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.     

Molecular Design and Synthesis of a Novel Substrate for Assaying Lysozyme Activity

A novel substrate {Galβ1,4GlcNAcβ1,4GlcNAc-β-pNP [Gal(GlcNAc)₂-β-pNP]} for assaying lysozyme activity has been designed using docking simulations and enzymatic synthesis via β-1,4-galactosyltransferase-mediated transglycosylation from UDP-Gal as the donor to (GlcNAc)₂-β-pNP as the acceptor. Hydrolysis of the synthesized Gal(GlcNAc)₂-β-pNP and related compounds using hen egg-white lysozyme (HEWL) demonstrated that the substrate was specifically cleaved to Gal(GlcNAc)₂ and p-nitrophenol (pNP). A combination of kinetic studies and docking simulation was further conducted to elucidate the mode of substrate binding. The results demonstrate that Gal(GlcNAc)₂-β-pNP selectively binds to a subsite of lysozyme to liberate the Gal(GlcNAc)₂ and pNP products. The work therefore describes a new colorimetric method for quantifying lysozyme on the basis of the determination of pNP liberated from the substrate.     

Enzymatic synthesis of primeverosides using transfer reaction by Trichoderma longibrachiatum xylanase

Enzymatic synthesis of two phenyl xylopyranosyl glucopyranosides, through transfer reaction by Trichoderma longibrachiatum endoxylanase, was achieved in the presence of n-hexane used as solvent, phenyl glucoside (10 mM) as acceptor and xylan (2 g/l) as donor. Kinetic study showed that only one compound, identified by ¹H and ¹³C NMR and heteronuclear 2D (¹H–¹³C) chemical shift correlation as phenyl primeveroside (phenyl 6-O-β-xylopyranosyl-1-β-d-glucopyranoside), was synthesized when the reaction time was beyond 1 h. Benzyl and hexyl primeverosides were obtained under the same conditions. When several phenyl glucoside concentrations, from 5 to 50 mM, were used with 2 g/l of xylan, a phenyl primeveroside isomer, identified as phenyl 4-O-β-xylopyranosyl-β-d-glucopyranoside, accumulated in the medium whereas the production of phenyl primeveroside decreased. Only phenyl primeveroside was produced when several xylan concentrations from 2 to 10 g/l were used with 10 mM of phenyl glucoside and its concentration in the reaction mixture increased with the increase of xylan concentration.     

Extraction of water-soluble xylan from wheat bran and utilization of enzymatically produced xylooligosaccharides by Lactobacillus, Bifidobacterium and Weissella spp.

Xylan was extracted from wheat bran after heat pretreatment in water using either an autoclave or a microwave oven. Xylooligosaccharides (XOS) were produced from the xylan using the thermostable xylanase RmXyn10A and the potential prebiotic properties of XOS were studied in vitro with different human gut bacteria: Lactobacillus brevis (DSMZ 1269), Bifidobacterium adolescentis (ATCC 15703) and two strains of recently isolated lactic acid bacteria from the species pair Weissella cibaria/confusa. The highest yield of (arabino)xylan with the heat pretreatment was obtained at 185 °C for 10 min. Higher temperature led to fewer arabinose substitutions present on the backbone which in turn resulted in a slightly more efficient enzymatic hydrolysis by RmXyn10A.     

Acetylated Xylan Degradation by Glycoside Hydrolase Family 10 and 11 Xylanases from the White-rot Fungus Phanerochaete chrysosporium

Endo-type xylanases are key enzymes in microbial xylanolytic systems, and xylanases belonging to glycoside hydrolase (GH) families 10 or 11 are the major enzymes degrading xylan in nature. These enzymes have typically been characterized using xylan prepared by alkaline extraction, which removes acetyl sidechains from the substrate, and thus the effect of acetyl groups on xylan degradation remains unclear. Here, we compare the ability of GH10 and 11 xylanases, PcXyn10A and PcXyn11B, from the white-rot basidiomycete Phanerochaete chrysosporium to degrade acetylated and deacetylated xylan from various plants. Product quantification revealed that PcXyn10A effectively degraded both acetylated xylan extracted from Arabidopsis thaliana and the deacetylated xylan obtained by alkaline treatment, generating xylooligosaccharides. In contrast, PcXyn11B showed limited activity towards acetyl xylan, but showed significantly increased activity after deacetylation of the xylan. Polysaccharide analysis using carbohydrate gel electrophoresis showed that PcXyn11B generated a broad range of products from native acetylated xylans extracted from birch wood and rice straw, including large residual xylooligosaccharides, while non-acetylated xylan from Japanese cedar was readily degraded into xylooligosaccharides. These results suggest that the degradability of native xylan by GH11 xylanases is highly dependent on the extent of acetyl group substitution. Analysis of 31 fungal genomes in the Carbohydrate-Active enZymes database indicated that the presence of GH11 xylanases is correlated to that of carbohydrate esterase (CE) family 1 acetyl xylan esterases (AXEs), while this is not the case for GH10 xylanases. These findings may imply co-evolution of GH11 xylanases and CE1 AXEs.     

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.     

Structural characterization of barley β-glucan extracted using a novel fractionation technique

A barley β-glucan concentrate prepared according to a novel technology was further purified and subjected to detailed structural characterization by NMR spectroscopy. β-Glucan was hydrolysed with β-glucan-4-glucanohydrolase (lichenase). Fractions of hydrolysate were collected using an HPLC-fraction collector. Intact β-glucan and the major fractions collected were subjected to MALDI-TOF–MS and NMR analyses. The two major oligosaccharides produced by lichenase hydrolysis of purified barley β-glucan were identified as β-d-Glc p-(1 → 4)- β-d-Glc p-(1 → 3)-β-d-Glc p and β-d-Glc p-(1 → 4)-β-d-Glc p-(1 → 4)-β-d-Glc p-(1 → 3)-β-d-Glc p based on ¹³C and ¹H NMR data. Spectrums were similar to those documented for barley β-glucan in the literature.     

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.     

Control of pH by CO 2 Pressurization for Enzymatic Saccharification of Ca(OH) 2 -Pretreated Rice Straw in the Presence of CaCO 3

The aim of this study was to investigate the effect of pH control by CO ₂ pressurization on the enzymatic hydrolysis of herbaceous feedstock in the calcium capturing by carbonation (CaCCO) process for fermentable sugar production. The pH of the slurry of 5 % (w/w) Ca(OH) ₂ -pretreated/CO ₂ -neutralized rice straw could be controlled between 5.70 and 6.38 at 50 °C by changing the CO ₂ partial pressure ( p CO ₂ ) from 0.1 to 1.0 MPa. A mixture of fungal enzyme preparations, namely, Trichoderma reesei cellulases/hemicellulases and Aspergillus niger β-glucosidase, indicated that pH 5.5–6.0 is optimal for solubilizing sugars from Ca(OH) ₂ -pretreated rice straw. Enzymatic saccharification of pretreated rice straw under various p CO ₂ conditions revealed that the highest soluble sugar yields were obtained at p CO ₂ 0.4 MPa and over, which is consistent with the expected pH at the p CO ₂ without enzymes and demonstrates the effectiveness of pH control by CO ₂ pressurization.     

α-Anomer-selective glucosylation of (+)-catechin by the crude enzyme, showing glucosyl transfer activity, of Xanthomonas campestris WU-9701

α-Anomer-selective glucosylation of (+)-catechin was carried out using the crude enzyme, showing α-glucose transferring activity, of Xanthomonas campestris WU-9701 with maltose as a glucosyl donor. When 60 mg of (+)-catechin and 50 mg of the enzyme (5.25 units as maltose hydrolysing activity) were incubated in 10 ml of 10 mM citrate-Na₂HPO₄ buffer (pH 6.5) containing 1.2 M maltose at 45°C, only one (+)-catechin glucoside was selectively obtained as a product. The (+)-catechin glucoside was identified as (+)-catechin 3′-O-α- -glucopyranoside (α-C-G) by ¹³C-NMR, ¹H-NMR and two-dimensional HMBC analysis. The reaction at 45°C for 36 h under the optimum conditions gave 12 mM α-C-G, 5.4 mg/ml in the reaction mixture, and the maximum molar conversion yield based on the amount of (+)-catechin supplied reached 57.1%. At 20°C, the solubility in pure water of α-C-G, of 450 mg/ml, was approximately 100 fold higher than that of (+)-catechin, of 4.6 mg/ml. Since α-C-G has no bitter taste and a slight sweet taste compared with (+)-catechin which has a very bitter taste, α-C-G may be a desirable additive for foods, particularly sweet foods.     

Formation and stability of α-lactalbumin–lysozyme spherical particles: Involvement of electrostatic forces

The formation and the subsequent stability of spherical microparticles resulting from the self-assembly between two oppositely charged proteins, lysozyme (LYS) and apo α-lactalbumin (apo α-LA), at pH 7.5 and 45 °C were studied under different physico-chemical conditions—ionic strength, type of salts, type of buffer. Increasing the ionic strength reduced the ability of the two proteins to interact together and to form microspheres. The formation of such microparticles was completely abolished at an ionic strength of 100 mM. Increasing salt concentration also allowed destabilisation and dissociation of formed microspheres in salt-dependent manner as assessed by turbidity experiments and microscopic observations. Microparticles were destabilised with either NaCl, MgCl₂ or CaCl₂, but the latter showed the greatest destabilising effect. The higher efficiency of calcium ions in the destabilisation experiment could be attributed to the presence of specific calcium binding site on α-LA. Interestingly, complete disappearance of formed particles was not reached even after adding salt concentrations as high as 250 mM of NaCl or 7.5 mM of divalent cations. Our results suggest that electrostatic interactions are clearly involved in the first events of the two-protein assembly and spherical particles building. Moreover, non-electrostatic forces are also involved in maintaining the integrity and stability of formed microparticles.     

Comparative NMR relaxometry of gels of amylomaltase-modified starch and gelatin

With the aim of generating gelatin-like starch gel functionality, starches extracted from normal potato, high amylose potato, maize, waxy maize, wheat and pea and oxidized potato starch were modified with amylomaltase (AM) (4-α-glucanotransferase; E.C. 2.4.1.25) from Thermus thermophilus. Gel characteristics after storage for 1 and 10 days at 20 °C of 12.0% gels were assessed by monitoring proton relaxation for the resulting 51 enzyme-modified starches and two gelatins using low-field ¹H nuclear magnetic resonance (LF NMR) relaxometry. Discrete and distributed exponential analysis of the Carr–Purcell–Meiboom–Gill (CPMG) LF NMR relaxation data revealed that the pastes and gels contained one water component and that the spin–spin relaxation time constants (T₂) and distributions differed with respect to starch type and enzyme modification. Typically, AM modification resulted in starches with decreased T₂ relaxation time and a more narrow T₂ distribution indicating a more homogeneous water population. In contrast, treatment with a branching enzyme (BE) (EC 2.4.1.18) combined with AM increased T₂ relaxation time and a broadened T₂ distribution. As evaluated by the principal component analysis (PCA), long chains of amylopectin generated hard gels and decreased T₂ relaxation time at both day 1 and day 10. Especially at day 10, T₂ relaxation time could be predicted from the amylopectin chain length (CL) distribution. Reconstructed amylopectin CL distribution required to emulate gelatin LF NMR data suggest the importance of combined fractions of long (DP 60–80) and short (DP 10–25) amylopectin chains.     

Structural features of the polysaccharide gum from Acacia glomerosa

Acacia glomerosa, Benth. (Vulgares Series) exudates a clear gum which produces gels easily. The physico-chemical data and sugar composition are very close to gum arabic from Acacia senegal, except that A. glomerosa gum contains a high nitrogen content. A series of degraded products was prepared by acid hydrolysis and Smith-degradation. The characterization of the degraded products by partial hydrolysis, sugar composition and by the application of uni- and bidimensional spectroscopy led to know interesting structural features of the polysaccharide isolated from A. glomerosa gum. This polysaccharide, as that from A. senegal gum, consists of 1,3-β- -galactopyranosyl backbone. There are side-chains of 1,3-β- -galactopyranosyl oligosaccharides attached to position six of the galactan main chain. Arabinose (furanosyl and pyranosyl) residues may be up to four units long because it was necessary to prepare four polysaccharides to remove them from the gum structure. Uronic acid residues were difficult to remove as has been observed in other Acacia gums.     

The effect of MgCl2 on the kinetics of the Maillard reaction in both aqueous and dehydrated systems

The modification of Maillard reaction kinetics induced by MgCl₂ was evaluated in both liquid and dehydrated model systems with special emphasis on the interactions of the salt with water and/or the sugars. In liquid trehalose systems, browning is accelerated by the presence of MgCl₂ due to the increased sugar hydrolysis and to the reduction of water mobility caused by the salt (shown by the decrease in ¹H NMR relaxation times T₂), counteracting the inhibitory effect of water on the reaction. In water restricted trehalose systems, MgCl₂ inhibited the Maillard reaction. The salt–sugar interactions, manifested by the delayed sugar crystallization, decreased the reaction rate by affecting the reactivity of reducing sugars. Molecular and supramolecular effects in the presence of MgCl₂ have been observed in the present work, and must be taken into account considering high technological interest in finding strategies to either inhibit or enhance the Maillard reaction depending on the application.     

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.     

Non-binding property of cathepsin L to myosin

Disodium pyrophosphate at 10 mM concentration, was effective in dissociating myosin and actin from actomyosin in walleye pollock (Theragra chalcogramma) surimi and red bulleye (Priacanthus macracanthus) surimi. After Sepharose 2B gel filtration, cathepsin L contained in the actomyosin was obviously non-binding to myosin. Actomyosin from carp (Cyprinus carpio) muscle was not dissociated in pyrophosphate solution in the absence of MgCl₂ and it was successfully dissociated by 10 mM pyrophosphate in the presence of 2 mM MgCl₂. Cathepsin L in carp actomyosin was shown to be much more complicated than that in the above two surimis. After Sepharose 2B gel filtration, there were two activity peaks of cathepsin L in carp, one almost corresponding with actomyosin, the other obviously separated from actomyosin. Both of the peaks were non-binding to myosin.     

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.     

The effects of different inorganic salts,buffer systems,and desalting of Varthemia crude water extract on DPPH radical scavenging activity

The DPPH radical-scavenging activity of 25 inorganic salts, two buffer systems, and crude water extract of aerial parts of Varthemia (Varthemia iphionoides) before and after resins purification were investigated. Eight of the 25 inorganic salts tested quenched the DPPH radical colour. Na₂S₂O₃ and FeCl₂ showed markedly high DPPH colour-quenching activity, with inhibition of 65.3% and 47.7% respectively, at a concentration of 10 μg/ml. Four salts slightly increased the intensity of DPPH radical colour. The rest of tested salts, acetate buffer, and phosphate buffer at a concentration less than 0.1 mM did not affect DPPH radical colour. The DPPH radical-scavenging activity of BHT and catechol was considerably affected by the concentration of phosphate buffer (pH 7.0), and by acetate buffer (pH 5.0) at concentrations more than 0.01 mM in the case of BHT only. The DPPH radical-scavenging activity of a crude water extract of aerial parts of Varthemia iphionoides was much higher than that of an extract desalted by cation-exchange resin, indicating that iron ions apparently elevated the DPPH radical-scavenging activity of the extract. Therefore, desalting of plant extracts is important in order to obtain the true value of DPPH radical-scavenging activity.     

Isolation and evaluation of the antioxidant activity of phenolic constituents of the Garcinia brasiliensis epicarp

A new glycosylated biflavonone, morelloflavone-4′″-O-β-d-glycosyl, and the known compounds 1,3,6,7-tetrahydroxyxanthone, morelloflavone (fukugetin) and morelloflavone-7″-O-β-d-glycosyl (fukugeside) were isolated from the epicarp of Garcinia brasiliensis collected in Brazil. The structures of these compounds were established using ¹H and ¹³C NMR, COSY, gHMQC and gHMBC spectroscopy. The compounds exhibited antioxidant activity. The greatest potency was displayed by morelloflavone (2), with IC₅₀ = 49.5 mM against DPPH and absorbance of 0.583 at 400 μg/mL for the reduction of Fe³⁺. The weakest potency was displayed by 1,3,6,7-tetrahydroxyxanthone (1), with IC₅₀ = 148 mM against DPPH and absorbance of 0.194 at 400 μg/mL for the reduction of Fe³⁺.