Characterization of a unique pH-dependent amylosucrase from Deinococcus cellulosilyticus.

The amylosucrase (ASase, EC 2.4.1.4) utilizes sucrose as the sole substrate to catalyze multifunctional reactions. It can naturally synthesize α-1,4-linked glucans such as amylose as well as sucrose isomers with more favorable properties than sucrose with a lower intestinal digestibility and non-cariogenic properties. The amino acid sequence of the asase gene from Deinococcus cellulosilyticus (DceAS) exhibits low homology with those of other ASases from other Deinococcus species. In this study, we cloned and expressed DceAS and demonstrated its high activity at pH 6 and pH 8 and maintained stability. It showed higher polymerization activity at pH 6 than at pH 8, but similar isomerization activity and produced more turanose and trehalulose at pH 6 than at pH 8 and produced more isomaltulose at pH 8. Furthermore, the molecular weight of DceAS was 226.6 kDa at pH 6 and 145.5 kDa at pH 8, indicating that it existed as a trimer and dimer, respectively under those conditions. Additionally, circular dichroism spectra showed that the DceAS secondary structure was different at pH 6 and pH 8. These differences in reaction products at different pHs can be harnessed to naturally produce sucrose alternatives that are more beneficial to human health.

Isomerization of 6-O-substituted glucose and fructose under neutral pH conditions and subsequent ß-elimination reactions.

Isomaltose (6-O-α-d-glucopyranosyl-d-glucose) and isomaltulose (palatinose; 6-O-ß-d-glucopyranosyl-d-fructose) were heated to 90 °C in 100 mM sodium phosphate buffer (pH 7.5). Aldose-ketose isomerization between isomaltose, isomaltulose, and epi-isomaltulose was observed in the early stage of the reaction, alongside the release of a small amount of glucose. The total concentration of these disaccharides gradually decreased as the heating time increased. However, this decrease did not correlate with the amount of glucose or fructose released, suggesting that the releases of these monosaccharides were not caused by the hydrolysis of glycosidic linkages. A slight decrease in the pH of the reaction solution was attributed to the formation of two organic acids, 6-O-ß-d-glucopyranosyl-3-deoxy-d-arabino-hexonic acid (1) and 6-O-ß-d-glucopyranosyl-3-deoxy-d-ribo-hexonic acid (2). These compounds were formed from the ß-elimination of the hydroxyl group at the C-3 of fructose, leaving a substituted glucose residue at the C-6 position, followed by keto-enol tautomerization and benzilic acid rearrangement. Although approximately 30% of 1 and 2 were degraded after 360 min of heating at 90 °C in 100 mM sodium phosphate, a little release of glucose was observed, indicating no hydrolysis of the glucoside bond at C-6. Besides 1 and 2, time-dependent changes in the NMR spectra of the reaction mixture in water indicated the formation of formic acid and the presence of species possibly resulting from the ß-elimination of the hydroxyl group from 3- and 4-ulose. The glucose released by heating isomaltose and isomaltulose may be generated via tautomerizations of keto-enols between the C-4 and C-5 positions and cleavage of 6-O-glycosidic linkage via ß-elimination.

Tuning the catalytic performances of a sucrose isomerase for production of isomaltulose with high concentration.

Obtaining a sucrose isomerase (SIase) with high catalytic performance is of great importance in industrial production of isomaltulose (a reducing sugar). In order to obtain such SIase mutant, a high-throughput screening system in microtiter plate format was developed based on a widely used 2,4-dinitrosalicylic acid (DNS) method for determination of reducing sugar. An SIase from Erwinia sp. Ejp617 (ErSIase) was selected to improve its catalytic efficiency. After screening of ~ 8000 mutants from a random mutagenesis library, Q209 and R456 were identified as beneficial positions. Saturation mutagenesis of the two positions resulted in a double-site mutant ErSIase_Q209S-R456H that showed the highest catalytic efficiency, and its specific activity reached 684 U/mg that is 17.5-fold higher than that of the wild-type ErSIase. By employing the lyophilized Escherichia coli (E. coli) cells harboring ErSIase_Q209S-R456H, a high space-time yield (STY = 3.9 kg/(L·d)) was achieved toward 600 g/L sucrose. Furthermore, the in silico analysis suggested that the hydrogen bond network was improved and steric hindrance was reduced due to the beneficial substitutions.Key points• A sucrose isomerase mutant with high catalytic efficiency was obtained.• The highest space-time yield was achieved toward high-concentration sucrose.• The optimized H-bond network contributed to the enhanced catalytic efficiency.

Structural insights reveal the second base catalyst of isomaltose glucohydrolase.

Glycoside hydrolase family 15 (GH15) inverting enzymes contain two glutamate residues functioning as a general acid catalyst and a general base catalyst, for isomaltose glucohydrolase (IGHase), Glu178 and Glu335, respectively. Generally, a two-catalytic residue-mediated reaction exhibits a typical bell-shaped pH-activity curve. However, IGHase is found to display atypical non-bell-shaped pH-kcat and pH-kcat /Km profiles, theoretically better-fitted to a three-catalytic residue-associated pH-activity curve. We determined the crystal structure of IGHase by the single-wavelength anomalous dispersion method using sulfur atoms and the cocrystal structure of a catalytic base mutant E335A with isomaltose. Although the activity of E335A was undetectable, the electron density observed in its active site pocket did not correspond to an isomaltose but a glycerol and a ß-glucose, cryoprotectant, and hydrolysis product. Our structural and biochemical analyses of several mutant enzymes suggest that Tyr48 acts as a second catalytic base catalyst. Y48F mutant displayed almost equivalent specific activity to a catalytic acid mutant E178A. Tyr48, highly conserved in all GH15 members, is fixed by another Tyr residue in many GH15 enzymes; the latter Tyr is replaced by Phe290 in IGHase. The pH profile of F290Y mutant changed to a bell-shaped curve, suggesting that Phe290 is a key residue distinguishing Tyr48 of IGHase from other GH15 members. Furthermore, F290Y is found to accelerate the condensation of isomaltose from glucose by modifying a hydrogen-bonding network between Tyr290-Tyr48-Glu335. The present study indicates that the atypical Phe290 makes Tyr48 of IGHase unique among GH15 enzymes.

Simple and efficient preparation of high-purity trehalulose from the waste syrup of isomaltulose production using solid-phase extraction followed by hydrophilic interaction chromatography.

A simple and efficient method was developed for the preparation of high-purity trehalulose from the waste syrup of isomaltulose production. The waste syrup was pre-treated with C18 solid-phase extraction, where 98% decolorization and 97% reducing sugar recovery were obtained, followed by hydrophilic interaction liquid chromatography separation on a cysteine-bonded zwitterionic column. Under optimized conditions, trehalulose was separated from isomaltulose isomer and prepared on a semi-preparative scale with >99% purity. The structure of the prepared trehalulose was subsequently confirmed by nuclear magnetic resonance, and three tautomers of trehalulose (α-D-glucosylpyranosyl-1,1-ß-D-fructopyranose, α-D-glucosylpyranosyl-1,1-ß-D-fructofuranose, and α-D-glucosylpyranosyl-1,1-α-D-fructofuranose) were detected and completely characterized by 13 C NMR spectroscopy for the first time in this study. The tautomerization of α-D and ß-D type transition was observed by hydrophilic interaction liquid chromatography on an AdvanceBio Glycan Mapping column, with smaller particle size (2.7 µm). Furthermore, the prepared trehalulose was applied as a standard for trehalulose quantification during the sucrose conversion by Klebsiella sp. LX3. The combination of solid-phase extraction and hydrophilic interaction liquid chromatography offers a new avenue for the preparation of sugar isomers from complex natural or fermentation products.

NMR analysis and molecular dynamics conformation of α-1,6-linear and α-1,3-branched isomaltose oligomers as mimetics of α-1,6-linked dextran.

The conformational preferences of several α-1,6-linear and α-1,3-branched isomalto-oligosaccharides were investigated by NMR and MD-simulations. Right-handed helical structure contributed to the solution geometry in isomaltotriose and isomaltotetraose with one nearly complete helix turn and stabilizing intramolecular hydrogen bonds in the latter by MD-simulation. Decreased helix contribution was observed in α-1,3-glucopyranosyl- and α-1,3-isomaltosyl-branched saccharide chains. Especially the latter modification was predicted to cause a more compact structure consistent with literature rheology measurements as well as with published dextranase-resistant α-1,3-branched oligosaccharides. The findings presented here are significant because they shed further light on the conformational preference of isomalto-oligosaccharides and provide possible help for the design of dextran-based drug delivery systems or for the targeted degradation of capsular polysaccharides by dextranases in multi-drug resistant bacteria.

Characterization of a recombinant sucrose isomerase and its application to enzymatic production of isomaltulose.

OBJECTIVE: To characterize a recombinant isomerase that can catalyze the isomerization of sucrose into isomaltulose and investigate its application for the enzymatic production of isomaltulose. RESULTS: A sucrose isomerase gene from Erwinia sp. Ejp617 was synthesized and expressed in Escherichia coli BL21(DE3). The enzymatic characterization revealed that the optimal pH and temperature of the purified sucrose isomerase were 6.0 and 40 °C, respectively. The enzyme activity was slightly activated by Mn2+and Mg2+, but partially inhibited by Ca2+, Ba2+, Cu2+, Zn2+ and EDTA. The kinetic parameters of Km and Vmax for sucrose were 69.28 mM and 118.87 U/mg, respectively. The time course showed that 240.9 g/L of isomaltulose was produced from 300 g/L of sucrose, and the yield reached 80.3% after bioreaction for 180 min. CONCLUSIONS: This recombinant enzyme showed excellent capability for biotransforming sucrose to isomaltulose at the substrate concentration of 300 g/L. Further investigations should be carried out focusing on selection of suitable heterologous expression system with the aim to improve its expression level.

Expression of a novel α-glucosidase from Aspergillus neoniger in Pichia pastoris and its efficient recovery for synthesis of isomaltooligosaccharides.

A gene conferring α-glucosidase (AG) with high transglycosylation activity from Aspergillus neoniger (a non-niger strain belonging to section Nigri) was cloned and expressed in Pichia pastoris. As the cDNA construction retained intronic portions due to alternative splicing, the exonic portions of the gene were stitched using restriction digestion and overlap extension PCR. Pre-determined open-loop exponential feeding strategies were evaluated for methanol dosage to improve the recombinant enzyme synthesis during high-cell density cultivation in 5 L bioreactor. Specific growth rate of 0.1 h-1 resulted in the highest enzyme activity of 182.3 mU/mL in the supernatant, whereas the activity of 3.8 U/g dry cell weight was obtained in the cell pellet. There was negligible enzyme activity in the cell lysate, indicating that approximately 80 % accumulation of total enzyme is in the periplasm. Later, this unreleased fraction was extracted to 90 % yield using 25 mM cysteine. The enzyme was purified and validated using western blot analysis and MS/MS profile. The SDS PAGE analysis revealed three bands corresponding to 80, 38, and 33 kDa indicating the multimeric nature of the enzyme. Thus, obtained enzyme was utilized in synthesis of a potential prebiotic molecule, isomaltooligosaccharides (IMOs), which can be used as a sweetener and bulk filler in the food industry. This is the first report to demonstrate challenges in cloning and expression of transglycosylating α-glucosidase from Aspergillus neoniger.

Economical production of isomaltulose from agricultural residues in a system with sucrose isomerase displayed on Bacillus subtilis spores.

A safe, efficient, environmentally friendly process for producing isomaltulose is needed. Here, the biocatalyst, sucrose isomerase (SIase) from Erwinia rhapontici NX-5, displayed on the surface of Bacillus subtilis 168 spores (food-grade strain) was applied for isomaltulose production. The anchored SIase showed relatively high bioactivity, suggesting that the surface display system using CotX as the anchoring protein was successful. The stability of the anchored SIase was also significantly better. Thermal stability analysis showed that 80% of relative activity was retained after incubation at 40 °C and 45 °C for 60 min. To develop an economical industrial fermentation medium, untreated beet molasses (30 g/L) and cold-pressed soybean powder (50 g/L) were utilised as the main broth components for SIase pilot-scale production. Under the optimal conditions, the productive spores converted 92% of sucrose after 6 h and the conversion rate was 45% after six cycles. Isomaltulose production with this system using the agricultural residues, untreated beet molasses and soybean powder, as substrates is cost-effective and environmentally friendly and can help to overcome issues due to the genetic background.

Sucrose isomers as alternative sweeteners: properties, production, and applications.

In the daily diet, sweeteners play an indispensable role. Among them, sucrose, a widely occurring disaccharide in nature, is a commonly used sweetener. However, the intake of sucrose can cause a rapid increase in blood glucose, which leads to a number of health problems. Therefore, there is an urgent need for possible alternatives to sucrose. Currently, four naturally occurring sucrose isomers, trehalulose, turanose, leucrose, and isomaltulose are considered to be possible alternatives to sucrose due to their suitable sweetness, potential physiological benefits, and feasible production processes. This review covers the properties of these alternative sweeteners, including their structure, sweetness, hydrolysis rate, toxicology, and cariogenicity, and exhibits their potential applications in chronic diseases management, anti-inflammatory supplement, prebiotic dietary supplement, and stabilizing agent. The biosynthesis of these sucrose isomers using carbohydrate-active enzymes and their industrial production processes are also systematically summarized.

Whole Conversion of Soybean Molasses into Isomaltulose and Ethanol by Combining Enzymatic Hydrolysis and Successive Selective Fermentations.

Isomaltulose is mainly produced from sucrose by microbial fermentation, when the utilization of sucrose contributes a high production cost. To achieve a low-cost isomaltulose production, soy molasses was introduced as an alternative substrate. Firstly, α-galactosidase gene from Rhizomucor miehei was expressed in Yarrowia lipolytica, which then showed a galactosidase activity of 121.6 U/mL. Under the effects of the recombinant α-galactosidase, most of the raffinose-family oligosaccharides in soy molasses were hydrolyzed into sucrose. Then the soy molasses hydrolysate with high sucrose content (22.04%, w/w) was supplemented into the medium, with an isomaltulose production of 209.4 g/L, and the yield of 0.95 g/g. Finally, by virtue of the bioremoval process using Pichia stipitis, sugar byproducts in broth were transformed into ethanol at the end of fermentation, thus resulting in high isomaltulose purity (97.8%). The bioprocess employed in this study provides a novel strategy for low-cost and efficient isomaltulose production from soybean molasses.

Effects of isomaltooligosaccharide and Bacillus supplementation on sow performance, serum metabolites, and serum and placental oxidative status.

This study investigated the effects of isomaltooligosaccharide (IMO) and Bacillus supplementation on sow performance, serum metabolites, and serum and placental oxidative status. Multiparous gestating sows (n = 130) with similar body conditions were randomly allocated to five groups (n = 26) receiving a basal diet (CON group) or a basal diet supplemented with 0.5% IMO (IMO group); 0.5% IMO and 0.02% Bacillus subtilis (IMO + S group); 0.5% IMO and 0.02% Bacillus licheniformis (IMO + L group); or 0.5% IMO, 0.02% Bacillus subtilis, and 0.02% Bacillus licheniformis (IMO + S+L group). There were no significant differences in the litter sizes among all dietary groups. The average piglet birth weight was improved in all treatment groups, and the placental efficiency was greater in the IMO + S and IMO + S+L groups than in the CON group (P < 0.05). The IMO + S+L group had increased the low-density lipoprotein cholesterol and reduced the total cholesterol in umbilical venous serum (P <  0.05). Additionally, the malondialdehyde concentrations were greater in umbilical venous serum of piglets in all treatment groups relative to that in the CON piglets (P <  0.05). The placental total antioxidant capacity was increased in the IMO+L and IMO+S+L groups (P <  0.05). Furthermore, the growth hormone concentration in umbilical venous serum was greater (P <  0.05) in all treatment groups. Overall, IMO and Bacillus supplementation during late gestation resulted in a changed metabolism of sows, improved the placental antioxidant capacity, and increased the growth hormone concentrations in umbilical venous serum, which ultimately improved the piglet birth weight and placental efficiency.

Heterologous Expression of a Thermostable α-Glucosidase from Geobacillus sp. Strain HTA-462 by Escherichia coli and Its Potential Application for Isomaltose⁻Oligosaccharide Synthesis.

Isomaltose-oligosaccharides (IMOs), as food ingredients with prebiotic functionality, can be prepared via enzymatic synthesis using α-glucosidase. In the present study, the α-glucosidase (GSJ) from Geobacillus sp. strain HTA-462 was cloned and expressed in Escherichia coli BL21 (DE3). Recombinant GSJ was purified and biochemically characterized. The optimum temperature condition of the recombinant enzyme was 65 °C, and the half-life was 84 h at 60 °C, whereas the enzyme was active over the range of pH 6.0-10.0 with maximal activity at pH 7.0. The α-glucosidase activity in shake flasks reached 107.9 U/mL and using 4-Nitrophenyl ß-D-glucopyranoside (pNPG) as substrate, the Km and Vmax values were 2.321 mM and 306.3 U/mg, respectively. The divalent ions Mn2+ and Ca2+ could improve GSJ activity by 32.1% and 13.8%. Moreover, the hydrolysis ability of recombinant α-glucosidase was almost the same as that of the commercial α-glucosidase (Bacillus stearothermophilus). In terms of the transglycosylation reaction, with 30% maltose syrup under the condition of 60 °C and pH 7.0, IMOs were synthesized with a conversion rate of 37%. These studies lay the basis for the industrial application of recombinant α-glucosidase.

NMR solution geometry of saccharides containing the 6-O-(α-D-glucopyranosyl)-α/ß-D-glucopyranose (isomaltose) or 6-O-(α-D-galactopyranosyl)-α/ß-D-glucopyranose (melibiose) core.

The solution geometries of D-Glcp, Me-D-Glcp, 6-O-Me-D-Glcp, Me-6-O-Me-D-Glcp, D-Glcp-(α-1,6)-D-Glcp (isomaltose), D-Glcp-(α-1,6)-D-Glcp-(α-1,6)-D-Glcp (isomaltotriose), D-Galp-(α-1,6)-D-Glcp (melibiose), D-Galp-(α-1,6)-D-Glcp-(α-1,2)-D-Fruf (raffinose), and D-Galp-(α-1,6)-D-Galp-(α-1,6)-D-Glcp-(α-1,2)-D-Fruf (stachyose) in water are described by NMR spectroscopy, molecular dynamic simulations and quantum mechanical calculations. Overall, a change in anomeric configuration at the reducing end and/or anomeric substitution (methylation) changed the conformational space of the terminal CH2OH group significantly. Conformational analysis of the free monosaccharides matched literature results very well. Dihedral angle histograms weighted against published Karplus equations yielded excellent matches of experimental J-values in some cases but significant deviations in other. The anomeric hemiacetal configuration appeared to have a significant remote influence on the conformational space of the α-1,6-glycosidic linkage. Rigid glycosidic φ-conformations (g+) combined with mostly st-conformations for glycosidic ψ-angles from computations matched experimental nuclear Overhauser enhancements in all cases. While the investigated Glcp-α-1,6-Glcp linkages were nearly identical in φ/ψ-conformation, differences were apparent in the Galp-α-1,6-Galp linkage of stachyose. Of twenty-one crystal structures, a total of fourteen had ligand conformations corresponding to the most abundant or second-most abundant solution geometry determined in this study.

Evaluation of the Reported Rates of Severe Hypersensitivity Reactions Associated with Ferric Carboxymaltose and Iron (III) Isomaltoside 1000 in Europe Based on Data from EudraVigilance and VigiBase™ between 2014 and 2017.

INTRODUCTION: Hypersensitivity reactions (HSRs) are among the known adverse events of intravenous (i.v.) iron products. Of these, particularly severe HSRs such as anaphylaxis are of great clinical concern due to their life-threatening potential. METHODS: This was a retrospective pharmacoepidemiological study with a case-population design evaluating the number of reported severe HSRs following administration of the two i.v. iron products-ferric carboxymaltose and iron (III) isomaltoside 1000-in relation to exposure in European countries from January 2014 to December 2017. Exposure to both products was estimated using IQVIA MIDAS sales data in European countries. Information on spontaneously reported severe HSRs was obtained from and analysed separately for the two established safety surveillance databases EudraVigilance and VigiBase™ using the MedDRA® Preferred Terms anaphylactic reaction, anaphylactic shock, anaphylactoid reaction and anaphylactoid shock associated with administration of either product. RESULTS: Between 2014 and 2017, the reporting rate of severe HSRs per 100,000 defined daily doses (100 mg dose equivalents of iron) varied from 0.3 to 0.5 for ferric carboxymaltose and from 2.4 to 5.0 for iron (III) isomaltoside 1000. The reporting rate ratio for iron (III) isomaltoside 1000 versus ferric carboxymaltose was between 5.6 (95% CI 3.5-9.0) and 16.2 (95% CI 9.4-27.8). CONCLUSIONS: Findings suggest that iron (III) isomaltoside 1000 is associated with a higher reporting rate of severe HSRs related to estimated exposure than ferric carboxymaltose in European countries. Future research investigating the occurrence of severe HSRs associated with i.v. ferric carboxymaltose and iron (III) isomaltoside 1000 is needed to broaden the evidence for benefit-risk assessment.

Effects of Mono-, Di-, and Tri-Saccharides on the Stability and Crystallization of Amorphous Sucrose.

Amorphous sucrose is a component of many food products but is prone to crystallize over time, thereby altering product quality and limiting shelf-life. A systematic investigation was conducted to determine the effects of two monosaccharides (glucose and fructose), five disaccharides (lactose, maltose, trehalose, isomaltulose, and cellobiose), and two trisaccharides (maltotriose and raffinose) on the stability of amorphous sucrose in lyophilized two-component sucrose-saccharide blends exposed to different relative humidity (RH) and temperature environmental conditions relevant for food product storage. Analyses included X-ray diffraction, differential scanning calorimetry, microscopy, and moisture content determination, as well as crystal structure overlays. All lyophiles were initially amorphous, but during storage the presence of an additional saccharide tended to delay sucrose crystallization. All samples remained amorphous when stored at 11% and 23% RH at 22 °C, but increasing the RH to 33% RH and/or increasing the temperature to 40 °C resulted in variations in crystallization onset times. Monosaccharide additives were less effective sucrose crystallization inhibitors relative to di- and tri-saccharides. Within the group of di- and tri-saccharides, effectiveness depended on the specific saccharide added, and no clear trends were observed with saccharide molecular weight and other commonly studied factors such as system glass transition temperature. Molecular level interactions, as evident in crystal structure overlays of the added saccharides and sucrose and morphological differences in crystals formed, appeared to contribute to the effectiveness of a di- or tri-saccharide in delaying sucrose crystallization. In conclusion, several di- and tri-saccharides show promise for use as additives to delay the crystallization kinetics of amorphous sucrose during storage at moderate temperatures and low RH conditions. PRACTICAL APPLICATION: Amorphous sucrose is desirable in a variety of food products, wherein crystallization can be problematic for texture and shelf-life. This study documents how different mono-, di-, and tri-saccharides influence the crystallization of sucrose. Monosaccharide additives were less effective sucrose crystallization inhibitors relative to di- and tri-saccharides. These findings increase the understanding of how different mono-, di-, and tri-saccharide structures and their solid-state properties influence the crystallization of amorphous sucrose and show that several di- and tri-saccharides have potential for use as sucrose crystallization inhibitors.

Synthesis of Isomalto-Oligosaccharides by Pichia pastoris Displaying the Aspergillus niger α-Glucosidase.

We explored the ability of an Aspergillus niger α-glucosidase displayed on P. pastoris to act as a whole-cell biocatalyst (Pp-ANGL-GCW61) system to synthesize isomalto-oligosaccharides (IMOs). IMOs are a mixture that includes isomaltose (IG2), panose (P), and isomaltotriose (IG3). In this study, the IMOs were synthesized by a hydrolysis-transglycosylation reaction in an aqueous system of maltose. In a 2 mL reaction system, the IMOs were synthesized with a conversion rate of approximately 49% in 2 h when 30% maltose was utilized under optimal conditions by Pp-ANGL-GCW61. Additionally, the 0.5-L reaction system was conducted in a 2-L stirred reactor with a conversion rate of approximately 44% in 2 h. Moreover, the conversion rate was relatively stable after the whole-cell catalyst was reused three times. In conclusion, Pp-ANGL-GCW61 has a high reaction efficiency and operational stability, which makes it a powerful biocatalyst available for industrial scale synthesis.

Effect of fructo-oligosaccharide and isomalto-oligosaccharide addition on baking quality of frozen dough.

The baking quality of frozen doughs containing different levels of fructo-oligosaccharides (FO) or isomalto-oligosaccharides (IMO) (3-9%, w/w flour), and stored for 0-8weeks at -18°C, was examined. The addition of FO or IMO increased the proof volume of the dough and the loaf volume of bread prepared from frozen dough. A 6% addition of FO or IMO was optimum, giving the highest proof volume and bread loaf volume, but a higher concentration than 6% induced low baking quality including lower proof volume and bread loaf volume. The bread crumb was moister and softer after the addition of FO or IMO before, and even after, frozen storage. Darker crumb colour was observed in the bread after the addition of FO or IMO. The oligosaccharides added to the frozen dough were effective in improving the quality of bread made from frozen dough, except for resulting in a darker bread crumb.

A glycoside hydrolase family 31 dextranase with high transglucosylation activity from Flavobacterium johnsoniae.

Glycoside hydrolase family (GH) 31 enzymes exhibit various substrate specificities, although the majority of members are α-glucosidases. Here, we constructed a heterologous expression system of a GH31 enzyme, Fjoh_4430, from Flavobacterium johnsoniae NBRC 14942, using Escherichia coli, and characterized its enzymatic properties. The enzyme hydrolyzed dextran and pullulan to produce isomaltooligosaccharides and isopanose, respectively. When isomaltose was used as a substrate, the enzyme catalyzed disproportionation to form isomaltooligosaccharides. The enzyme also acted, albeit inefficiently, on p-nitrophenyl α-D-glucopyranoside, and p-nitrophenyl α-isomaltoside was the main product of the reaction. In contrast, Fjoh_4430 did not act on trehalose, kojibiose, nigerose, maltose, maltotriose, or soluble starch. The optimal pH and temperature were pH 6.0 and 60 °C, respectively. Our results indicate that Fjoh_4430 is a novel GH31 dextranase with high transglucosylation activity.

Production of keto-disaccharides from aldo-disaccharides in subcritical aqueous ethanol.

Isomerization of disaccharides (maltose, isomaltose, cellobiose, lactose, melibiose, palatinose, sucrose, and trehalose) was investigated in subcritical aqueous ethanol. A marked increase in the isomerization of aldo-disaccharides to keto-disaccharides was noted and their hydrolytic reactions were suppressed with increasing ethanol concentration. Under any study condition, the maximum yield of keto-disaccharides produced from aldo-disaccharides linked by ß-glycosidic bond was higher than that produced from aldo-disaccharides linked by α-glycosidic bond. Palatinose, a keto-disaccharide, mainly underwent decomposition rather than isomerization in subcritical water and subcritical aqueous ethanol. No isomerization was noted for the non-reducing disaccharides trehalose and sucrose. The rate constant of maltose to maltulose isomerization almost doubled by changing solvent from subcritical water to 80 wt% aqueous ethanol at 220 °C. Increased maltose monohydrate concentration in feed decreased the conversion of maltose and the maximum yield of maltulose, but increased the productivity of maltulose. The maximum productivity of maltulose was ca. 41 g/(h kg-solution).