β-Galactosidase (Aspergillus niger)

Background: In recent years there has been a surge in the number of commercially available lactose‐free variants of a wide variety of products. This presents an analytical challenge for the measurement of the residual lactose content in the presence of high levels of mono‐, di‐, and oligosaccharides. Results: In the current work, we describe the development of a novel enzymatic low‐lactose determination method termed LOLAC (low lactose), which is based on an optimized glucose removal pre‐treatment step followed by a sequential enzymatic assay that measures residual glucose and lactose in a single cuvette. Sensitivity was improved over existing enzymatic lactose assays through the extension of the typical glucose detection biochemical pathway to amplify the signal response. Selectivity for lactose in the presence of structurally similar oligosaccharides was provided by using a β-galactosidase with much improved selectivity over the analytical industry standards from Aspergillus oryzae and Escherichia coli (EcLacZ), coupled with a ‘creep’ calculation adjustment to account for any overestimation. The resulting enzymatic method was fully characterized in terms of its linear range (2.3-113 mg per 100 g), limit of detection (LOD) (0.13 mg per 100 g), limit of quantification (LOQ) (0.44 mg per 100 g) and reproducibility (≤ 3.2% coefficient of variation (CV)). A range of commercially available lactose‐free samples were analyzed with spiking experiments and excellent recoveries were obtained. Lactose quantitation in lactose‐free infant formula, a particularly challenging matrix, was carried out using the LOLAC method and the results compared favorably with those obtained from a United Kingdom Accreditation Service (UKAS) accredited laboratory employing quantitative high performance anion exchange chromatography - pulsed amperometric detection (HPAEC‐PAD) analysis. Conclusion: The LOLAC assay is the first reported enzymatic method that accurately quantitates lactose in lactose‐free samples.

Procedures for the measurement of starch, starch damage (gelatinised starch), resistant starch and the amylose/amylopectin content of starch, β-glucan, fructan, glucomannan and galactosyl-sucrose oligosaccharides (raffinose, stachyose and verbascose) in plant material, animal feeds and foods are described. Most of these methods have been successfully subjected to interlaboratory evaluation. All methods are based on the use of enzymes either purified by conventional chromatography or produced using molecular biology techniques. Such methods allow specific, accurate and reliable quantification of a particular component. Problems in calculating the actual weight of galactosyl-sucrose oligosaccharides in test samples are discussed in detail.

An American Association of Cereal Chemists/AOAC collaborative study was conducted to evaluate the accuracy and reliability of an enzyme assay kit procedure for measurement of total starch in a range of cereal grains and products. The flour sample is incubated at 95 degrees C with thermostable alpha-amylase to catalyze the hydrolysis of starch to maltodextrins, the pH of the slurry is adjusted, and the slurry is treated with a highly purified amyloglucosidase to quantitatively hydrolyze the dextrins to glucose. Glucose is measured with glucose oxidase-peroxidase reagent. Thirty-two collaborators were sent 16 homogeneous test samples as 8 blind duplicates. These samples included chicken feed pellets, white bread, green peas, high-amylose maize starch, white wheat flour, wheat starch, oat bran, and spaghetti. All samples were analyzed by the standard procedure as detailed above; 4 samples (high-amylose maize starch and wheat starch) were also analyzed by a method that requires the samples to be cooked first in dimethyl sulfoxide (DMSO). Relative standard deviations for repeatability (RSD(r)) ranged from 2.1 to 3.9%, and relative standard deviations for reproducibility (RSD(R)) ranged from 2.9 to 5.7%. The RSD(R) value for high amylose maize starch analyzed by the standard (non-DMSO) procedure was 5.7%; the value was reduced to 2.9% when the DMSO procedure was used, and the determined starch values increased from 86.9 to 97.2%.

The elucidation of the structural characteristics of polysaccharides from natural sources is generally difficult owing to their structural complexity and heterogeneity. In our previous study, an immuno-stimulatory polysaccharide (RGP-AP-I) was isolated from Korean red ginseng (Panax ginseng C.A. Meyer). The present study aims to elucidate the structural characteristics of RGP-AP-I. Sequential enzyme hydrolysis was performed using four specific glycosylases, and chemical cleavage via β-elimination was carried out to determine the fine structure of RGP-AP-I. The degraded fragments were chemically identified using various chromatographic and spectrometric analyses, including HPLC-UVD, GC–MS, and tandem mass spectrometry. The results indicated that RGP-AP-I comprises a rhamnogalacturonan I (RG-I) backbone with repeating disaccharide units [→2)-Rhap-(1 → 4)-GalAp-(1→] and three side chains substituted at the C(O)4 position of the rhamnose residue in the backbone. The three side chains were identified as a highly branched α-(1 → 5)-arabinan, a branched β-(1 → 4)-galactan, and an arabino-β-3,6-galactan. Our results represent the first findings regarding the fine structure of the immuno-stimulatory polysaccharide RG-AP-I isolated from red ginseng.

The charophycean green algae (CGA or basal streptophytes) are of particular evolutionary significance because their ancestors gave rise to land plants. One outstanding feature of these algae is that their cell walls exhibit remarkable similarities to those of land plants. Xyloglucan (XyG) is a major structural component of the cell walls of most land plants and was originally thought to be absent in CGA. This study presents evidence that XyG evolved in the CGA. This is based on a) the identification of orthologs of the genetic machinery to produce XyG, b) the identification of XyG in a range of CGA and, c) the structural elucidation of XyG, including uronic acid-containing XyG, in selected CGA. Most notably, XyG fucosylation, a feature considered as a late evolutionary elaboration of the basic XyG structure and orthologs to the corresponding biosynthetic enzymes are shown to be present in Mesotaenium caldariorum.

Potato cell walls (PCW) are a low value by-product from the potato starch industry. Valorisation of PCW is hindered by its high water-binding capacity (WBC). The composition of polysaccharides and interactions between these entities, play important roles in regulating the WBC in the cell wall matrix. Here, we show that in vivo exo-truncation of RG-I β-(1→4)-D-galactan side-chains decreased the WBC by 6-9%. In contrast, exo-truncation of these side-chains increased the WBC by 13% in vitro. We propose that degradation of RG-I galactan side-chains altered the WBC of PCW, due to cell wall remodelling and loosening that affected the porosity. Our findings reinforce the view that RG-I galactan side-chains play a role in modulating WBC, presumably by affecting polysaccharide architecture (spacing) and interactions in the matrix. Better understanding of structure-function relationships of pectin macromolecules is needed before cell wall by-products may be tailored to render added-value in food and biobased products.

Rhamnogalacturonan I (RG-I), one of the pectic components of the plant cell wall, is composed of a backbone of repeating disaccharide units of rhamnose and galacturonic acid, and side chains, such as galactans, arabinans, and arabinogalactans. The activity of RG-I galactosyltransferase, which transfers galactosyl residues to rhamnosyl residues in the RG-I backbone, has not been detected until now. Here, we detected galactosyltransferase activity in azuki bean epicotyls using fluorogenic RG-I oligosaccharide acceptors. This enzyme prefers oligosaccharides with a degree of polymerization more than 9. The enzyme activity was detected in the Golgi apparatus, which is the site of pectin synthesis. In vitro hyperactivation of this enzyme was also observed. Moreover, enzyme activity was increased up to 40-fold in the presence of cationic surfactants or polyelectrolytes.

A rapid mass spectrometry method for determining the anomeric configuration of the sugar at the reducing end of an oligosaccharide was demonstrated. The method was employed to identify the nascent anomeric configuration (i.e., before significant mutarotation occurs) of oligosaccharides released by carbohydrate-active enzymes, which enabled determination of the enzyme mechanism. This method was validated by applying it to various enzymes, including α-glucosidase, β-glucosidases, endoglycoceramidase II, β-galactosidase, and β-amylase.

Lactose conversion by lactic acid bacteria is of high industrial relevance and consistent starter culture quality is of outmost importance. We observed that Lactococcus lactis using the high-affinity lactose-phosphotransferase system excreted galactose towards the end of the lactose consumption phase. The excreted galactose was re-consumed after lactose depletion. The lacS gene, known to encode a lactose permease with affinity for galactose, a putative galactose–lactose antiporter, was upregulated under the conditions studied. When transferring cells from anaerobic to respiration-permissive conditions, lactose-assimilating strains exhibited a long and non-reproducible lag phase. Through systematic preculture experiments, the presence of galactose in the precultures was correlated to short and reproducible lag phases in respiration-permissive main cultivations. For starter culture production, the presence of galactose during propagation of dairy strains can provide a physiological marker for short culture lag phase in lactose-grown cultures.

The conversion of whey permeates to galacto-oligosaccharides (GOS) was studied by the enzymatic action of β-galactosidase from Aspergillus oryzae in a continuous flow packed-bed reactor. A novel method of enzyme immobilization involving covalent immobilization of β-galactosidase on 3-aminopropyl triethoxysilane(3-APTES)-modified glass beads was developed by the cross-linking method. The pH and temperature dependence of the enzymatic efficiency of the glass bead-immobilized enzyme was compared with that of the free enzyme. Increased pH and thermal stabilities were observed for the immobilized enzyme versus the free enzyme. The reusability of the enzyme immobilized packed-bed reactor was studied and only about 4.6% of GOS yield was lost after 8 reuses. Repeated cycle reactions were also carried out to improve the formation of GOS. The results showed that the GOS formation increased and a maximum GOS yield of 39.3% with 56.4% lactose conversion was obtained after the 2nd cycle of passing.

Considering the potential applications of partially degalactosylated xyloglucans as a drug delivery vehicle and reconstruction of tissues, the aim of this study was to investigate whether degalactosylated xyloglucans are immunologically active. The effects of the degalactosylated xyloglucan from seeds of Copaifera langsdorffii (XGCd), Hymenaea courbaril (XGJd), and Tamarindus indica (XGTd) on murine peritoneal macrophages in vitro were evaluated. XGCd, XGJd, and XGTd stimulated NO production in a dose-dependent manner reaching ~280% for XGTd at 50 µg/mL. Regarding cytokines production, XGJd at 50 µg/mL increased IL-1β level by ~100% and XGCd (10 µg/mL) enhanced IL-6 level by 40%. At 10 µg/mL, XGTd increased TNF- α and IL-1β levels by 104 and 2370%, respectively, as compared to the control group. For IL-6, XGTd enhanced this cytokine production by 80% at all concentrations tested. XGTd exhibited the most intensive effects on the production of pro-inflammatory mediators by peritoneal macrophages. All degalactosylated xyloglucans evaluated showed not to be biologically inert. Thus, this finding is relevant for groups that are investigating the use of degalactosylated xyloglucan from T. indica for drug delivery and reconstruction of tissues. The effects observed could contribute to potentiate the immune system against infections or toxicity to tumor cells.

Gelation kinetics of aqueous solutions of xyloglucan (XG) extracted from H. courbaril seeds were investigated, in-situ, during enzymatic removal of galactose units by oscillatory shear rheological measurements, at different XG and enzyme (β-galactosidase) concentrations. Increasing the enzyme concentration (C_enz) led to an increase of the gelation rate. Master curves of the evolution of the storage shear modulus at different C_enz could be formed by time-C_enz superposition showing that C_enz influenced the kinetics, but not the gelation process and the final gel stiffness. The behaviour of gels formed by XG with different molar mass (M_w), prepared by endoglucanase hydrolysis, was evaluated as a function of the temperature. It was found that cooling led to a decrease of the crosslink density causing a decrease of the gel stiffness. The decrease of the crosslink density was sufficient to depercolate the network formed by relatively small XG with M_w = 10⁵ g mol⁻¹, but gels formed by XG with Mw ≥ 8 × 10⁵ g mol⁻¹ persisted down to 10°C. It is shown that the melting temperature and the gel stiffness at high temperatures can be controlled independently by varying the molar mass and the concentration of XG chains.

Prebiotics are selectively fermented by the gastrointestinal microflora, resulting in benefits to human health. The seed mucilage of Hyptis suaveolens contains neutral and acidic polysaccharides in a ratio of 1:1. The neutral polysaccharides consist of galactose, glucose and mannose whereas the acidic polysaccharides contain fucose, xylose and 4-O-methylglucuronic acid -residues. The growth of probiotics in the presence of total, acidic or neutral polysaccharides and oligosaccharides was tested using turbidity measurements. The majority (11 out of 14) of the tested probiotic strains significantly grew in the neutral fraction. Growth occurred with some time delay, but may be longer lasting than with other lower molecular prebiotics. The extent of growth increased with neutral polysaccharides from H. suaveolens corresponding to the externally available galactose units (20%). In conclusion, neutral poly- and oligosaccharides from H. suaveolens have a prebiotic potential characterized by a delayed but long lasting effect.

An improved method for direct determination of available carbohydrates in low-level products has been developed and validated for a low-carbohydrate soy infant formula. The method involves modification of an existing direct determination method to improve specificity, accuracy, detection levels, and run times through a more extensive enzymatic digestion to capture all available (or potentially available) carbohydrates. The digestion hydrolyzes all common sugars, starch, and starch derivatives down to their monosaccharide components, glucose, fructose, and galactose, which are then quantitated by high-performance anion-exchange chromatography with photodiode array detection. Method validation consisted of specificity testing and 10 days of analyzing various spike levels of mixed sugars, maltodextrin, and corn starch. The overall RSD was 4.0 across all sample types, which contained within-day and day-to-day components of 3.6 and 3.4, respectively. Overall average recovery was 99.4 (n = 10). Average recovery for individual spiked samples ranged from 94.1 to 106 (n = 10). It is expected that the method could be applied to a variety of low-carbohydrate foods and beverages.

A Brazilian source of galactoxyloglucan was obtained from seeds of Hymenaea courbaril and investigated before (JN) and after the hydrolytic removal of galactose (JH). The native polysaccharide contained glucose, xylose and galactose in a ~ 4.0:2.7:1 molar ratio. Gelation occurred when ~ 51% of the galactose residues was removed from the xyloglucan by fungal β-galactosidase. The rheological properties of JN and JH were analyzed. Dynamic oscillatory measurements characterized JN as a viscoelastic solution and JH as a true gel. JH achieved the maximum storage modulus G′ at temperatures above 40°C and was found to increase with the increasing concentration. The gel point temperatures of JH fraction were determined at the cross-over point of G′ and G″ and by calculating the inflexion point of the G′ plot.

An arabinogalactan protein (F2) was isolated in 1.5% yield from the seeds of Ribes nigrum L. (Grossulariaceae) by aqueous extraction and a one-step anion exchange chromatography on DEAE-Sephacel with 24% galactose, 43% arabinose, and 20% xylose as main carbohydrate residues. Methylation analysis revealed the presence of a 1,3-/1,3,6-galactose backbone, side chains from arabinose in different linkages, and terminal xylose residues. The polysaccharide which turned out to be an arabinogalactan protein had a molecular weight of >10₆ Da and deaggregated under chaotropic conditions. The cellular dehydrogenase activities (MTT and WST-1 tests) of human skin cells (fibroblasts, keratinocytes) as well as the proliferation rate of keratinocytes (BrdU incorporation ELISA) were significantly stimulated by the polymer at 10 and 100 µg/mL. F2 had no influence on differentiation status of keratinocytes and did not exhibit any cytotoxic potential (LDH test). The biological activity of F2 was not dependent on the high molecular weight. Influence of the polysaccharide on the gene expression of specific growth factors, growth factor receptors, signal proteins and marker proteins for skin cell proliferation, and differentiation by RT-PCR could not be shown. Gene array investigations indicated an increased expression of various genes encoding for catabolic enzymes, DNA repair, extracellular matrix proteins, and signal transduction factors. Removal of terminal arabinose residues by α-L-arabinofuranosidase did not influence the activity toward skin cells, while the treatment with β-D-galactosidase yielded an inactive polysaccharide. The FITC-labeled polysaccharide was incorporated in a time-dependent manner into human fibroblasts (laser scanning microscopy) via endosomal transport. This internalization of the polysaccharide was inhibited by Cytochalasin B.

Attractive color is one of the most important sensory characteristics of fruit and berry products, and elderberry juice is widely used as natural colorant. When pectinase preparations were used in the production of elderberry juice for clarification, a concomitant decrease of anthocyanins and thus a color loss were observed. This paper demonstrates that this is due to side glycosidase activities contained in commercial pectinase preparations from Aspergillus sp. Using LC-MS, sequential deglycosylation of cyanidin-3-sambubioside, cy-3-glucoside, cy-3-sambubioside-5-glucoside, and cy-3,5-diglucoside was found to be catalyzed by specific glycosidases contained in the pectinase preparations. There was no big difference in the deglycosylation rate between monoglucosidic or diglucosidic anthocyanins. However, the degradation rate was decreased when rutinose was attached to cyanidin, whereas the structure of the aglycone itself had almost no influence. Pure β-glucosidases from Agrobacterium species and Aspergillus niger and the β-glucosidase N188 from A. niger did not show any conversion of anthocyanins, indicating the presence of specific glycosidases. Thus, an activity gel based assay was developed to detect anthocyanin-specific glycosidase activity in enzyme preparations, and according to LC-MS peptide mass mapping of digested bands, homologies to a β-glucosidase from Aspergillus kawachii were found.

A library of phenyl β-glycosides of xylogluco-oligosaccharides was synthesized via a chemoenzymatic approach to produce new, specific substrates for xyloglucanases. Tamarind xyloglucan was completely hydrolyzed to four, variably galactosylated component oligosaccharides based on Glc₄ backbones, using a Trichoderma endo-glucanase mixture. Oligosaccharide complexity could be further reduced by β-galactosidase treament. Subsequent per-O-acetylation, α-bromination, phase-transfer glycosylation, and Zemplén deprotection yielded phenyl glycosides of XXXG and XLLG oligosaccharides with a broad range of aglycon pK_a values. Kinetic and product analysis of the action of the archetypal plantendo-xyloglucanase, Tropaeolum majus NXG1, on these compounds indicated that formation of the glycosyl−enzyme intermediate was rate-limiting in the case of phenol leaving groups with pK_a values of >7, leading exclusively to substrate hydrolysis. Conversely, substrates with aglycon pK_a values of 5.4 gave rise to a significant amount of transglycosylation products, indicating a change in the relative rates of formation and breakdown of the glycosyl−enzyme intermediate for these faster substrates. Notably, comparison of the initial rates of XXXG-Ar and XLLG-Ar conversion indicated that catalysis by TmNXG1 was essentially insensitive to the presence of galactose in the negative subsites for all leaving groups. More broadly, analysis of a selection of enzymes from CAZy families GH 5, 12, and 16 indicated that the phenyl glycosides are substrates for anomeric configuration-retaining endo-xyloglucanases but are not substrates for strict xyloglucan endo-transglycosylases (XETs). The relative activities of the GH 5, 12, and 16 endo-xyloglucanases toward GGGG-CNP, XXXG-CNP, and XLLG-CNP reflected those observed using analogous high molar mass polysaccharides. These new chromogenic substrates may thus find wide application in the discovery, screening, and detailed kinetic analysis of new xyloglucan-active enzymes.

The substrate specificity of the xyloglucanase Cel74A from Hypocrea jecorina (Trichoderma reesei) was examined using several polysaccharides and oligosaccharides. Our results revealed that xyloglucan chains are hydrolyzed at substituted Glc residues, in contrast to the action of all known xyloglucan endoglucanases (EC 3.2.1.151). The building block of xyloglucan, XXXG (where X is a substituted Glc residue, and G is an unsubstituted Glc residue), was rapidly degraded to XX and XG (K_cat = 7.2 s^-1 and K_m = 120 µm at 37°C and pH 5), which has only been observed before with the oligoxyloglucan-reducing-end-specific cellobiohydrolase from Geotrichum (EC 3.2.1.150). However, the cellobiohydrolase can only release XG from XXXGXXXG, whereas Cel74A hydrolyzed this substrate at both chain ends, resulting in XGXX. Differences in the length of a specific loop at subsite + 2 are discussed as being the basis for the divergent specificity of these xyloglucanases.

The enzymatic degradation of the plant cell wall is central both to the natural carbon cycle and, increasingly, to environmentally friendly routes to biomass conversion, including the production of biofuels. The plant cell wall is a complex composite of cellulose microfibrils embedded in diverse polysaccharides collectively termed hemicelluloses. Xyloglucan is one such polysaccharide whose hydrolysis is catalyzed by diverse xyloglucanases. Here we present the structure of the Clostridium thermocellum xyloglucanase Xgh74A in both apo and ligand-complexed forms. The structures, in combination with mutagenesis data on the catalytic residues and the kinetics and specificity of xyloglucan hydrolysis reveal a complex subsite specificity accommodating seventeen monosaccharide moieties of the multibranched substrate in an open substrate binding terrain.