D-Glucuronic/D-Galacturonic Acid Assay Kit

Background: The level of available carbohydrates in our diet is directly linked to two major diseases; obesity and Type II diabetes. Despite this, to date there is no method available to allow direct and accurate measurement of available carbohydrates in human and animal foods. Objective: The aim of this research was to develop a method that would allow simple and accurate measurement of available carbohydrates, defined as non-resistant starch, maltodextrins, maltose, isomaltose, sucrose, lactose, glucose, fructose and galactose. Method: Non-resistant (digestible) starch is hydrolysed to glucose and maltose by pancreatic α-amylase and amyloglucosidase at pH 6.0 with shaking or stirring at 37°C for 4 h. Sucrose, lactose, maltose and isomaltose are completely hydrolyzed by specific enzymes to their constituent monosaccharides, which are then measured using pure enzymes in a single reaction cuvette. Results: A method has been developed that allows the accurate measurement of available carbohydrates in all cereal, vegetable, fruit, food, and feed products, including dairy products. Conclusions: A single-laboratory validation was performed on a wide range of food and feed products. The inter-day repeatability (%RSDr) was <3.58% (w/w) across a range of samples containing 44.1 to 88.9% available carbohydrates. The LOD and LOQ obtained were 0.054% (w/w) and 0.179% (w/w), respectively. The method is all inclusive, specific, robust and simple to use. Highlights: A unique method has been developed for the direct measurement of available carbohydrates, entailing separate measurement of glucose, fructose and galactose; information of value in determining the glycemic index of foods.

Plant polysaccharides have attracted much attention because of their various biological activities. The structure characterization of polysaccharide from durian seed and its neuroprotective effects against Alzheimer's disease in a transgenic Caenorhabditis elegans model were conducted in this study. A water-soluble polysaccharide was obtained using atmospheric pressure plasma treatment, and named DSPP-1. DSPP-1 was composed of rhamnose, galactose and galacturonic acid and its molecular weight was 3.765×10⁵ Da. The study in vitro showed that DPPH radical scavenging activity of DSPP-1 was 79.20% and the inhibitory rate on Aβ_1-42 aggregation was 24.65%. In vivo results showed that DSPP-1 could decrease abnormal Aβ 1-42 aggregation to delay the paralysis process of AD-nematodes. Moreover, DSPP-1 significantly improved the antioxidant enzyme activities and reduced lipid peroxidation in AD-nematodes. Taken together, these results indicated that DSPP-1 could be used as a potential natural source for the prevention and treatment of AD.

Bacterial Cellulose (BC) is a biopolymer with numerous applications. The growth of BC-producing bacteria, Komagataeibacter intermedius, could be stimulated by Dekkera bruxellensis, however, the effect on BC yield needs further investigation. This study investigates BC production and biochemical changes in the K. intermedius-D. bruxellensis co-culture system. D. bruxellensis was introduced at various concentrations (10³ and 10⁶ CFU/mL) and inoculation times (days 0 and 3). BC yield was ~24% lower when D. bruxellensis was added at 10³ CFU/mL compared to K. intermedius alone (0.63 ± 0.11 g/L). The lowest BC yield was observed when 10³ CFU/mL yeast was added on day 0, which could be compromised by higher gluconic acid production (10.08 g/L). In contrast, BC yields increased by ~88% when 10⁶ CFU/mL D. bruxellensis was added, regardless of inoculation time. High BC yield might correlate with faster sugar consumption or increased ethanol production when 10⁶ CFU/mL D. bruxellensis was added on day 0. These results suggest that cell concentration and inoculation time have crucial impacts on species interactions in the co-culture system and product yield.

Guignardia bidwellii, indigenous to North America, is a significant pathogen of grapes long known in Hungary, infecting only the growing green parts of the vine (leaves, petioles, shoots, and bunches). In the absence of adequate plant protection and extreme weather conditions such as a predominantly humid, warm year, black rot of grapes can be expected. The pathogen can cause high yield losses due to grape rot and reduce wine quality if the infection is severe. The evolution of certain biogenic amine compounds were investigated under the influence of grape black rot. The results obtained showed that they were present in low concentrations from an oenological point of view. Polyphenol composition was consistent with the literature, blackening affected mainly the concentration of catechin. Black rot fungus does not produce β-glucosidase enzyme. In terms of resveratrol content, black rot has no particular effect. However, like Botrytis cinerea, it produces glycerol and, proportionally, gluconic acid in lower concentrations. It can be concluded that black rot of grapes does not cause health problems when introduced into wine processing.

Although there are chemotherapeutic efforts in place for Type 2 diabetes mellitus (T2DM), there is a need for novel strategies (including natural products) to manage T2DM. Fucoidan, a sulphated polysaccharide was extracted from Ecklonia radiata. The integrity of the fucoidan was confirmed by structural analysis techniques such as FT-IR, NMR and TGA. In addition, the fucoidan was chemically characterised and tested for cell toxicity. The fucoidan was investigated with regards to its potential to inhibit α-amylase and α-glucosidase. The fucoidan was not cytotoxic and inhibited α-glucosidase (IC₅₀ 19 µg/mL) more strongly than the standard commercial drug acarbose (IC₅₀ 332 µg/mL). However, the fucoidan lacked potency against α-amylase. On the other hand, acarbose was a more potent inhibitor of α-amylase (IC₅₀ of 109 µg/mL) than α-glucosidase. Due to side effects associated with the use of acarbose, a combination approach using acarbose and fucoidan was investigated. The combination showed synergistic inhibition (>70%) of α-glucosidase compared to when the drugs were used alone. The medicinal implication of this synergism is that a regimen with a reduced acarbose dose may be used, thus minimising side effects to the patient, while achieving the desired therapeutic effect for managing T2DM.

The environmental yeast Cryptococcus neoformans is the most common cause of deadly fungal meningitis in primarily immunocompromised populations. A number of factors contribute to cryptococcal pathogenesis. Among them, inositol utilization has been shown to promote C. neoformans development in nature and invasion of central nervous system during dissemination. The mechanisms of the inositol regulation of fungal virulence remain incompletely understood. In this study, we analyzed inositol-induced capsule growth and the contribution of a unique inositol catabolic pathway in fungal development and virulence. We found that genes involved in the inositol catabolic pathway are highly induced by inositol, and they are also highly expressed in the cerebrospinal fluid of patients with meningoencephalitis. This pathway in C. neoformans contains three genes encoding myo-inositol oxygenases that convert myo-inositol into d-glucuronic acid, a substrate of the pentose phosphate cycle and a component of the polysaccharide capsule. Our mutagenesis analysis demonstrates that inositol catabolism is required for C. neoformans virulence and deletion mutants of myo-inositol oxygenases result in altered capsule growth as well as the polysaccharide structure, including O-acetylation. Our study indicates that the ability to utilize the abundant inositol in the brain may contribute to fungal pathogenesis in this neurotropic fungal pathogen.

The aim of this work was to investigate the use of isobutanol as organic solvent for the efficient delignification and fractionation of beechwood through the OxiOrganosolv process in the absence of any catalyst. The results demonstrate that cellulose-rich solid pulp produced after pretreatment is a source of fermentable sugars that can be easily hydrolyzed and serve as a carbon source in microbial fermentations for the production of omega-3 fatty acids and D-lactic acid. The C5 sugars are recovered in the aqueous liquid fractions and comprise a fraction rich in xylo-oligosaccharides with prebiotic potential. The maximum production of optically pure D-lactic from Lactobacillus delbrueckii sp. bulgaricus reached 51.6 g/L (0.57 g/g_biomass), following a simultaneous saccharification and fermentation strategy. Crypthecodenium cohnii accumulated up to 52.1 wt% lipids with a DHA content of 54.1 %, while up to 43.3 % hemicellulose recovery in form of oligosaccharides was achieved in the liquid fraction.

Bacteroidetes are efficient degraders of complex carbohydrates, much thanks to their use of polysaccharide utilization loci (PULs). An integral part of PULs are highly specialized carbohydrate-active enzymes, sometimes composed of multiple linked domains with discrete functions-multicatalytic enzymes. We present the biochemical characterization of a multicatalytic enzyme from a large PUL encoded by the gut bacterium Bacteroides eggerthii. The enzyme, BeCE15A-Rex8A, has a rare and novel architecture, with an N-terminal carbohydrate esterase family 15 (CE15) domain and a C-terminal glycoside hydrolase family 8 (GH8) domain. The CE15 domain was identified as a glucuronoyl esterase (GE), though with relatively poor activity on GE model substrates, attributed to key amino acid substitutions in the active site compared to previously studied GEs. The GH8 domain was shown to be a reducing-end xylose-releasing exo-oligoxylanase (Rex), based on having activity on xylooligosaccharides but not on longer xylan chains. The full-length BeCE15A-Rex8A enzyme and the Rex domain were capable of boosting the activity of a commercially available GH11 xylanase on corn cob biomass. Our research adds to the understanding of multicatalytic enzyme architectures and showcases the potential of discovering novel and atypical carbohydrate-active enzymes from mining PULs.

Premature skin aging occurs due to the increased formation of reactive oxygen species (ROS), which causes oxidative stress, DNA damage, and collagen degradation. This study investigates the protective effect of Scenedesmus dimorphus polysaccharides (SDP) as an antioxidant and anti-aging agent on an aging rat model induced by D-galactose (D-gal). This study used 48 male Wistar rats divided into six groups: (1) normal control, (2) polysaccharide control, (3) aging control induced by D-gal 0.25 mg/g bw/days, (4) drug control (vitamin E) treated twice with SDP in an aging rat model, (5) D-gal + SDP (0.2 mg/g bw), and (6) D-gal + SDP (0.8 mg/g bw) treated with oral treatment and observed for periods of 2, 4, and 8 weeks. Giving SDP at a dose of 0.8 mg/g bw can increase superoxide dismutases and catalase activity and reduce malondialdehyde after 8 weeks of observation, which is better than giving vitamin E. The treatment of SDP can stimulate collagen synthesis and reduce advanced glycation end products. Histopathology shows an increase in the area of fibrocollagen proportions and deposition from the matrix on giving SDP, which is better than vitamin E, since SDP can repair skin tissue. Thus, SDP can be useful as an antioxidant and anti-aging agent in an aging Wistar rats model.

In this work, a new methodological approach, relying on the high specificity of enzymes in a complex mixture, was developed to estimate the composition of bioactive polysaccharides produced by microalgae, directly in algal cultures. The objective was to set up a protocol to target oligomers commonly known to be associated with exopolysaccharides’ (EPS) nutraceutical and pharmaceutical activities (i.e., rhamnose, fucose, acidic sugars, etc.) without the constraints classically associated with chromatographic methods, while maintaining a resolution sufficiently high to enable their monitoring in the culture system. Determination of the monosaccharide content required the application of acid hydrolysis (2 M trifluoroacetic acid) followed by NaOH (2 M) neutralization. Quantification was then carried out directly on the fresh hydrolysate using enzyme kits corresponding to the main monosaccharides in a pre-determined composition of the polysaccharides under analysis. Initial results showed that the enzymes were not sensitive to the presence of TFA and NaOH, so the methodology could be carried out on fresh hydrolysate. The limits of quantification of the method were estimated as being in the order of the log of nanograms of monosaccharides per well, thus positioning it among the chromatographic methods in terms of analytical performance. A comparative analysis of the results obtained by the enzymatic method with a reference method (high-performance anion-exchange chromatography) confirmed good recovery rates, thus validating the closeness of the protocol. Finally, analyses of raw culture media were carried out and compared to the results obtained in miliQ water; no differences were observed. The new approach is a quick, functional analysis method allowing routine monitoring of the quality of bioactive polysaccharides in algal cultures grown in photobioreactors.

Background: Efficient degradation of lignocellulosic biomass has become a major bottleneck in industrial processes which attempt to use biomass as a carbon source for the production of biofuels and materials. To make the most effective use of the source material, both the hemicellulosic as well as cellulosic parts of the biomass should be targeted, and as such both hemicellulases and cellulases are important enzymes in biorefinery processes. Using thermostable versions of these enzymes can also prove beneficial in biomass degradation, as they can be expected to act faster than mesophilic enzymes and the process can also be improved by lower viscosities at higher temperatures, as well as prevent the introduction of microbial contamination. Results: This study presents the investigation of the thermostable, dual-function xylanase-glucuronoyl esterase enzyme CkXyn10C-GE15A from the hyperthermophilic bacterium Caldicellulosiruptor kristjanssonii. Biochemical characterization of the enzyme was performed, including assays for establishing the melting points for the different protein domains, activity assays for the two catalytic domains, as well as binding assays for the multiple carbohydrate-binding domains present in CkXyn10C-GE15A. Although the enzyme domains are naturally linked together, when added separately to biomass, the expected boosting of the xylanase action was not seen. This lack of intramolecular synergy might suggest, together with previous data, that increased xylose release is not the main beneficial trait given by glucuronoyl esterases. Conclusions: Due to its thermostability, CkXyn10C-GE15A is a promising candidate for industrial processes, with both catalytic domains exhibiting melting temperatures over 70°C. Of particular interest is the glucuronoyl esterase domain, as it represents the first studied thermostable enzyme displaying this activity.

Despite significant efforts recently made to improve the recovery yield of food pigments from natural sources, the development of green and sustainable biotechnological approaches is currently under investigation. Within the context of circular economy, food wastes represent a cheap source for the recovery of valuable compounds including food ingredients. In this study, a conservative approach consisting in a tailored enzyme-assisted extraction protocol for the recovery of carotenoid-containing chromoplasts from unsold tomatoes in which lycopene is stable and protected against oxidation, has now been developed. A tailored enzymatic mix based on polygalacturonase, pectin lyase, cellulase and xylanase, has been designed taking into account the polysaccharide composition of the tomato cell wall. The optimal process conditions for enhancing the recovery of carotenoids from tomatoes i.e.; temperature, pH, enzymatic mix of total dosage and processing time, have been investigated. The suitable temperature and pH identified by the RSM analysis, were found to be: 45-55°C at pH 5-5.5. The treatment carried out using the total dosage of 25 U/g for 180 min was the most convenient for maximizing the recovery yield [about 4.30 ± 0.08 (mg_Lyc/Kg_tomato)/U as carotenoid-containing chromoplasts and about 5.43 ± 0.04 (mg_Lyc/Kg_tomato)/U as total carotenoids].

In this paper, lemon and fennel wastes were recovered and used as secondary-raw polysaccharide sources. These polysaccharides were exploited as natural plasticizers of sodium alginate (A) based films, in order to improve sodium alginate performances, limited by its fragility, extending its potential application in a cost effective and eco-friendly way. Different green processes, such as maceration (MAC), ultrasound assisted extraction (UAE) and microwave assisted extraction (MAE), were carried out for obtaining high yield of lemon and fennel polysaccharides (LP and FP). Actually, HPAE-PAD and TLC analyses evidenced the presence of xyslose, galactose, glucose and rhamnose monomers and galacturonic acid, typical of polysaccharides like pectin and xyloglucan chains. These findings were confirmed by NMR and FTIR spectroscopic analyses. Moreovers, gel filtration chromatography assessed the high molecular weight of recovered polysaccharides, particularly of FP waste fraction. The extracted polysaccharides were used as eco-friendly and cost-effective plasticizers of sodium alginate films (AFP and ALP). DSC analysis evidenced a significant decreasing of glass transition temperature of the polymer, tensile tests showed an enlightened rising of elongation at break and TGA analysis showed a faster degradation kinetics of AFP and ALP films, as expected in a plasticized system.

Anthocyanins determine the color and potential health-promoting properties of red fruit juices, but the juices contain remarkably less anthocyanins than the fruits, which is partly caused by the interactions of anthocyanins with the residues of cell wall polysaccharides like pectin. In this study, pectin was modified by ultrasound and enzyme treatments to residues of polysaccharides and oligosaccharides widely differing in their molecular weight. Modifications decreased viscosity and degrees of acetylation and methylation and released smooth and hairy region fragments. Native and modified pectin induced different effects on the concentrations of individual anthocyanins after short-term and long-term incubation caused by both hydrophobic and hydrophilic interactions. Results indicate that both pectin and anthocyanin structure influence these interactions. Linear polymers generated by ultrasound formed insoluble anthocyanin complexes, whereas oligosaccharides produced by enzymes formed soluble complexes with protective properties. The structure of the anthocyanin aglycone apparently influenced interactions more than the sugar moiety.

Glucuronoyl esterases (GEs) catalyze the cleavage of ester linkages found between lignin and glucuronic acid moieties on glucuronoxylan in plant biomass. As such, GEs represent promising biochemical tools in industrial processing of these recalcitrant resources. However, details on how GEs interact with their natural substrates are sparse, calling for thorough structure-function studies. Presented here is the structure and biochemical characterization of a GE, TtCE15A, from the bacterium Teredinibacter turnerae, a symbiont of wood-boring shipworms. To gain deeper insight into enzyme-substrate interactions, inhibition studies were performed with both the WT TtCE15A and variants in which we, by using site-directed mutagenesis, substituted residues suggested to have key roles in binding to or interacting with the aromatic and carbohydrate structures of its uronic acid ester substrates. Our results support the hypothesis that two aromatic residues (Phe-174 and Trp-376), conserved in bacterial GEs, interact with aromatic and carbohydrate structures of these substrates in the enzyme active site, respectively. The solved crystal structure of TtCE15A revealed features previously not observed in either fungal or bacterial GEs, with a large inserted N-terminal region neighboring the active site and a differently positioned residue of the catalytic triad. The findings highlight key interactions between GEs and complex lignin-carbohydrate ester substrates and advance our understanding of the substrate specificities of these enzymes in biomass conversion.

The effects of different plant seed mucilage (PSM) extracts, namely chia seed (CSM) and flaxseed (FSM), on the kinetics of δ-glucono-lactone induced acidification and gelation phenomena of whey proteins (5% w/w WPI) were investigated. The rheological and microstructural properties of mixed whey protein-PSM (0.05-0.75% w/w) cold-set gels produced at 30 or 37°C were studied by means of oscillatory rheology and confocal microscopy. On exceeding 0.125% of PSM, a significant reduction of the gelation time due to the formation of loosely entangled whey protein soluble aggregates was observed. The impact of PSM on the gelation rates was closely related to the PSM type and concentration. CSM addition induced a gradual reduction of maximal gelation rate over the entire concentration range tested. On the other hand, FSM conferred a steep impedance of the gelation when exceeded 0.375%, which was associated with the occurrence of segregative phase separation. Fitting the elastic modulus - gelation time data to a model adapted to the Flory-Stockmayer theory, it was demonstrated that the presence of PSM inhibits the whey protein crosslinking capacity under both tested acidification regimes, leading to the formation of shorter protein crosslinks and therefore, to lower gel stiffness. However, the formation rate of elastically active chain networks was found to be increasing for CSM and FSM contents up to 0.5 and 0.25% respectively, suggesting a synergistic acid gel structuring effect of PSM under these conditions.

The glucuronoyl esterases (GEs) that have been identified so far belong to family 15 of the carbohydrate esterases in the CAZy classification system and are presumed to target ester bonds between lignin alcohols and (4-O-methyl-)D-glucuronic acid residues of xylan. Few GEs have been cloned, expressed and characterised to date. Characterisation has been done on a variety of synthetic substrates; however, the number of commercially available substrates is very limited. We identified novel putative GEs from a wide taxonomic range of fungi and expressed the enzymes originating from Acremonium alcalophilum and Wolfiporia cocos as well as the previously described PcGE1 from Phanerochaete chrysosporium. All three fungal GEs were active on the commercially available compounds benzyl glucuronic acid (BnGlcA), allyl glucuronic acid (allylGlcA) and to a lower degree on methyl glucuronic acid (MeGlcA). The enzymes showed pH stability over a wide pH range and tolerated 6-h incubations of up to 50°C. Kinetic parameters were determined for BnGlcA. This study shows the suitability of the commercially available model compounds BnGlcA, MeGlcA and allylGlcA in GE activity screening and characterisation experiments. We enriched the spectrum of characterised GEs with two new members of a relatively young enzyme family. Due to its biotechnological significance, this family deserves to be more extensively studied. The presented enzymes are promising candidates as auxiliary enzymes to improve saccharification of plant biomass.

Glucuronic acid and/or 4-O-methyl-glucuronic acid (GlcA/MeGlcA) are substituents of the main xylans present in hardwoods, conifers, and many cereal grains. α-Glucuronidases from glycoside hydrolase family GH115 can target GlcA/MeGlcA from both internally and terminally substituted regions of xylans. The current study describes the first GH115 α-glucuronidase, AxyAgu115A, from the alkaliphilic organism Amphilbacillus xylanus. AxyAgu115A was active in a wide pH range, and demonstrated better performance in alkaline condition compared to other characterized GH115 α-glucuronidases, which generally show optimal activity in acidic conditions. Specifically, its relative activity between pH 5.0 and pH 8.5 was above 80%, and was 35% of maximum at pH 10.5; although the enzyme lost 30% and 80% relative residual activity after 24-h pre-incubation at pH 9 and pH 10, respectively. AxyAgu115A was also similarly active towards glucuronoxylan as well as comparatively complex xylans such as spruce arabinoglucurunoxylan. Accommodation of complex xylans was supported by docking analyses that predicted accessibility of AxyAgu115A to branched xylo-oligosaccharides. MeGlcA release by AxyAgu115A from each xylan sample was increased by up to 30% by performing the reaction at pH 11.0 rather than pH 4.0, revealing applied benefits of AxyAgu115A for xylan recovery and processing.

4-O-Methyl-D-glucuronic acid (MeGlcA) is a side-residue of glucuronoarabinoxylan and can form ester linkages to lignin, contributing significantly to the strength and rigidity of the plant cell wall. Glucuronoyl esterases (4-O-methyl-glucuronoyl methylesterases, GEs) can cleave this ester bond, and therefore may play a significant role as auxiliary enzymes in biomass saccharification for the production of biofuels and biochemicals. GEs belong to a relatively new family of carbohydrate esterases (CE15) in the CAZy database (www.cazy.org), and so far around ten fungal GEs have been characterized. To explore additional GE enzymes, we used a genome mining strategy. BLAST analysis with characterized GEs against approximately 250 publicly accessible fungal genomes identified more than 150 putative fungal GEs, which were classified into eight phylogenetic sub-groups. To validate the genome mining strategy, 21 selected GEs from both ascomycete and basidiomycete fungi were heterologously produced in Pichia pastoris. Of these enzymes, 18 were active against benzyl D-glucuronate demonstrating the suitability of our genome mining strategy for enzyme discovery.

The kinetic characteristics of Rhizopus oryzae endo-polygalacturonase, RPG1, hydrolyzing galacturonic acid oligomers (GalpA)_n were determined. RPG1 generates (GalpA)₃ as a dominant product of polygalacturonic acid and (GalpA)_4-6 hydrolysis. The enzyme can hydrolyze (GalpA)₃, but hydrolysis occurs at a significantly lower rate relative to oligomers with a higher degree of polymerization. Hydrolysis of the α-1,4 glycosidic bond by RPG1 is an endothermic process with a δH_app, of 1.03±0.04 kcal/mol. Determination of kinetic constants by isothermal titration calorimetry showed that for oligomers (GalpA)_3-6, the K_m decreased and the K_cat increased as the length of the (GalpA) oligomer increased. Fixed time point assays followed by chromatographic analysis provided apparent K_cat values similar to those found using isothermal titration calorimetry. Assays to determine to what extent the enzyme is subject to product inhibition demonstrated that the enzyme is competitively inhibited by (GalpA)₂ when using (GalpA)₄ as substrate. The apparent K_i of 767 µM is significantly higher than the K_m values obtained for the series of galacturonic acid oligomers.