Search Results (98)

Alginate, a major polysaccharide in brown algae, is vital for the carbon cycling of the ocean ecosystem and holds promise for biotechnological applications. Marine Bacteroidetes, known for the ability to degrade complex polysaccharides, play an important role in the ocean carbon cycle; however, the detailed alginate degradation pattern remains to be further explored. In this study, an alginate utilization locus was identified in the genome of a new marine Bacteroidetes, Roseihalotalea indica gen. nov. sp. nov. TK19036^T, and encodes two new alginate lyases, RiAlyPL6 and RiAlyPL17, which play potential roles in the degradation and utilization of alginate. RiAlyPL6 and RiAlyPL17 have distinct degradation products and substrate preferences, revealing the adaptation of the strain to utilize alginate with different M/G ratios. Based on the results in this paper, we have proposed a model for the degradation and utilization mechanism of alginate in Roseihalotalea indica gen. nov. sp. nov. TK19036^T. All in all, our research provides a new insight into the alginate mechanisms within marine Roseihalotalea, and the two novel alginate lyases are excellent candidates for preparation and application. Full article

The objective of this study was the evaluation of fungal solid-state fermentation (SSF) for the production of alginate lyase and extraction of uronic acids from Sargassum sp. For this purpose, the fungi Trichoderma asperellum, Aspergillus oryzae, and Rhizopus oryzae were applied (alone or combined) to Sargassum sp. biomass through SSF (10⁷ spores g_biomass⁻¹, 30 °C, and 7 days of treatment). In general, individual SSF with all three fungi degraded the biomass, achieving a marked synergy in the production of cellulase, laminarinase, and alginate lyase activities (especially for the last one). Trichoderma was the most efficient species in producing laminarinase, whereas Rhizophus was the best option for producing alginate lyase. However, when dual combinations were tested, the maximal values of alginate lyase activities were reached (13.4 ± 0.2 IU g_biomass⁻¹ for Aspergillus oryzae and Rhizopus oryzae). Remarkably, uronic acids were the main monomeric units from algal biomass solubilization, achieving a maximum yield of 14.4 mg_uronic g_biomass⁻¹, with the A + R condition being a feasible, eco-friendly alternative to chemical extraction of this monomer. Additionally, the application of all the fungal pretreatments drastically decreased the total phenolic content (TPC) in the biomass from 369 mg L⁻¹ to values around 44–84 mg L⁻¹, minimizing the inhibition for possible subsequent biological processes in which the residual solid can be used. Full article

Alginate lyases are critical enzymes in hydrolyzing alginate into alginate oligosaccharides (AOS), which are bioactive compounds known for their antioxidant properties and ability to lower serum glucose and lipid concentrations. However, elucidating catalytic mechanisms and discovering enzymes with enhanced catalytic efficiency remain long-term challenges. Here, we report AlgL2491, a novel bifunctional and cold-adapted alginate lyase from Pseudoalteromonas carrageenovora ASY5, belonging to the polysaccharide lyase family 18. This enzyme uniquely cleaves both polyguluronic (polyG) and polymannuronic (polyM), predominantly releasing disaccharides, trisaccharides, and tetrasaccharides after 12 h of hydrolysis. The enzyme achieves peak catalytic efficiency at 35 °C and pH 7.5, with activity increasing 5.5-fold in 0.5 M of NaCl. Molecular dynamics simulations demonstrate that salt ions enhance structural stability by minimizing conformational fluctuations and strengthening interdomain interactions, providing mechanistic insights into its salt-activated behavior. The alginate oligosaccharides (AOS) exhibit excellent free radical-scavenging activities of 86.79 ± 0.31%, 83.42 ± 0.18%, and 71.28 ± 2.27% toward hydroxyl, ABTS, and DPPH radicals, with IC50 values of 8.8, 6.74, and 9.71 mg/mL, respectively. These findings not only reveal the salt-activation mechanism of AlgL2491 and highlight the potential value of its hydrolysate in antioxidant activity but also provide a sustainable industrial solution in industrial-scale AOS production directly from marine biomass, eliminating the need for energy-intensive desalination of alginate, which may inform future biocatalyst design for marine polysaccharide valorization. Full article

Kelp (Laminaria japonica) is renowned for its rich content of flavor-enhancing amino acids and nucleotides; however, approximately 40% of kelp, including the thin edges and root areas, is discarded during its processing due to its inferior taste. To recycle these kelp byproducts, we have cultivated a functional microbial consortium through continuous enrichment. Analysis via 16S rRNA sequencing has shown that during the three fed-batch fermentation stages of kelp waste, the microbial community was predominantly and consistently composed of three phyla: Halanaerobiaeota, Bacteroidota, and Proteobacteria. At the genus level, Halanaerobium emerged as the dominant player, exhibiting a trend of initial increase followed by a decline throughout the fermentation process. Enzymes such as alginate lyases and both acidic and neutral proteases were found to play crucial roles in the degradation of kelp residues into sauces. Notably, electronic tongue analysis revealed that the fermented kelp sauce demonstrated strong umami characteristics. Furthermore, four novel umami peptides, EIL, STEV, GEEE, and SMEAVEA, from kelp were identified for the first time, with their umami effect largely attributed to strong hydrogen bond interactions with the T1R1–T1R3 umami receptors. In conclusion, this study proposed a sustainable method for kelp by-product utilization, with implications for other seaweed processing. Full article

Alginate lyase degrades alginate through the β-elimination mechanism to produce alginate oligosaccharides (AOS) with notable biochemical properties and diverse biological activities. However, its poor thermostability limits large-scale industrial production. In this study, we employed a rational computational design strategy combining computer-aided evolutionary coupling analysis and ΔΔG_fold evaluation to enhance both the thermostability and catalytic activity of the alginate lyase VxAly7B-CM. Among ten single-point mutants, the E188N and S204G mutants exhibited increases in T_m from 47.0 °C to 48.9 °C and 50.2 °C, respectively, with specific activities of 3701.02 U/mg and 2812.01 U/mg at 45 °C. Notably, the combinatorial mutant E188N/S204G demonstrated a ΔT_m of 5 °C and an optimal reaction temperature up to 50 °C, where its specific activity reached 3823.80 U/mg—a 31% increase. Moreover, its half-life at 50 °C was 38.4 h, which is 7.0 times that of the wild-type enzyme. Protein structural analysis and molecular dynamics simulations suggested that the enhanced catalytic performance and thermostability of the E188N/S204G mutant may be attributed to optimized surface charge distribution, strengthened hydrophobic interactions, and increased tertiary structure stability. Overall, our findings provided valuable insights into enzyme stabilization strategies and supported the industrial production of functional AOS. 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

Alginate lyases are of great importance in biotechnological and industrial processes, yet research on these enzymes from Mesonia genus bacteria is still limited. In this study, a novel PL6 family alginate lyase, MhAly6, was cloned and characterized from the deep-sea bacterium Mesonia hitae R32. The enzyme, composed of 797 amino acids, contains both PL6 and GH28 catalytic domains. A phylogenetic analysis revealed its classification into subfamily 1 of the PL6 family. MhAly6 showed optimal activity at 45 °C and pH 9.0, retaining over 50% activity after 210 min of incubation at 40 °C, highlighting its remarkable thermal stability. The enzyme exhibited degradation activity toward sodium alginate, Poly M, and Poly G, with the highest affinity for its natural substrate, sodium alginate, producing alginate oligosaccharides (AOSs) with degrees of polymerization (DP) ranging from 2 to 7. Molecular docking identified conserved catalytic sites (Lys241/Arg262) and Ca²⁺ binding sites (Asn202/Glu234/Glu236), while the linker and GH28 domain played an auxiliary role in substrate binding. Antioxidant assays revealed that the MhAly6-derived AOSs showed potent radical-scavenging activity, achieving 80.64% and 95.39% inhibition rates against DPPH and ABTS radicals, respectively. This work not only expands our understanding of alginate lyases from the Mesonia genus but also highlights their biotechnological potential for producing functional AOSs with antioxidant properties, opening new avenues for their applications in food and pharmaceuticals. Full article

In this study, we identified AlgVR7, a novel bifunctional alginate lyase from Vibrio rumoiensis and characterized its biochemical properties and substrate specificity. Sequence alignment analysis inferred the key residues K267, H162, N86, E189, and T244 for AlgVR7 catalysis, and it is derived from the PL7 family; exhibited high activity towards sodium alginate, polyM (PM), and polyG (PG); and can also degrade polygalacturonic acid (PGA) efficiently, with the highest affinity and catalytic efficiency for the MG block of the substrate. The optimal temperature and pH for AlgVR7 were determined to be 40 °C and pH 8, respectively. The enzyme activity of AlgVR7 was maximum at 40 °C, 40% of the enzyme activity was retained after incubation at 60 °C for 60 min, and enzyme activity was still present after 60 min incubation. AlgVR7 activity was stimulated by 100 Mm NaCl, indicating a halophilic nature and suitability for marine environments. Degradation products analyzed using ESI-MS revealed that the enzyme primarily produced trisaccharides and tetrasaccharides. At 40 °C and pH 8.0, its K_m values for sodium alginate, PM, and PG were 16.67 μmol, 13.12 μmol, and 22.86 μmol, respectively. Structural analysis and molecular docking studies unveiled the key catalytic residues involved in substrate recognition and interaction. Glu167 was identified as a critical residue for the PL7_5 subfamily, uniquely playing an essential role in alginate decomposition. Overall, AlgVR7 exhibits great potential as a powerful bifunctional enzyme for the efficient preparation of alginate oligosaccharides, with promising applications in biotechnology and industrial fields. Full article

This research introduced a strategy to fabricate sub-millimeter-diameter artificial liver tissue by extruding a combination of a liver decellularized extracellular matrix (dECM), alginate, endothelial cells, and hepatocytes. Vascularization remains a critical challenge in liver tissue engineering, as replicating the liver’s intricate vascular network is essential for sustaining cellular function and viability. Seven scaffold groups were evaluated, incorporating different cell compositions, scaffold materials, and structural configurations. The hepatocyte and endothelial cell scaffold treated with alginate lyase demonstrated the highest diffusion rate, along with enhanced albumin secretion (2.8 µg/mL) and urea synthesis (220 µg/mL) during the same period by day 10. A dense and interconnected endothelial cell network was observed as early as day 4 in the lyased coculture group. Furthermore, three-week implantation studies in rats showed a stable integration to the host with no adverse effects. This approach offers significant potential for advancing functional liver tissue replacements, combining accelerated diffusion, enhanced albumin secretion, improved urea synthesis, dense vascular network formation, and stable implantation outcomes. Full article

Algin is the most abundant substance in alga. Alginate lyase degrades algin and produces algin monosaccharides, disaccharides, and oligosaccharides, which are widely used in bioenergy, food, medicine, and other fields. In this study, one Exiguobacterium strain isolated from rotten kelp exhibited a robust ability to degrade the alga. The sequencing of this strain revealed the presence of three different types of algin alginate lyase. Nevertheless, the expression of three genes in Escherichia coli revealed a lower alginate lyase activity compared to that of the original strain. After codon optimization, the gene with the highest activity of the three was successfully expressed in Pichia pastoris to produce recombinant EbAlg664. The activity of the recombinant enzyme in 5 L high-density fermentation reached 1306 U/mg protein, 3.9 times that of the original Exiguobacterium strain. The results of the enzymatic analysis revealed that the optimal temperature and the pH range of recombinant EbAlg664 were narrower compared to the original strain. Additionally, the presence of Cu²⁺ and Co²⁺ enhanced the enzymatic activity, whereas Mg²⁺ and Fe³⁺ exhibited inhibitory effects on the recombinant alginate lyase. The study offers a theoretical and practical foundation for the industrial-scale production of engineered Pichia pastoris with high alginate lyase activity. 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

Alginate oligosaccharides (AOs), derived from alginate degradation, exhibit diverse biological activities and hold significant promise in various fields. The enzymatic preparation of AOs relies on alginate lyases, which offers distinct advantages. In contrast to the conventional use of sodium alginate derived from brown algae as the substrate for the enzymatic preparation of AOs, AO preparation directly from brown algae is more appealing due to its time and energy efficiency. Thus, the identification of potent alginate lyases and cost-effective brown algae substrates is crucial for optimizing AO production. Herein, we identified and characterized an alginate lyase, AlyP18, capable of efficiently decomposing algae, from a marine bacterium Pseudoalteromonas agarivorans A3 based on secretome analysis. AlyP18 is a mesothermal, endo-type and bifunctional alginate lyase with high enzymatic activity. Two brown algae substrates, Laminaria japonica roots and Macrocystis pyrifera, were used for the AO preparation by AlyP18. Upon optimization of AlyP18 hydrolysis parameters, the substrate degradation efficiency and AO production reached 53% and ~32% for L. japonica roots, respectively, and 77% and ~46.5% for M. pyrifera. The generated AOs primarily consisted of dimers to pentamers, with trimers and tetramers being dominant. This study provides an efficient alginate lyase and alternative brown algal feedstock for the bioconversion of high-value AOs from brown algae. Full article

Here, we report on a bifunctional alginate lyase (Vnalg7) expressed in Pichia pastoris, which can degrade natural Undaria pinnatifida into unsaturated guluronic acid di- and trisaccharide without pretreatment. The enzyme activity of Vnalg7 (3620.00 U/mL-culture) was 15.81-fold higher than that of the original alg (228.90 U/mL-culture), following engineering modification. The degradation rate reached 52.75%, and reducing sugar reached 30.30 mg/mL after combining Vnalg7 (200.00 U/mL-culture) and 14% (w/v) U. pinnatifida for 6 h. Analysis of the action mode indicated that Vnalg7 could degrade many substrates to produce a variety of unsaturated alginate oligosaccharides (AOSs), and the minimal substrate was tetrasaccharide. Site-directed mutagenesis showed that Glu²³⁸, Glu²⁴¹, Glu³¹², Arg²³⁶, His³⁰⁷, Lys⁴¹⁴, and Tyr⁴¹⁸ are essential catalytic sites, while Glu³³⁴, Glu³⁴⁴, and Asp³¹¹ play auxiliary roles. Mechanism analysis revealed the enzymatic degradation pattern of Vnalg7, which mainly recognizes and attacks the third glycosidic linkage from the reducing end of oligosaccharide substrate. Our findings provide a novel alginate lyase tool and a sustainable and commercial production strategy for value-added biomolecules using seaweeds. Full article