4-O-Methyl Modifications of Glucuronic Acids in Xylans Are Indispensable for Substrate Discrimination by GH67 α-Glucuronidase from Bacillus halodurans C-125

A GH67 α-glucuronidase gene derived from Bacillus halodurans C-125 was expressed in E. coli to obtain a recombinant enzyme (BhGlcA67). Using the purified enzyme, the enzymatic properties and substrate specificities of the enzyme were investigated. BhGlcA67 showed maximum activity at pH 5.4 and 45 °C. When BhGlcA67 was incubated with birchwood, oat spelts, and cotton seed xylan, the enzyme did not release any glucuronic acid or 4-O-methyl-glucuronic acid from these substrates. BhGlcA67 acted only on 4-O-methyl-α-D-glucuronopyranosyl-(1→2)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranose (MeGlcA³Xyl₃), which has a glucuronic acid side chain with a 4-O-methyl group located at its non-reducing end, but did not on β-D-xylopyranosyl-(1→4)-[4-O-methyl-α-D-glucuronopyranosyl-(l→2)]-β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-xylop- yranose (MeGlcA³Xyl₄) and α-D-glucuronopyranosyl-(l→2)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-xylopyranose (GlcA³Xyl₃). The environment for recognizing the 4-O-methyl group of glucuronic acid was observed in all the crystal structures of reported GH67 glucuronidases, and the amino acids for discriminating the 4-O-methyl group of glucuronic acid were widely conserved in the primary sequences of the GH67 family, suggesting that the 4-O-methyl group is critical for the activities of the GH67 family.     

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.     

1,6-α-L-Fucosidases from Bifidobacterium longum subsp. infantis ATCC 15697 Involved in the Degradation of Core-fucosylated N -Glycan

Bifidobacterium longum subsp. infantis ATCC 15697 possesses five α-L-fucosidases, which have been previously characterized toward fucosylated human milk oligosaccharides containing α1,2/3/4-linked fucose [Sela et al.: Appl. Environ. Microbiol., 78, 795-803 (2012)]. In this study, two glycoside hydrolase family 29 α-L-fucosidases out of five (Blon_0426 and Blon_0248) were found to be 1,6-α-L-fucosidases acting on core α1,6-fucose on the N-glycan of glycoproteins. These enzymes readily hydrolyzed p-nitrophenyl-α-L-fucoside and Fucα1-6GlcNAc, but hardly hydrolyzed Fucα1-6(GlcNAcβ1-4)GlcNAc, suggesting that they de-fucosylate Fucα1-6GlcNAcβ1-Asn-peptides/proteins generated by the action of endo-β- N-acetylglucosaminidase. We demonstrated that Blon_0426 can de-fucosylate Fucα1-6GlcNAc-IgG prepared from Rituximab using Endo-CoM from Cordyceps militaris. To generate homogenous non-fucosylated N-glycan-containing IgG with high antibody-dependent cellular cytotoxicity (ADCC) activity, the resulting GlcNAc-IgG has a potential to be a good acceptor substrate for the glycosynthase mutant of Endo-M from Mucor hiemalis. Collectively, our results strongly suggest that Blon_0426 and Blon_0248 are useful for glycoprotein glycan remodeling.     

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 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.     

Microencapsulation of β-galactosidase with different biopolymers by a spray-drying process

The aim of this work was to investigate the possibility of producing microparticles containing β-galactosidase, using different biopolymers (arabic gum, chitosan, modified chitosan, calcium alginate and sodium alginate) as encapsulating agents by a spray-drying process. This study focused on the enzyme β-galactosidase, due to its importance in health and in food processing. Encapsulation of β-galactosidase can increase the applicability of this enzyme in different processes and applications. A series of β-galactosidase microparticles were prepared, and their physicochemical structures were analyzed by laser granulometry analysis, zeta potential analysis, and by scanning electron microscopy (SEM). Microparticles with a mean diameter around 3 μm have been observed, for all the biopolymers tested. The microparticles formed with chitosan or arabic gum presented a very rough surface; on the other hand, the particles formed with calcium or sodium alginate or modified chitosan presented a very smooth surface. The activity of the enzyme was studied by spectrophotometric methods using the substrate ONPG (O-nitrophenyl-β,d-galactopyranoside). The microencapsulated β-galactosidase activity decreases with all the biopolymers. The relative enzyme activity is 37, 20, 20 and 13%, for arabic gum, modified chitosan, calcium alginate and sodium alginate, respectively, when compared with the free enzyme activity. The enzyme microparticles formed with arabic gum shows the smallest decrease of Vmax, followed by the calcium alginate, sodium alginate, and modified chitosan.     

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.     

Hydrocolloids from coffee: physicochemical and functional properties of an arabinogalactan–protein fraction from green beans

The arabinogalactan–protein (AGP) fraction of green coffee beans accounts for 15% of the dry bean. A procedure was developed to solubilise most of the AGP content of the beans so that its properties as a hydrocolloid could be investigated. An AGP fraction was partially purified from green arabica coffee beans, its rheological properties characterised and compared to those of some commercially important hydrocolloids, particularly acacia gum. The coffee AGP fraction dissolved readily in water to give colourless clear solutions. The polymer was a polyelectrolyte with a high molecular weight (M_w 3.78×10⁶), characterised by a narrow polydispersity index (M_w/M_n 1.3). The intrinsic viscosity was close to that of acacia gum ([η]=0.23 dL g⁻¹), but a 1 wt% solution of coffee AGP was three times more viscous than acacia gum at the same concentration. Coffee AGP showed Newtonian flow for concentrations below 6 wt%, but above this concentration the flow behaviour entered a shear-thinning regime. The coffee AGP fraction possessed interesting foaming properties providing that the biopolymer concentration was high enough to initially stabilize the interface that is created. The high molecular weight of coffee AGP combined with its globular structure conferred upon it a high ability to retain water within a foam thin film. However, the structure of the interfacial film was less effective than that of acacia gum to entrap efficiently the gas into the foam. In summary, coffee AGP shows some interesting rheological features which suggest that coffee beans could be used as an alternative source of the class of surface-active polymers which find many commercial applications.     

Structural Analysis of Novel Low-Digestible Sucrose Isomers Synthesized from D-Glucose and D-Fructose by Thermal Treatment

The synthesis of the saccharide β-D-fructopyranosyl-(2→6)-D-glucopyranose, which was isolated from Super Ohtaka^®, has recently been reported. During the synthesis of this saccharide, the formation of two novel saccharides from D-glucose and D-fructose was observed. The present study aimed to confirm the structures of the two disaccharides synthesized from D-glucose and D-fructose by thermal treatment. Furthermore, various properties of the saccharides were investigated. Both saccharides were isolated from the reaction mixture by carbon-Celite column chromatography and an HPLC system and were determined to be novel sucrose-isomers, β-D-fructopyranosyl-(2↔1)-β-D-glucopyranoside (1) and β-D-fructofuranosyl-(2↔1)-β-D-glucopyranoside (2), by MALDI-TOF MS and NMR analyses. Both saccharides showed low digestibility in vitro, and the sweetness of saccharide 2 was 0.45 times that of sucrose.     

Analysis of Transglucosylation Products of Aspergillus niger α-Glucosidase that Catalyzes the Formation of α-1,2- and α-1,3-Linked Oligosaccharides

According to whole-genome sequencing, Aspergillus niger produces multiple enzymes of glycoside hydrolases (GH) 31. Here we focus on a GH31 α-glucosidase, AgdB, from A. niger . AgdB has also previously been reported as being expressed in the yeast species, Pichia pastoris ; while the recombinant enzyme (rAgdB) has been shown to catalyze tranglycosylation via a complex mechanism. We constructed an expression system for A. niger AgdB using Aspergillus nidulans . To better elucidate the complicated mechanism employed by AgdB for transglucosylation, we also established a method to quantify glucosidic linkages in the transglucosylation products using 2D NMR spectroscopy. Results from the enzyme activity analysis indicated that the optimum temperature was 65 °C and optimum pH range was 6.0–7.0. Further, the NMR results showed that when maltose or maltopentaose served as the substrate, α-1,2-, α-1,3-, and small amount of α-1,1-β-linked oligosaccharides are present throughout the transglucosylation products of AgdB. These results suggest that AgdB is an α-glucosidase that serves as a transglucosylase capable of effectively producing oligosaccharides with α-1,2-, α-1,3-glucosidic linkages.     

Hydrocolloids with emulsifying capacity. Part 1 – Emulsifying properties and interfacial characteristics of conventional (Acacia senegal (L.) Willd. var. senegal) and matured (Acacia (sen) SUPER GUM™) Acacia senegal

The emulsifying properties and interfacial behaviour of conventional arabic gum (Acacia Senegal, control gum) and after thermal maturation (EM1 and EM2) were studied. All gums presented good oil in water emulsifying capacities. However, after 20–60 high-pressure homogenization passes, matured gums formed more homogenous emulsions with smaller mean oil droplets (0.4 μm) than the control gum (0.65 μm). Modification of pH from 4.5 to 7.0 did not affect sample characteristics. The film-forming capability of all samples at air–water interface was studied using adsorbed and spread techniques. The greater the amount and molecular mass of the arabinogalactan protein (AGP) component in the gum, the better the interfacial properties (tension drop kinetic and interfacial tension final values). Good dilatational elasticity was found for all three gums samples, corresponding to highly elastic interfacial films. The spread films of matured gum EM2 rearranged during compression, passing through liquid expanded, liquid condensed into solid states. Conversely, control and EM1 gums only formed liquid expanded films and smaller surface coverage than EM2. Analytical interfacial results confirm the improved emulsifying properties of matured gums. The properties of the AGP molecules in the gums could be responsible for these differences.     

Effect of C-6 Methylol Groups on Substrate Recognition of Glucose/Xylose Mixed Oligosaccharides by Cellobiose Dehydrogenase from the Basidiomycete Phanerochaete chrysosporium

Cellobiose dehydrogenase (CDH) is a flavocytochrome catalyzing oxidation of the reducing end of cellobiose and cellooligosaccharides, and has a key role in the degradation of cellulosic biomass by filamentous fungi. Here, we use a lineup of glucose/xylose-mixed β-1,4-linked disaccharides and trisaccharides, enzymatically synthesized by means of the reverse reaction of cellobiose phosphorylase and cellodextrin phosphorylase, to investigate the substrate recognition of CDH. We found that CDH utilizes β-D-xylopyranosyl-(1→4)-D-glucopyranose (Xyl-Glc) as an electron donor with similar K _m and k _cat values to cellobiose. β-D-Glucopyranosyl-(1→4)-D-xylopyranose (Glc-Xyl) shows a higher K _m value, while xylobiose does not serve as a substrate. Trisaccharides show similar behavior; i.e., trisaccharides with cellobiose and Xyl-Glc units at the reducing end show similar kinetics, while the enzyme was less active towards those with Glc-Xyl, and inactive towards those with xylobiose. We also use docking simulation to evaluate substrate recognition of the disaccharides, and we discuss possible molecular mechanisms of substrate recognition by CDH.     

Microbial α-L-Rhamnosidases of Glycosyl Hydrolase Families GH78 and GH106 Have Broad Substrate Specificities toward α-L-Rhamnosyl- and α-L-Mannosyl-Linkages

α-L-Rhamnosidases (α-L-Rha-ases, EC 3.2.1.40) are glycosyl hydrolases (GHs) that hydrolyze a terminal α-linked L-rhamnose residue from a wide spectrum of substrates such as heteropolysaccharides, glycosylated proteins, and natural flavonoids. As a result, they are considered catalysts of interest for various biotechnological applications. α-L-rhamnose (6-deoxy-L-mannose) is structurally similar to the rare sugar α-L-mannose. Here we have examined whether microbial α-L-Rha-ases possess α-L-mannosidase activity by synthesizing the substrate 4-nitrophenyl α-L-mannopyranoside. Four α-L-Rha-ases from GH78 and GH106 families were expressed and purified from Escherichia coli cells. All four enzymes exhibited both α-L-rhamnosyl-hydrolyzing activity and weak α-L-mannosyl-hydrolyzing activity. SpRhaM, a GH106 family α-L-Rha-ase from Sphingomonas paucimobilis FP2001, was found to have relatively higher α-L-mannosidase activity as compared with three GH78 α-L-Rha-ases. The α-L-mannosidase activity of SpRhaM showed pH dependence, with highest activity observed at pH 7.0. In summary, we have shown that α-L-Rha-ases also have α-L-mannosidase activity. Our findings will be useful in the identification and structural determination of α-L-mannose-containing polysaccharides from natural sources for use in the pharmaceutical and food industries.     

Characterization of Three Fungal Isomaltases Belonging to Glycoside Hydrolase Family 13 That Do not Show Transglycosylation Activity

α-1,6-Glucosidase (isomaltase) belongs to glycoside hydrolase (GH) families 13 and 31. Genes encoding 3 isomaltases belonging to GH family 13 were cloned from filamentous fungi, Aspergillus oryzae (agl1), A. niger (agdC),and Fusarium oxysporum (foagl1), and expressed in Escherichia coli. The enzymes hydrolyzed isomaltose and α-glucosides preferentially at a neutral pH, but did not recognize maltose, trehalose, and dextran. The activity of AgdC and Agl1 was inhibited in the presence of 1 % glucose, while Foagl1 was more tolerant to glucose than the other two enzymes were. The three fungal isomaltases did not show transglycosylation when isomaltose was used as the substrate and a similar result was observed for AgdC and Agl1 when p-nitrophenyl-α-glucoside was used as the substrate.     

The effect of sterilizing doses of γ-irradiation on the molecular weight and emulsification properties of gum arabic

Experiments were performed on raw, kibbled and spray-dried gum arabic. Microbiological tests showed that following a radiation dose of 10 kGy the contaminating micro-organisms were effectively inactivated. The average molecular weight of kibbled and spray dried gum arabic was shown by viscosity measurements to be somewhat reduced as the result of γ-irradiation. No significant radiation-induced changes in the molecular weight of raw gum arabic could be detected. The yields of radiolytically produced strand breaks (breaks/100 eV of radiation absorbed) have been calculated for spray-dried [G(strand breaks) = 4.1 × 10⁻¹] and for kibbled [G(strand breaks) = 2.9 × 10⁻¹]. Gel permeation chromatography confirmed this order of radiation stability and indicated that gum arabic consists of at least three major components, the larger molecular weight fraction appearing to be the more radiation sensitive. There was no measurable change in the total hexuronic acid content (20%) of any of the samples on γ-irradiation. Finally, measurements showed no adverse effect of γ-irradiation, even up to doses of 30 kGy, on the ability of the gum arabic to stabilize emulsions. The extent of chemical change induced by irradiation with sterilizing doses has no practical adverse significance and demonstrates that this technique is suitable for processing raw gum arabic samples.     

Preparation and Analysis of α-1,6 Glucan as a Slowly Digestible Carbohydrate

Carbohydrate materials that produce lower postprandial blood glucose increase are required for diabetic patients. To develop slowly digestible carbohydrates, the effect of degree of polymerization (DP) of α-1,6 glucan on its digestibility was investigated in vitro and in vivo. We prepared four fractions of α-1,6 glucan composed primarily of DP 3–9, DP 10–30, DP 31–150, and DP 151+ by fractionating a dextran hydrolysate. An in vitro experiment using digestive enzymes showed that the glucose productions of DP 3–9, DP 10–30, DP 31–150, and DP 151+ were 70.3, 53.4, 28.2, and 19.2 % in 2 h, and 92.1, 83.9, 39.6, and 33.3 % in 24 h relative to dextrin, respectively. An in vivo glycemic response showed that the incremental area under the curve (iAUC) of blood glucose levels of α-1,6 glucan with DP 3–9, DP 10–30, DP 31–150, and DP 151+ were 99.5, 84.3, 65.4, and 40.1 % relative to dextrin, respectively. These results indicated that α-1,6 glucan with higher DP had stronger resistance to digestion and produced a smaller blood glucose response. DP 10–30 showed significantly lower maximum blood glucose levels than dextrin; however, no significant difference was observed in iAUC, indicating that DP 10–30 was slowly digestible. In addition, α-1,6 glucan was also produced using an enzymatic reaction with dextrin dextranase (DDase). This produced similar results to DP 10–30. The DDase product can be synthesized from dextrin at low cost. This glucan is expected to be useful as a slowly digestible carbohydrate source.     

Rheological behavior of aqueous dispersions of cashew gum and gum arabic: effect of concentration and blending

Rheological properties of cashew gum (CG) and gum arabic (AR), the exudate polysaccharides from Anacardium occidentale L. and Acacia, at different solutions (0.4–50% w/v) were studied. The intrinsic viscosity, [η], of CG in water at 20°C was ≈0.1 dl g⁻¹, while that of AR was ≈0.6 dl g⁻¹. The apparent viscosity of the unheated and the heated (at 80°C for 30 min) CG and AR solutions showed a progressive increase with increasing concentration. The flow curves of blends with equal viscosity solutions of AR/CG: 25/75, 50/50 and 75/25, showed no major interaction. The apparent viscosity (η_a) vs. shear rate data for both the AR and CG dispersions (4–50% w/v) exhibited shear-thinning characteristics at low shear rates (< about 10 s⁻¹) and Newtonian plateaus at shear rates >100 s⁻¹, and the Sisko model described well the η_a vs. data of all the dispersions.     

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.