Search Results (101)

Pectin is a dynamic and complex polysaccharide that forms a substantial proportion of the primary plant cell wall and middle lamella of forage ingested by grazing ruminants. Pectin methylesterases (PMEs) are enzymes that belongs to the carbohydrate esterase family 8 (CE8) and catalyze the demethylesterification of pectin, a key polysaccharide in cell walls. Here we present the crystal structure of the catalytic domain of PmeC5 that is associated with a gene from Butyrivibrio fibrisolvens D1^T that encodes a large secreted pectinesterase family protein (2089 aa) determined to a resolution of 1.33 Å. Protein in silico modelling of the secreted pectinesterase confirmed the presence of an additional pectate lyase (PL9) and adhesin-like domains. The structure of PmeC5 was the characteristic right-handed parallel β-helical topology and active site residues of Asp231, Asp253, and Arg326 typical of the enzyme class. PmeC5 is a large modular enzyme that is characteristic of rumen B. fibrisolvens megaplasmids and plays a central role in degrading plant cell wall components and releasing methanol in the rumen environment. Such secreted PMEs are significant contributors to plant fiber digestion and methane production, making them attractive targets for both methane mitigation strategies and livestock productivity enhancement. Full article

Background/Objectives: Alginate and its oligosaccharides (AOS) are widely used in the food industry all over the world. However, how they are fermented by the human gut microbiota has not been fully elucidated. Here, we aim to explore the structure–property relationships of the fermentation of these carbohydrates by the human gut microbiota. Methods: High-performance liquid chromatography, 16S rRNA gene amplicon high-throughput sequencing, whole genome sequencing, and metabolome analysis were used to study the fermentation of alginate and AOS by the human gut microbiota. Results and Conclusions: Low-molecular-weight alginate and AOS were more fermentable than alginate. Moreover, fermentation of AOS with a molecular weight (Mw) of 0.8 kDa produced higher amounts of acetate and butyrate than that with a Mw of 0.3 kDa. B. xylanisolvens was a keystone species responsible for the fermentation. Additionally, each B. xylanisolvens strain was characterized with a unique capability for AOS fermentation. Specifically, B. xylanisolvens P19-10, a bacterium isolated from healthy human colon, exhibited the best fermentation capacity. Genomic analysis suggested that B. xylanisolvens P19-10 was armed with a plethora of carbohydrate-active enzymes. Additionally, the polysaccharide lyase family 6_1 was identified as a candidate enzyme responsible for the utilization of AOS. Moreover, fermentation of AOS by B. xylanisolvens P19-10 was associated with significant changes in bacterial metabolites and metabolic pathways. Future perspectives: Our study provides novel mechanistic insights into the fermentation of alginate and AOS by human gut microbiota, which has applications for the development of new carbohydrate-based nutraceuticals and foods. Full article

Soda lakes are extreme saline–alkaline environments that harbor metabolically versatile microbial communities with significant biotechnological potential. This study employed shotgun metagenomics (NovaSeq PE150) to investigate the functional diversity and metabolic potential of microbial communities in Ethiopia’s Chitu and Shala Lakes. An analysis of gene content revealed 554,609 and 525,097 unique genes in Chitu and Shala, respectively, in addition to a substantial fraction (1,253,334 genes) shared between the two, underscoring significant functional overlap. Taxonomic analysis revealed a diverse phylogenetic composition, with bacteria (89% in Chitu Lake, 92% in Shala Lake) and archaea (4% in Chitu Lake, 0.8% in Shala Lake) as the dominant domains, alongside eukaryotes and viruses. Predominant bacterial phyla included Pseudomonadota, Actinomycetota, and Gemmatimonadota, while Euryarchaeota and Nitrososphaerota were prominent among archaea. Key genera identified in both lakes were Nitriliruptor, Halomonas, Wenzhouxiangella, Thioalkalivibrio, Aliidiomarina, Aquisalimonas, and Alkalicoccus. Functional annotation using the KEGG, eggNOG, and CAZy databases revealed that the identified unigenes were associated with various functions. Notably, genes related to amino acid, carbohydrate, and energy metabolism (KEGG levels 1–2) were predominant, indicating that conserved core metabolic functions are essential for microbial survival in extreme conditions. Higher-level pathways included quorum sensing, two-component signal transduction, and ABC transporters (KEGG level 3), facilitating environmental adaptation, stress response, and nutrient acquisition. The eggNOG annotation revealed that 13% of identified genes remain uncharacterized, representing a vast untapped reservoir of novel enzymes and biochemical pathways with potential applications in biofuels, bioremediation, and synthetic biology. This study identified 375 unique metabolic pathways, including those involved in pyruvate metabolism, xenobiotic degradation, lipid metabolism, and oxidative stress resistance, underscoring the microbial communities’ ability to thrive under fluctuating salinity and alkalinity. The presence of carbohydrate-active enzymes (CAZymes), such as glycoside hydrolases, polysaccharide lyases, and oxidoreductases, highlights their role in biomass degradation and carbon cycling. Enzymes such as alkaline proteases (Apr), lipases (Lip), and cellulases further support the lakes’ potential as sources of extremophilic biocatalysts. These findings position soda lakes as reservoirs of microbial innovation for extremophile biotechnology. Future research on unannotated genes and enzyme optimization promises sustainable solutions in bioenergy, agriculture, and environmental management. Full article

Lactic acid bacteria (LAB) is a collective term for bacteria capable of producing lactic acid from fermentable carbohydrates. Despite their widespread presence in the gastrointestinal tracts of humans and animals, where they play important physiological roles, functional analysis of specific strains from particular sources requires further enrichment. The objective of this study was to explore the differences between Pediococcus acidilactici OM681363 and Lacticaseibacillus paracasei ON606241, both isolated from the rumen of Chinese Holstein dairy cows, using whole-genome sequencing. The results indicate that P. acidilactici OM681363 contained three CRISPR fragments and numerous enzymes involved in carbohydrate degradation. Additionally, P. acidilactici OM681363 possessed more genes related to fiber degradation, especially cellobiose, and the sole carbon source experiment also confirmed this. However, it lacked genes associated with polysaccharide lyase. In contrast, L. paracasei ON606241 was found to be more specialized in breaking down non-fiber carbohydrates, producing more acetic and lactic acids. Overall, P. acidilactici OM681363 may have a greater capacity to degrade complex carbohydrates, while L. paracasei ON606241 appears to specifically target non-fiber carbohydrates. Full article

Osmotic stress impacts the cell wall properties in plants. This study aimed to elucidate the mechanisms involved in cell wall remodeling in etiolated (dark-grown) rice (Oryza sativa L.) shoots grown under polyethylene glycol (PEG)-induced osmotic stress conditions. Shoot growth was inhibited by 70% by the treatment with 60 mM PEG for 2 days. However, when the stressed seedlings were transferred to a solution without PEG, their shoot growth rate increased significantly. A measurement of the cell wall mechanical properties revealed that the cell walls of the stressed shoots became looser and more extensible than those of unstressed shoots. Among the cell wall constituents, the amounts of cell wall-bound phenolic acids, such as ferulic acid (FA), p-coumaric acid (p-CA), and diferulic acid (DFA), per shoot and per unit of matrix polysaccharide content were significantly reduced in the stressed shoots compared to those in the unstressed shoots. Concerning the formation of cell wall-bound phenolic acids, the activity of cell wall-bound peroxidase (CW-PRX) per unit of cell wall content, which is responsible for the coupling reaction of FA to produce DFA, was 3.5 times higher in stressed shoots than in unstressed shoots, while the activity was reduced by 20% on a shoot basis in stressed shoots compared to that in unstressed shoots. The expression levels of the major class III peroxidase genes in stressed shoots were either comparable to or slightly lower than those in unstressed shoots. Conversely, the phenylalanine ammonia-lyase (PAL) activity, which contributes to the biosynthesis of FA and p-CA, was reduced by 55% and 30% on a shoot and unit-of-protein-content basis, respectively, in stressed shoots compared to that in unstressed shoots. The expression levels of abundantly expressed PAL genes decreased by 14–46% under osmotic stress. Moreover, the gene expression levels of specific BAHD acyltransferases, which are responsible for the addition of FA and p-CA to form ester-linked moieties on cell wall constituents, decreased by 15–33% under osmotic stress. These results suggest that the downregulation of the expression of specific PAL and BAHD acyltransferase genes in osmotically stressed rice shoots is responsible for a reduction in the formation of cell wall-bound phenolic acid monomers. This, in turn, may result in a decrease in the levels of DFAs. The reduction in the formation of DFA-mediated cross-linking structures within the cell wall may contribute to an increase in the mechanical extensibility of the cell wall. The remodeling of cell walls in an extensible and loosened state could assist in maintaining the growth capacity of etiolated rice shoots grown under osmotic stress and contribute to rapid growth recovery following the alleviation of osmotic stress. Full article

The recovery of polysaccharides (PS) from red grape marc and white grape pomace by enzymatic degradation of their cell walls is an interesting green extraction technique that preserves the structure and bioactivity of PS. The type and dose of enzyme, and the liquid/solid (L/S) ratio in PS extraction were studied using four commercial enzymes. Four different doses per enzyme were used, with tartaric acid as solvent and L/S ratios of 1.3/1 and 4/1 for 24 h at 20 °C, compared with a control. The highest dose of enzyme E1, polygalacturonase + pectin lyase + pectin-methyl-esterase (with the highest activity) was the most effective in the degradation of high and medium molecular weight PS. At the lower L/S ratio, the fact that the highest dose of E1 degraded a higher percentage of high and medium molecular weight PS in the marc was explained by the difference in cell wall deconstruction between pomace and marc. The highest total PS purity was achieved in pomace with E1 at the maximum dose in both ratios, and in marc at the 1.3/1 ratio. The extraction efficiency of total PS was low for all enzymes. In the future, extraction with E1 combined with other green extraction techniques will be studied. Full article

Sugar beet pulp is a byproduct of white sugar production, and it is quite significant in terms of volume. Every year, tens of millions of tons of beet pulp are produced around the world. However, only a fraction of it is currently used, mainly as animal feed. The composition of beet pulp includes plant polysaccharides, such as cellulose, arabinan, and pectin. Through the process of enzymatic hydrolysis, these polysaccharides are converted into technical C6/C5 sugars, which can be further used as a substrate for the microbial synthesis of various substances, including biofuels, organic acids, and other green chemistry molecules. The current study was designed with a primary objective that focused on the development of a strain that had the potential for enhanced productivity and the capacity to produce enzymes suitable for beet pulp hydrolysis. The pelA and abfA genes, which encode pectin lyase and arabinofuranosidase, respectively, in the fungus Penicillium canescens (VKPM F-178), were cloned and successfully expressed in the recipient strain Penicillium verruculosum B1-537 (VKPM F-3972D). New recombinant strains were created using the expression system of the mycelial fungus P. verruculosum B1-537, which is capable of simultaneously producing pectin lyase and arabinofuranosidase, as well as homologous cellulases. The screening of strains for increased enzymatic activity towards citrus pectin, sugar beet branched arabinan, and microcrystalline cellulose revealed that a B4 clone of P. verruculosum exhibited the greatest potential in sugar beet pulp cake hydrolysis. This clone was selected as the basis for the creation of a new enzyme preparation with enhanced pectin lyase, arabinase, and cellulase activities. The component composition of the enzyme preparation was determined, and the results indicated that the enzyme content comprised approximately 11% pectin lyase, 40% arabinofuranosidase, and 40% cellulases. The primary products of the enzymatic hydrolysis of the unpretreated beet pulp cake were arabinose and glucose. The degree of arabinan and cellulose conversion was observed to be up to 50% and 80%, respectively, after a period of 48 to 72 h of hydrolysis. The new B4 preparation was observed to be highly efficacious in the hydrolysis of beet cake at elevated concentrations of solids (up to 300 g/L) within the reaction mixture. The newly developed strain, as a producer of pectin lyase, arabinofuranosidase, and cellulase complexes, has the potential to be utilized for the bioconversion of sugar beet processing wastes and for the efficient generation of highly concentrated solutions of technical sugars for further implementation in processes of microbial synthesis. Full article

Green algae, particularly Ulva species, are rich in complex polysaccharides, such as ulvan, which have significant potential for biotechnological applications. However, the biochemical properties of ulvan depolymerised products remain underexplored. The enzymatic depolymerisation of ulvan has garnered attention owing to its cost advantages over alternative methods. Nevertheless, the biochemical characterisation of ulvan lyases, specifically those belonging to the polysaccharide lyase family 25 (PL25), is limited. In this study, we identified and biochemically characterised a novel PL25 ulvan lyase, PaUL25, which functions optimally at pH 10. Additionally, we explored the alpha (α)-glucosidase inhibitory properties of ulvan depolymerised products. PaUL25 exhibited optimum activity at 35 °C in Tris-HCl buffer (pH 10). Moreover, enzyme activity was enhanced by more than 150% in the presence of Mn²⁺ metal ions at and below concentrations of 10 mM. The endolytic action of PaUL25 produced ulvan oligosaccharides with degrees of polymerisation of 2 and 4 as its end products. Partially and completely hydrolysed ulvan oligosaccharides exhibited α-glucosidase inhibitory activity, with half inhibitory concentration IC₅₀ values of 3.21 ± 0.13 and 2.51 ± 0.19 mg/mL, respectively. These findings expand our understanding of PL25 and highlight the pharmaceutical potential of ulvan oligosaccharides, particularly as antidiabetic agents. Full article

Hemp fibers, recognized for their breathability, specific strength, and ultraviolet resistance, are widely utilized in textile manufacturing and composite materials. Bio-degumming is a promising alternative technology to traditional chemical degumming that can be used to produce hemp fibers due to its eco-friendly nature. However, its lower efficiency has hindered its widespread adoption. The unclear and complex structure of the gums leads to a poor understanding on the enzyme types required for bio-degumming, thereby restricting improvements in its efficiency. In this study, the morphological characteristics, polysaccharide composition, and branched structure of hemp stem, roving fibers, and refined fibers were investigated using scanning electron microscopy and laser scanning confocal microscopy in combination with immunofluorescence techniques, with a view to identify the enzymes necessary for the efficient bio-degumming of hemp. The results revealed that the gums were primarily located in the middle lamella, phloem parenchyma, and certain xylem tissues. These tissues showed chunk-like, fence-like, and plate-like shapes, respectively, and tightly wrapped around the fiber bundles. In these tissues, pectin comprised low-esterified homogalacturonan, along with rhamnogalacturonan carrying galactan and arabinan branches. Xylan exhibited acetyl, arabinose, and glucuronic acid branches, while mannan displayed acetyl and galactose branches. Partial xylan and mannan were masked by pectin, and the branching structures impeded their enzymatic removal. As a consequence, the necessary enzymes and their synergistic effects for effective hemp roving degumming were elucidated. Pectin degradation was facilitated by pectate lyase and rhamnogalacturonan-degrading enzymes. Xylan and mannan were effectively removed by endo-xylanase and endo-mannanase, a process necessitating the synergistic action of branched-chain-degrading enzymes, including the esterase, α-L-arabinofuranosidase, α-galactosidase, and α-glucuronidase. This study provided practical strategies to enhance the efficiency of hemp bio-degumming. Full article

Marine bacteria are crucial sources of alginate lyases, which play an essential role in alginate oligosaccharide (AOS) production. This study reports the biochemical characteristics of a new species of the Microbulbifer genus, Microbulbifer sp. HZ11. The strain HZ11 is Gram-negative, aerobic, flagellate-free, and rod-shaped. The genome of strain HZ11 is a 4,248,867 bp circular chromosome with an average GC content of 56.68%. HZ11 can degrade alginate and other polysaccharides. The carbohydrate-active enzyme (CAZyme) genes account for 4.57% of the total protein-coding genes of HZ11. Its alginate metabolism process is consistent with the characteristics of the polysaccharide utilization locus (PUL) system. The alginate lyase produced by strain HZ11 showed the highest activity at 50 °C, pH 8.5, and 0.1 M NaCl. The substrate preference was as follows: sodium alginate > poly mannuronic acid > poly guluronic acid. The thin layer chromatography (TLC) results revealed that the main enzymatic degradation products were monosaccharides or AOSs with a degree of polymerization (DP) of 2–3. These results help clarify the metabolism and utilization mechanism of alginate by marine bacteria and provide a theoretical reference for its application in the degradation of alginate and other polysaccharides. Full article

Alginate, a polysaccharide found in brown seaweeds, has regularly gained attention for its potential use as a source of bioactive compounds. However, it is structurally complex with a high molecular weight, limiting its application. Alginate oligosaccharides (AOS) are small, soluble fragments, making them more bioavailable. Alginate hydrolysis by enzymes is the preferred method for AOS production. Commercially available alginate lyases are limited, expensive, and sometimes exhibit unsatisfactory activity, making the search for novel alginate lyases with improved activity indispensable. The aims of this study were to codon-optimise, synthesise, express, purify, and characterise a recombinant alginate lyase, AL2, from Flammeovirga sp. strain MY04 and to compare it to a commercial alginate lyase. Expression was successfully performed using Escherichia coli ArcticExpress (DE3) RP cells, and the protein was purified through affinity chromatography. The recombinant enzyme was characterised by pH optimum studies, and temperature optimum and stability experiments. The optimal reaction conditions for AL2 were pH 9.0 and 37 °C, while for the commercial enzyme, the optimal conditions were pH 8.0 and 37 °C. At optimal reaction conditions, the specific activity of AL2 was 151.6 ± 12.8 µmol h⁻¹ mg⁻¹ protein and 96.9 ± 13.1 µmol h⁻¹ mg⁻¹ protein for the commercial alginate lyase. Moreover, AL2 displayed impressive activity in breaking down alginate into AOS. Hence, AL2 shows potential for use as an industrial enzyme for the hydrolysis of alginate into alginate oligosaccharides. Additional studies should be carried out to further characterise this enzyme, improve its purity, and optimise its activity. Full article

Relatively little is known about enzymes with broad substrate spectra, leading to limited applications and progress. Herein, we elucidate Aly16-1 of Streptomyces sp. strain CB16 as a novel multifunctional member of the eighth polysaccharide lyase (PL8) family, although it shared few sequence identities with the characterized enzymes. The recombinant enzyme rAly16-1 showed lyase activities against several acidic polysaccharides, including many glycosaminoglycan types, xanthan, and alginate. It was mannuronate (M)-preferred, endolytic, and optimal at 50 °C and pH 6.0. The smallest substrate was an ∆M-terminal (∆: unsaturated monosaccharide) trisaccharide, and the minimal product was ∆. In the final alginate digestions by rAly16-1, the fractions larger than disaccharides were ∆G-terminal (G: guluronate), while the disaccharides were mainly ∆M, showing an oligosaccharide-yielding property under the succession law. However, when degrading various oligosaccharides, rAly16-1 continued producing ∆M from the non-reducing end even when the substrates increased their sizes, quite different from the elucidated alginate lyases with variable alginate-degrading modes. Thus, co-determined by its M-preference, Aly16-1 is novel for its ∆M-yielding property in oligosaccharide preparations. Additionally, rAly16-1 can be applied in sequencing unsaturated trisaccharides, whether ∆M- or ∆G-terminal. This study provides novel insights into the characteristics and applications of a multifunctional enzyme within the PL8 family for resource explorations. Full article

Immobilized enzyme reactors (IMERs) are critical tools for developing novel oligosaccharides based on the enzymatic catalysis of polysaccharides. In this paper, a novel glucuronan lyase from Peteryoungia rosettiformans was produced, purified, and then immobilized on a CIM^® CDI disk for cleaving glucuronan. The results showed that around 63.6% of glycuronan lyases (800.9 μg) were immobilized on the disk. The V_max values of immobilized glucuronan lyases did not significantly change (56.9 ± 4.7 μM∙min⁻¹), while the K_m values (0.310 ± 0.075 g∙L⁻¹) increased by 2.5 times. It is worth noting that immobilized glucuronan lyases overcame the catalytic inhibition of free enzymes observed under high glucuronan concentrations (0.5–2 g∙L⁻¹). circumscribed central composite design (CCCD) and response surface methodology (RSM) showed that glucuronan concentration, flow rate, and reaction time significantly affected the yield of oligoglucuronans. The degree of polymerization (DP) of degraded glucuronan ranged from DP 2–8 according to the results obtained by high performance anion exchange chromatography coupled with a pulsed amperometric detector (HPAEC-PAD). The IMER retained 50.9% activity after running 2373 column volumes of glucuronan. Finally, this glucuronan lyase reactor was tentatively connected to an immobilized laccase reactor to depolymerize, and gallic acid (GA) was added to glucuronan. Approximately 8.5 mg of GA was added onto 1 g of initial glucuronan, and the GA–oligoglucuronan conjugates showed notable antioxidant activity. Full article

Approximately one-third of the entire world’s food resources are deemed to be wasted. Palm kernel meal (PKM), a product that is extensively generated by the palm oil industry, exhibits a unique nutrient-rich composition. However, its recycling is seldom prioritized due to numerous factors. To evaluate the impact of enzymatic pretreatment and Lactobacillus plantarum and Lactobacillus reuteri fermentation upon the antioxidant activity of PKM, we implemented integrated metagenomics and metabolomics approaches. The substantially enhanced (p < 0.05) property of free radicals scavenging, as well as total flavonoids and polyphenols, demonstrated that the biotreated PKM exhibited superior antioxidant capacity. Non-targeted metabolomics disclosed that the Lactobacillus fermentation resulted in substantial (p < 0.05) biosynthesis of 25 unique antioxidant biopeptides, along with the increased (p < 0.05) enrichment ratio of the isoflavonoids and secondary metabolites biosynthesis pathways. The 16sRNA sequencing and correlation analysis revealed that Limosilactobacillus reuteri, Pediococcus acidilactici, Lacticaseibacillus paracasei, Pediococcus pentosaceus, Lactiplantibacillus plantarum, Limosilactobacillus fermentum, and polysaccharide lyases had significantly dominated (p < 0.05) proportions in PMEL, and these bacterial species were strongly (p < 0.05) positively interrelated with antioxidants peptides. Fermented PKM improves nutritional value by enhancing beneficial probiotics, enzymes, and antioxidants and minimizing anti-nutritional factors, rendering it an invaluable feed ingredient and gut health promoter for animals, multifunctional food elements, or as an ingredient in sustainable plant-based diets for human utilization, and functioning as a culture substrate in the food sector. Full article

Ulvan is a water-soluble sulfated polysaccharide extracted from the green algae cell wall. Compared with polysaccharides, oligosaccharides have drawn increasing attention in various industries due to their enhanced biocompatibility and solubility. Ulvan lyase degrades polysaccharides into low molecular weight oligosaccharides through the β-elimination mechanism. The elucidation of the structure, catalytic mechanism, and molecular modification of ulvan lyase will be helpful to obtain high value-added products from marine biomass resources, as well as reduce environmental pollution caused by the eutrophication of green algae. This review summarizes the structure and bioactivity of ulvan, the microbial origin of ulvan lyase, as well as its sequence, three-dimensional structure, and enzymatic mechanism. In addition, the molecular modification of ulvan lyase, prospects and challenges in the application of enzymatic methods to prepare oligosaccharides are also discussed. It provides information for the preparation of bioactive Ulva oligosaccharides through enzymatic hydrolysis, the technological bottlenecks, and possible solutions to address these issues within the enzymatic process. Full article