized the GH36 β-L-arabinopyranosidase BAD_1528 from Bifidobacterium adolescentis JCM1275. The recombinant BAD_1528 expressed in Escherichia coli had a hydrolytic activity toward p-nitrophenyl (pNP)-β-L-arabinopyranoside (Arap) and a weak activity toward pNP-α-D-galactopyranoside (Gal). The enzyme liberated L-arabinose efficiently not from any oligosaccharides or polysaccharides containing Arap-β1,3-linkages, but from the disaccharide Arap-β1,3-L-arabinose. However, we were unable to confirm the in vitro fermentability of Arap-β1,3-Ara in B. adolescentis strains. The enzyme also had a transglycosylation activity toward 1-alkanols and saccharides as acceptors.     

Epimerization and Decomposition of Kojibiose and Sophorose by Heat Treatment under Neutral pH Conditions

We evaluated the stabilities of kojibiose and sophorose when heated under neutral pH conditions. Kojibiose and sophorose epimerized at the C-2 position of glucose on the reducing end, resulting in the production of 2-O-α-D-glucopyranosyl-D-mannose and 2-O-β-D-glucopyranosyl-D-mannose, respectively. Under weak alkaline conditions, kojibiose was decomposed due to heating into its mono-dehydrated derivatives, including 3-deoxy-2,3-unsaturated compounds and bicyclic 3,6-anhydro compounds. Following these experiments, we propose a kinetic model for the epimerization and decomposition of kojibiose and sophorose by heat treatment under neutral pH and alkaline conditions. The proposed model shows a good fit with the experimental data collected in this study. The rate constants of a reversible epimerization of kojibiose at pH 7.5 and 90 °C were (1.6 ± 0.1) × 10⁻⁵ s⁻¹ and (3.2 ± 0.2) × 10⁻⁵ s⁻¹ for the forward and reverse reactions, respectively, and were almost identical to those [(1.5 ± 0.1) × 10⁻⁵ s⁻¹ and (3.5 ± 0.4) × 10⁻⁵ s⁻¹] of sophorose. The rate constant of the decomposition reaction for kojibiose was (4.7 ± 1.1) × 10⁻⁷ s⁻¹ whereas that for sophorose [(3.7 ± 0.2) × 10⁻⁶ s⁻¹] was about ten times higher. The epimerization reaction was not significantly affected by the variation in the buffer except for a borate buffer, and depended instead upon the pH value (concentration of hydroxide ions), indicating that epimerization occurred as a function of the hydroxide ion. These instabilities are an extension of the neutral pH conditions for keto-enol tautomerization that are often observed under strong alkaline conditions.     

Quantification of uncertainty using Bayesian and bootstrap models to simulate the impact of nitrogen fertilisation on β‐glucan levels in barley

BACKGROUND: β‐Glucans have enjoyed renewed interest as a functional food ingredient, with current attention focused on optimising β‐glucan levels in finished products without compromising final product quality. In order to measure the uncertainty about the level of β‐glucans in barley, two different statistical methods (Bayesian inference and Bootstrap technique) were applied to measured levels of β‐glucan in three different varieties of barley grain (n = 83). RESULTS: The resulting probability density distributions were similar for the full data set and also when applied to smaller sample sizes, highlighting the potential for either method in quantifying the total uncertainty in β‐glucan levels. Bayesian inference was used to model the effect of nitrogen treatment on β‐glucan and protein contents in barley. The model found that a low level of fertilisation (50 kg N ha^?1) did not have a significant effect on β‐glucan or protein content. However, fertilisation above this level did result in an increase in β‐glucan and protein levels, the effect seeming to plateau at 100 kg N ha^?1. In addition, the uncertainty distributions were significantly different for two consecutive years of data, highlighting the potential environmental influence on β‐glucan content. CONCLUSION: The model developed in this study could be a useful tool for processors to quantify the uncertainty about the initial level of β‐glucan in barley and to evaluate the influence of environmental factors, thus enabling them to formulate their ingredient base to optimise levels of β‐glucan without compromising final product quality. Copyright © 2009 Society of Chemical Industry     

Purification,Characterization, and cDNA Cloning of a Prominent β-Glucosidase from the Gut of the Xylophagous Cockroach Panesthia angustipennis spadica

Abstract: In this study, a β-glucosidase (PaBG1b) with high specific activity was purified from gut extracts of the wood-feeding cockroach Panesthia angustipennis spadica using Superdex 75 gel filtration chromatography and High-Trap phenyl hydrophobic chromatography. The protein was purified 14-fold to a single band identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis, with an apparent molecular mass of 56.7 kDa. The specific activity of the purified enzyme was 708 μmol/min/mg protein using cellobiose as substrate. To the best of our knowledge, this is the highest specific activity reported among β-glucosidases to date. The purified PaBG1b showed optimal activity at pH 5.0 and retained more than 65 % of the activity between pH 4.0 and 6.5. The activity was stable up to 50 °C for 30 min. Kinetic studies on cellobiose revealed that the K_m was 5.3 mM, and the V_max was 1,020 μmol/min/mg. The internal amino acid sequence of PaBG1b was analyzed, and two continuous sequences (a total of 39 amino acids) of the C-terminal region were elucidated. Based on these amino acid sequences, a full-length cDNA (1,552 bp) encoding 502 amino acids was isolated. The encoded protein showed high similarity to β-glucosidases from glycoside hydrolase family 1. Thus, the current study demonstrated the potential of PaBG1b for application in enzymatic biomass-conversion as a donor gene for heterologous recombination of cellulase-producing agents (fungi or bacteria) or an additive enzyme for cellulase products based on the high-performance of PaBG1b as a digestive enzyme in cockroaches.     

Synthesis of 3-Keto-levoglucosan Using Pyranose Oxidase and Its Spontaneous Decomposition via β-Elimination

3-Keto-levoglucosan (3ketoLG) has been postulated to be the product of a reaction catalyzed by levoglucosan dehydrogenase (LGDH), a bacterial enzyme involved in the metabolism of levoglucosan (LG). To investigate the LG metabolic pathway catalyzed by LGDH, 3ketoLG is needed. However, 3ketoLG has not been successfully isolated from the LGDH reaction. This study investigated the ability of pyranose oxidase to convert LG into 3ketoLG by oxidizing the C3 hydroxyl group. During the oxidation of LG, 3ketoLG was spontaneously crystallized in the reaction mixture. Starting with 500 mM LG, the isolation yield of 3ketoLG was 80 %. Nuclear magnetic resonance analyses revealed that a part of 3ketoLG dimerized in aqueous solution, explaining its poor solubility. Even under normal conditions, 3ketoLG was unstable in aqueous solution, with a half-life of 16 h at pH 7.0 and 30 °C. The decomposition proceeded through β-elimination of the C–O bonds at both C1 and C5, as evidenced by decomposition products. This instability explains the difficulty in obtaining 3ketoLG via the LGDH reaction.     

A predictive model of the effects of genotypic,pre‐ and postharvest stages on barley β‐glucan levels

BACKGROUND: β‐Glucan is a bioactive component of cereal grains that has many potential uses and health‐promoting benefits. Recent research has focused on improving the nutritional value of food by increasing human exposure to β‐glucan. This study looks at the development of a farm‐level baseline model (including scenario analysis) to evaluate the impact of pre‐ and postharvest stages (including genotypic factors, environmental conditions, agronomic factors and storage) on β‐glucan levels in barley. Monte Carlo simulation techniques were employed to model various stages in pre‐ and postharvest processes and to simulate the factors influencing the level of β‐glucan content in both hulled barley (H_B) and hull‐less barley (H_LB) genotypes. RESULTS: The baseline model found that the mean simulated level of β‐glucan was 40.99 and 56.77 g kg^?1 for H_B and H_LB genotypes respectively. A sensitivity analysis highlighted that genotype was the most important parameter in determining the final β‐glucan content (correlation coefficients of 0.66 and 0.78 for H_B and H_LB respectively), more so than any of the agronomic factors analysed. The scenario analysis highlighted the importance of harvest date (scenario 2) and storage conditions (scenario 3), with a potential 32.6 and 32.7% decrease in β‐glucan (compared with the baseline model) if harvesting is carried out early during physiological maturity (i.e. at growth stage 92) and a potential 20.1 and 19.5% increase in β‐glucan for H_B and H_LB respectively if storage time is minimised. CONCLUSION: This study predicted the influence of genotypic, pre‐ and postharvest operations on β‐glucan content and thus allows strategies to be identified to influence β‐glucan content in barley products. Copyright © 2008 Society of Chemical Industry     

Enhanced Azidolysis by the Formation of Stable Ser–His Catalytic Dyad in a Glycoside Hydrolase Family 10 Xylanase Mutant

Glycoside hydrolases require carboxyl groups as catalysts for their activity. A retaining xylanase from Streptomyces olivaceoviridis E-86 belonging to glycoside hydrolase family 10 possesses Glu128 and Glu236 that respectively function as acid/base and nucleophile. We previously developed a unique mutant of the retaining xylanase, N127S/E128H, whose deglycosylation is triggered by azide. A crystallographic study reported that the transient formation of a Ser–His catalytic dyad in the reaction cycle possibly reduced the azidolysis reaction. In the present study, we engineered a catalytic dyad with enhanced stability by site-directed mutagenesis and crystallographic study of N127S/E128H. Comparison of the Michaelis complexes of N127S/E128H with pNP-X₂ and with xylopentaose showed that Ser127 could form an alternative hydrogen bond with Thr82, which disrupts the formation of the Ser–His catalytic dyad. The introduction of T82A mutation in N127S/E128H produces an enhanced first-order rate constant (6 times that of N127S/E128H). We confirmed the presence of a stable Ser–His hydrogen bond in the Michaelis complex of the triple mutant, which forms the productive tautomer of His128 that acts as an acid catalyst. Because the glycosyl azide is applicable in the bioconjugation of glycans by using click chemistry, the enzyme-assisted production of the glycosyl azide may contribute to the field of glycobiology.     

Enzymatic Synthesis and Structural Confirmation of Novel Oligosaccharide,D-Fructofuranose-linked Chitin Oligosaccharide

Utilizing transglycosylation reaction catalyzed by β- N -acetylhexosaminidase of Stenotrophomonas maltophilia , β-D-fructofuranosyl-(2↔1)-α- N , N ´diacetylchitobioside (GlcNAc ₂ -Fru) was synthesized from N -acetylsucrosamine and N , N ´-diacetylchitobiose (GlcNAc ₂ ), and β-D-fructofuranosyl-(2↔1)-α- N , N ´, N ´´-triacetylchitotrioside (GlcNAc ₃ -Fru) was synthesized from GlcNAc ₂ -Fru and GlcNAc ₂ . Through purification by charcoal column chromatography, pure GlcNAc ₂ -Fru and GlcNAc ₃ -Fru were obtained in molar yields of 33.0 % and 11.7 % from GlcNAc ₂ , respectively. The structures of these oligosaccharides were confirmed by comparing instrumental analysis data of fragments obtained by enzymatic hydrolysis and acid hydrolysis of them with known data of these fragments.     

Effects of banana flour and β-glucan on the nutritional and sensory evaluation of noodles

The purpose of this study is to determine the nutritional and sensory attributes of the yellow alkaline noodle (YAN) prepared from 30% matured green banana (Musa acuminata × balbisiana Colla cv. Awak) flour (BF) and with addition of 10% oat β-glucan. The substitution of wheat flour with BF resulted in significantly (p < 0.05) higher total dietary fibre (TDF), and especially insoluble dietary fibre (IDF), resistant starch (RS) and total starch contents. Thirty percent of BF significantly (p < 0.05) improved the antioxidant properties (AP) of noodles in terms of the total phenolic (TP) content and inhibition of peroxidation. Noodle incorporated with 30% BF and added oat β-glucan showed the lowest GI and carbohydrate digestibility rate, and higher concentrations of essential minerals (magnesium, calcium, potassium and phosphorus) and proximate components, with the exception of crude fat, when compared to the control. Sensory evaluation indicated that the quality of the 30% BF-substituted noodle was comparable to the control.     

Effects of Tocols and β-Glucan on Serum Lipid Parameters in Chickens*

Cereal grain diets affect serum lipids by their soluble fibre and tocotrienols. Chickens were fed diets containing an oat bran fraction or waxy hulless barley that were enriched or depleted in β-glucan and/or tocotrienols. Serum cholesterol and triacylglycerides and enzymes of cholesterol metabolism were measured. Weight gains appeared to be lower in birds on oat bran fraction-containing diets and higher in those on barley-containing diets supplemented with β-glucanase. All diets containing oat bran fraction or barley lowered serum total cholesterol and low-density-lipoprotein (LDL) cholesterol relative to the corn control diet. LDL cholesterol was reduced more by oat bran fraction supplemented with tocotrienols than by either oat bran fraction or tocotrienols alone. LDL cholesterol levels were the same for all barley-based diets. Activities of β-hydroxy-β-methylglutaryl coenzyme A (HMG CoA) reductase and cholesterol 7α-hydroxylase were inversely affected by the diets. Oat bran fraction plus tocotrienols, barley and solvent-extracted barley decreased HMG CoA reductase by 50% and increased cholesterol 7α-hydroxylase by 100%; other diets caused lesser effects. It was concluded that both β-glucan and tocotrienols affected cholesterol levels and metabolism, and the effects were additive or less. Removal of β-glucan from barley diets abolished or diminished effects on enzyme activities but did not alter effects on cholesterol levels, indicating the possibility of another component in barley that affected cholesterol levels. © 1997 SCI.     

Effects of ε-polylysine and rosemary extract on quality attributes and microbial communities in vacuum-packaged large yellow croaker (Pseudosciaena crocea) during ice storage

The effects of vacuum package combined with 0.1% ε-polylysine and 0.2% rosemary extract (V + RP) on the quality attributes and microbial communities of large yellow croaker (Pseudosciaena crocea) during ice storage were investigated. The quality was evaluated by chemical characteristics (total volatile basic nitrogen (TVB-N), K-value and biogenic amines (BAs)), microbiological indexes (Total viable counts (TVC), Shewanella bacteria counts, Pseudomonas bacteria counts, Psychrophilic bacteria counts (PBC)), changes in microbial composition were analyzed using high-throughput sequencing. Results showed that the increase of TVB-N, K-value, microorganisms and BAs could be inhibited by V + RP. Psychrobacter and Pseudomonas were detected in all samples. Shewanella increases rapidly in the middle of storage. Vagococcus and Shewanella were related to the decomposition of ATP, the formation of BAs, and TVB-N, respectively. In conclusion, V + RP presented the optimal effects, which could extend the shelf life of large yellow croaker for another 9 days compared with the control.     

Polyelectrolyte screening effects on the dissolution of whey protein gels at high pH conditions

The literature reports an optimum NaOH concentration for the alkaline cleaning of whey deposits or gels; at NaOH concentrations higher than this optimum, cleaning proceeds much more slowly. Although this phenomenon is of great importance in the cleaning of dairy equipment, no conclusive physical explanation has yet been presented. In this study, we present strong evidence that the dissolution rate is affected by the equilibrium-swelling ratio in β-lactoglobulin (βLg) gels. The swelling ratio is greatly reduced in the presence of salts due to the polyelectrolyte screening effect of the cations. This has been observed in free-swelling βLg gels using gravimetrical analysis and in the uniaxial swelling of WPC gel deposits using fluid dynamic gauging. At high dissolution pH (>13.3), the high Na⁺ concentration reduces swelling in spite of the high surface charge of the protein. It is proposed that the reduction of the free volume inside the gel impedes the transport of the protein aggregates out of the NaOH penetration zone. We have also observed that the final dissolution rate of gels pre-soaked in 1 M NaOH or NaCl is similar, despite the difference in pH, and much lower than for untreated gels: the high Na⁺ concentration in the soaked gels hinders swelling, inhibiting the disentanglement of the protein clusters regardless of the high pH.     

GH-16 Type β-1,3-Glucanase from Lysobacter sp. MK9-1 Enhances Antifungal Activity of GH-19 Type Chitinase,and Its Glucan-binding Domain Binds to Fungal Cell-wall

The GH-16 type β-1,3-glucanase (BgluC16MK) gene of Lysobacter sp. MK9-1 was cloned to study its antifungal activities. BgluC16MK displays amino acid sequence similarity with GluC from L. enzymogenes strain N4-7. BgluC16MK includes a signal sequence, a catalytic domain and carbohydrate-binding module family 6-type β-glucan binding domain (B-GBD). The expression of the BgluC16MK gene in Escherichia coli without the signal sequence resulted in antifungal activity at a dose of 0.6-0.8 nmol/disk. However, BgluC16MK displayed antifungal activity at a dose of 0.025 nmol/disk in combination with Chi19MK. Substrate-specific assay revealed that purified BgluC16MK hydrolyzed insoluble curdlan more readily than the soluble substrate. Furthermore, to explore the binding selectivity of B-GBD of BgluC16MK, we constructed a fusion protein (B-GBD-GFP) using the B-GBD and green fluorescent protein. The activity of the fusion protein against various substrates indicates that B-GBD was selective for glucans with β-1,3-linkages. An additional study demonstrated the binding ability of B-GBD-GFP to the cell-wall of living fungi, such as T. reesei and Aspergillus oryzae. These findings suggest that BgluC16MK can be utilized to generate antifungal enzyme preparations and that the fusion protein B-GBD-GFP can be used to identify the fungal cell surface structure using β-glucans.