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Marine polysaccharide degrading enzymes are pivotal for catalyzing glycosidic linkages' breakdown in polysaccharides, yielding low-degree oligosaccharides and monosaccharides. These enzymes show substantial potential in diverse biotechnological applications. Marine polysaccharides and their derivatives exhibit anti-inflammatory, antiviral, anticoagulant, and anticancer properties, and are used in food additives with potential health benefits such as prebiotic oligosaccharides. Research explores pharmaceutical applications of marine polysaccharide degrading enzymes, including novel drug delivery and therapeutic bioactive oligosaccharide production. These enzymes are crucial for depolymerizing marine polysaccharides, providing energy and nutrients to microorganisms, and contributing to nutrient cycling and ecological balance. The breakdown of marine polysaccharides is vital for the global carbon cycle and helps regulate atmospheric carbon dioxide levels, impacting environmental sustainability. This comprehensive review delves into marine polysaccharide degrading enzymes, emphasizing carrageenase, agarase, chitinase, chitosanase and alginate lyase. Challenges such as low yields, poor stability, and high costs prompt solutions through metabolic engineering, enzyme immobilization, and engineering, enhancing resistance and efficiency. These advancements amplify the value of marine polysaccharidedegrading enzymes in biotechnological and environmental contexts. [ABSTRACT FROM AUTHOR]

Alginate lyases have countless potential for application in industries and medicine particularly as an appealing biocatalyst for the production of biofuels and bioactive oligosaccharides. Solid-state fermentation (SSF) allows improved production of enzymes and consumes less energy compared to submerged fermentation. Seaweeds can serve as the most promising biomass for the production of biochemicals. Alginate present in the seaweed can be used by alginate lyase–producing bacteria to support growth and can secrete alginate lyase. In this perspective, the current study was directed on the bioprocessing of brown seaweeds for the production of alginate lyase using marine bacterial isolate. A novel alginate–degrading marine bacterium Enterobacter tabaci RAU2C which was previously isolated in the laboratory was used for the production of alginate lyase using Sargassum swartzii as a low-cost solid substrate. Process parameters such as inoculum incubation period and moisture content were optimized for alginate lyase production. SSF resulted in 33.56 U/mL of alginate lyase under the static condition maintained with 75% moisture after 4 days. Further, the effect of different buffers, pH, and temperature on alginate lyase activity was also analyzed. An increase in alginate lyase activity was observed with an increase in moisture content from 60 to 75%. Maximum enzyme activity was perceived with phosphate buffer at pH 7 and 37 °C. Further, the residual biomass after SSF could be employed as biofertilizer for plant growth promotion based on the preliminary analysis. To our knowledge, this is the first report stating the usage of seaweed biomass as a substrate for the production of alginate lyase using solid-state fermentation. [ABSTRACT FROM AUTHOR]

This research aimed to assess the metabolic activities of Azotobacter vinelandii DT1 strain by assaying its alginate synthesizing enzymes and modifying genes, synthesize alginate from the agricultural residues, and eradicate the pathogenicity and risk associated with Pseudomonas aeruginosa alginate. Preliminary screening for alginate production revealed the presence of different alginate products. High-performance liquid chromatography (HPLC) confirmed the existence of varying alginate concentrations, such as sodium alginate (2.42754×10-1 g/ 100 ml), calcium alginate (1.09597×10-1 g/ 100 ml), acid alginate (1.39420×10-2 g/ 100 ml), alginate oligosaccharide (8.20576×10-2 g/100 ml), and potassium alginate (9.78836×10-2 g/ 100 ml). These were accompanied with the corresponding alginate synthesizing enzymes; mainly GDP-Mannose dehydrogenase (23.77± 0.13 U/ ml); glycosyltransferase (9.68± 0.53 U/ ml), phosphomannomutase (266.09± 0.16 U/ ml), mannose phosphate isomerase (95.87± 0.51 U/ ml), alginate lyase (24.50± 0.95 U/ ml), and mannuronan epimerase (49.93± 0.82 U/ ml). In this study, the expression of alginate-modifying genes such as alginate lyase and GDP-Mannose dehydrogenase amplicons of Azotobacter vinelandii DT1 strain corresponding to 766 bp, 43 ng and 600 bp, 33 ng molecular weights of the fast DNA marker; justified the synthesis of different alginate products. Azotobacter alginate synthesis using a low-cost substrate (i.e., corn cobs) and completely non-pathogenic bacteria (Azotobacter vinelandii DT1) may be desirable compared to the pathogenicity risks and poor jellying qualities linked to Pseudomonas alginate biosynthesis, its expensive production costs, and the adverse environmental effects of seaweed harvesting and processing.

Noha Nafee,1,2 Dina M Gaber,3 Alaa Abouelfetouh,4,5 Mustafa Alseqely,4 Martin Empting,6 Marc Schneider7 1Department of Pharmaceutics, College of Pharmacy, Kuwait University, Safat, 13110, Kuwait; 2Department of Pharmaceutics, Faculty of Pharmacy, Alexandria University, Alexandria, 21521, Egypt; 3Department of Pharmaceutics, Division of Pharmaceutical Sciences, College of Pharmacy, Arab Academy for Science, Technology and Maritime Transport, Alexandria, 1029, Egypt; 4Department of Microbiology and Immunology, Faculty of Pharmacy, Alexandria University, Alexandria, 21521, Egypt; 5Department of Microbiology and Immunology, Faculty of Pharmacy, Alamein International University, Alamein, 5060335, Egypt; 6Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Department of Antiviral and Antivirulence Drugs (AVID), Saarland University, Saarbrücken, 66123, Germany; 7Department of Pharmacy, Biopharmaceutics and Pharmaceutical Technology, Saarland University, Saarbrücken, 66123, GermanyCorrespondence: Noha Nafee, Department of Pharmaceutics, College of Pharmacy, Kuwait University, Safat, 13110, Kuwait, Tel +96524634914 ; +96560300583, Fax +96524636843, Email noha.nafee@ku.edu.kwIntroduction: Cystic fibrosis (CF) is associated with pulmonary Pseudomonas aeruginosa infections persistent to antibiotics.Methods: To eradicate pseudomonal biofilms, solid lipid nanoparticles (SLNs) loaded with quorum-sensing-inhibitor (QSI, disrupting bacterial crosstalk), coated with chitosan (CS, improving internalization) and immobilized with alginate lyase (AL, destroying alginate biofilms) were developed.Results: SLNs (140– 205 nm) showed prolonged release of QSI with no sign of acute toxicity to A549 and Calu-3 cells. The CS coating improved uptake, whereas immobilized-AL ensured > 1.5-fold higher uptake and doubled SLN diffusion across the artificial biofilm sputum model. Respirable microparticles comprising SLNs in carbohydrate matrix elicited aerodynamic diameters MMAD (3.54, 2.48 μm) and fine-particle-fraction FPF (65, 48%) for anionic and cationic SLNs, respectively. The antimicrobial and/or antibiofilm activity of SLNs was explored in Pseudomonas aeruginosa reference mucoid/nonmucoid strains as well as clinical isolates. The full growth inhibition of planktonic bacteria was dependent on SLN type, concentration, growth medium, and strain. OD measurements and live/dead staining proved that anionic SLNs efficiently ceased biofilm formation and eradicated established biofilms, whereas cationic SLNs unexpectedly promoted biofilm progression. AL immobilization increased biofilm vulnerability; instead, CS coating increased biofilm formation confirmed by 3D-time lapse confocal imaging. Incubation of SLNs with mature biofilms of P. aeruginosa isolates increased biofilm density by an average of 1.5-fold. CLSM further confirmed the binding and uptake of the labeled SLNs in P. aeruginosa biofilms. Considerable uptake of CS-coated SLNs in non-mucoid strains could be observed presumably due to interaction of chitosan with LPS glycolipids in the outer cell membrane of P. aeruginosa.Conclusion: The biofilm-destructive potential of QSI/SLNs/AL inhalation is promising for site-specific biofilm-targeted interventional CF therapy. Nevertheless, the intrinsic/extrinsic fundamentals of nanocarrier–biofilm interactions require further investigation.Keywords: alginate lyase, chitosan, solid lipid nanoparticles, cystic fibrosis, Pseudomonas aeruginosa, quorum-sensing inhibitors

Alginate lyases cleave the 1,4-glycosidic bond of alginate by eliminating sugar molecules from its bond. While earlier reported alginate lyases were primarily single catalytic domains, research on multi-module alginate lyases has been lfiguimited. This study identified VsAly7A, a multi-module alginate lyase present in Vibrio sp. QY108, comprising a "Pro-Asp-Thr(PDT)" fragment and two PL-7 catalytic domains (CD I and CD II). The "PDT" fragment enhances the soluble expression level and increases the thermostability and binding affinity to the substrate. Moreover, CD I exhibited greater catalytic efficiency than CD II. The incorporation of PDT-CD I resulted in an increase in the optimal temperature of VsAly7A, whereas CD II displayed a preference for polyG degradation. The multi-domain structure of VsAly7A provides a new idea for the rational design of alginate lyase, whilst the "PDT" fragment may serve as a fusion tag in the soluble expression of recombinant proteins. [ABSTRACT FROM AUTHOR]

Polysaccharide-degrading bacteria are key participants in the global carbon cycle and algal biomass recycling. Herein, a polysaccharide lyase-producing strain HB226069 was isolated from Sargassum sp. from Qingge Port, Hainan, China. Results of the phylogenetic of the 16S rRNA gene and genotypic analysis indicated that the isolate should be classified as Microbulbifer thermotolerans. The whole genome is a 4,021,337 bp circular chromosome with a G+C content of 56.5%. Analysis of the predicted genes indicated that strain HB226069 encoded 161 carbohydrate-active enzymes (CAZymes), and abundant putative enzymes involved in polysaccharide degradation were predicted, including alginate lyase, fucosidase, agarase, xylanase, cellulase, pectate lyase, amylase, and chitinase. Three of the putative polysaccharide lyases from PL7 and PL17 families were involved in alginate degradation. The alginate lyases of strain HB226069 showed the maximum activity of 117.4 U/mL at 50 °C, pH 7.0, and 0.05 M FeCl3, while exhibiting the best stability at 30 °C and pH 7.0. The Thin Layer Chromatography (TLC) and Electrospray Ionization Mass Spectrometry (ESI-MS) analyses indicated that the alginate oligosaccharides (AOSs) degraded by the partially purified alginate lyases contained oligosaccharides of DP2–DP5 and monosaccharide while reacting for 36 h. The complete genome of M. thermotolerans HB226069 enriches our understanding of the mechanism of polysaccharide lyase production and supports its potential application in polysaccharide degradation. [ABSTRACT FROM AUTHOR]

Alginate is derived from brown algae, which can be cultivated in large quantities. It can be broken down by alginate lyase into alginate oligosaccharides (AOSs), which exhibit a higher added value and better bioactivity than alginate. In this study, metagenomic technology was used to screen for genes that code for high-efficiency alginate lyases. The candidate alginate lyase gene alg169 was detected from Psychromonas sp. SP041, the most abundant species among alginate lyase bacteria on selected rotten kelps. The alginate lyase Alg169 was heterologously expressed in Escherichia coli BL21 (DE3), Ni-IDA-purified, and characterized. The optimum temperature and pH of Alg169 were 25 °C and 7.0, respectively. Metal ions including Mn2+, Co2+, Ca2+, Mg2+, Ni2+, and Ba2+ led to significantly increased enzyme activity. Alg169 exhibited a pronounced dependence on Na+, and upon treatment with Mn2+, its activity surged by 687.57%, resulting in the highest observed enzyme activity of 117,081 U/mg. Bioinformatic analysis predicted that Alg169 would be a double-domain lyase with a molecular weight of 65.58 kDa. It is a bifunctional enzyme with substrate specificity to polyguluronic acid (polyG) and polymannuronic acid (polyM). These results suggest that Alg169 is a promising candidate for the efficient manufacturing of AOSs from brown seaweed. [ABSTRACT FROM AUTHOR]

Dietary fiber metabolism by gut microorganisms plays important roles in host physiology and health. Alginate, the major dietary fiber of daily diet seaweeds, is drawing more attention because of multiple biological activities. To advance the understanding of alginate assimilation mechanism in the gut, we show the presence of unsaturated alginate oligosaccharides (uAOS)-specific alginate utilization loci (AUL) in human gut microbiome. As a representative example, a working model of the AUL from the gut microorganism Bacteroides clarus was reconstructed from biochemistry and transcriptome data. The fermentation of resulting monosaccharides through Entner-Doudoroff pathway tunes the metabolism of short-chain fatty acids and amino acids. Furthermore, we show that uAOS feeding protects the mice against dextran sulfate sodium-induced acute colitis probably by remodeling gut microbiota and metabolome. [ABSTRACT FROM AUTHOR]

Alginate oligosaccharides (AOS), products of alginate degradation by endotype alginate lyases, possess favorable biological activities and have broad applications. Although many have been reported, alginate lyases with homogeneous AOS products and secretory production by an engineered host are scarce. Herein, the alginate lyase AlyC7 from Vibrio sp. C42 was characterized as a trisaccharide-producing lyase exhibiting high activity and broad substrate specificity. With PelB as the signal peptide and 500 mM glycine as the additive, the extracellular production of AlyC7 in Escherichia coli reached 1122.8 U/mL after 27 h cultivation in Luria-Bertani medium. The yield of trisaccharides from sodium alginate degradation by the produced AlyC7 reached 758.6 mg/g, with a purity of 85.1%. The prepared AOS at 20 μg/mL increased the root length of lettuce, tomato, wheat, and maize by 27.5%, 25.7%, 9.7%, and 11.1%, respectively. This study establishes a robust foundation for the industrial and agricultural applications of AlyC7. [ABSTRACT FROM AUTHOR]

Marine macroalgae are increasingly recognized for their significant biological and economic potential. The key to unlocking this potential lies in the efficient degradation of all carbohydrates from the macroalgae biomass. However, a variety of polysaccharides (alginate, cellulose, fucoidan, and laminarin), are difficult to degrade simultaneously in a short time. In this study, the brown alga Saccharina japonica was found to be rapidly and thoroughly degraded by the marine bacterium Agarivorans albus B2Z047. This strain harbors a broad spectrum of carbohydrate-active enzymes capable of degrading various polysaccharides, making it uniquely equipped to efficiently break down both fresh and dried kelp, achieving a hydrolysis rate of up to 52%. A transcriptomic analysis elucidated the presence of pivotal enzyme genes implicated in the degradation pathways of alginate, cellulose, fucoidan, and laminarin. This discovery highlights the bacterium's capability for the efficient and comprehensive conversion of kelp biomass, indicating its significant potential in biotechnological applications for macroalgae resource utilization. [ABSTRACT FROM AUTHOR]

Abstract Background Ovarian cancer treatment is challenged by resistance and off-target effects. Melittin shows promise against cancer but is limited by its instability and harmful cellular interactions. Our study introduces SiO2–alginate–melittin nano-conjugates (SAMNs), incorporating alginate lyase to enhance melittin's release and mitigate alginate drawbacks. Methods We combined melittin with alginate and mesoporous silica, using alginate lyase to control melittin release. Effects on SKOV3 ovarian cancer cells were evaluated via viability, invasion, migration assays, ROS levels, apoptosis-related proteins, and mitochondrial function tests. Results SAMNs extended melittin’s cell control, reducing proliferation, invasion, and migration compared to free melittin. Alginate lyase facilitated controlled melittin release, decreasing off-target cytotoxicity. The only melittin group showed severe mitochondrial impairment, while the SAMNs and lyase groups had moderated impacts, indicating a dose-dependent effect on mitochondrial health and cell uptake. Conclusions SAMNs, especially with alginate lyase, offer an effective strategy for ovarian cancer treatment, optimizing melittin delivery while minimizing adverse effects of alginate. This approach enhances the therapeutic potential of melittin in combating ovarian cancer.

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Alginate lyase (AL) is a polysaccharide-degrading enzyme that can degrade alginate by hydrolyzing glycosidic bonds and produces unsaturated alginate oligosaccharides (AOSs). These AOSs have wide therapeutic and nutraceutical applications. However, to produce alginate oligosaccharides in a cost-effective manner is challenging due to the low availability and high cost of this degrading enzyme. Immobilization of the enzyme facilitates industrial applications owing to its stability, reusability, and cost-effectiveness. This study was focused on the enhancement of the properties of alginate lyase and improvement of the production of AOS. Alginate lyase was immobilized on magnetic nanoparticles (NPs) using glutaraldehyde as the crosslinker. The study showed that the maximum binding achieved between NPs and protein in the enzyme was 71% at a ratio of 1:150 NP:protein. As a result of immobilization, the optimum activity of free enzyme which was obtained at 37 °C and pH 7.4 changed to 45 °C and pH 9. Furthermore, the enzyme was thermostable at 45 °C for 3 h with up to 50% reusability for six consecutive cycles. Storage stability after 15 days showed ~67% relative hydrolysis of alginate. The free alginate lyase (25 IU) showed 76% raw biomass (seaweed) hydrolysis which is higher compared to 63% provided by the immobilized enzyme. As a result of efficient hydrolysis, AOSs with molecular weight profile of 370–1040 kDa were produced and detected using HPLC. [ABSTRACT FROM AUTHOR]

As the major component in the cell wall of brown algae, alginates are degradable by alginate lyases via b-elimination. Alginate lyases can be categorized into various polysaccharide lyase (PL) families, and PL7 family alginate lyases are the largest group and can be divided into six subfamilies. However, the major difference among different PL7 subfamilies is not fully understood. In this work, a marine alginate lyase, VaAly2, from Vibrio alginolyticus ATCC 17749 belonging to the PL7_5 subfamily was identified and characterized. It displayed comparatively high alginolytic activities toward different alginate substrates and functions as a bifunctional lyase. Molecular docking and biochemical analysis suggested that VaAly2 not only contains a key catalyzing motif (HQY) conserved in the PL7 family but also exhibits some specific characters limited in the PL7_5 subfamily members, such as the key residues and a long loop1 structure around the active center. Our work provides insight into a loop structure around the center site which plays an important role in the activity and substrate binding of alginate lyases belonging to the PL7_5 subfamily. [ABSTRACT FROM AUTHOR]

Alginate oligosaccharides are a kind of low molecular weight polymer composed of 2~10 monosaccharide molecules linked by the same or different glycosidic bonds. Alginate lyase is an important tool for the enzymatic preparation of AOS. AOS prepared by the enzymatic method is superior to AOS prepared by other methods in purity, performance and structure. In this paper, the origin, structure and enzymatic properties of alginate lyase were reviewed, and the applications of alginate lyase in aquaculture, such as feed additive, microalgae cultivation, immune regulation, antioxidant and antibacterial, were summarized. The research direction of alginate lyase and the application prospect of alginate lyase in aquaculture were prospected, which provided reference for the application of alginate lyase and AOS in aquaculture. [ABSTRACT FROM AUTHOR]

BACKGROUND: Alginate lyases are important tools for alginate biodegradation and oligosaccharide production, which have great potential in food and biofuel fields. The alginate polysaccharide utilization loci (PUL) typically encode a series of alginate lyases with a synergistic action pattern. Exploring valuable alginate lyases and revealing the synergistic effect of enzymes in the PUL is of great significance. RESULTS: An alginate PUL was discovered from the marine bacterium Wenyingzhuangia fucanilytica CZ1127T, and a repertoire of alginate lyases within it was cloned, expressed and characterized. The four alginate lyases in PUL demonstrated similar optimal reaction conditions: maximum enzyme activity at 35–50 °C and pH 8.0–9.0. The results of action pattern indicated that they were two PL7 endolytic bifunctional enzymes (Aly7A and Aly7B), a PL6 exolytic bifunctional enzyme (Aly6A) and a PL17 exolytic M‐specific enzyme (Aly17A). Ultra‐performance liquid chromatography–mass spectrometry was employed to reveal the synergistic effect of the four enzymes. The end products of Aly7A were further degraded by Aly7B and eventually generated oligosaccharides, from disaccharide to heptasaccharide. The oligosaccharide products were completely degraded to monosaccharides by Aly6A, but it was unable to directly degrade alginate. Aly17A could also produce monosaccharides by cleaving the M‐blocks of oligosaccharide products, as well as the M‐blocks of polysaccharides. The combination of these enzymes resulted in the complete degradation of alginate to monosaccharides. CONCLUSION: A new alginate PUL was mined and four novel alginate lyases in the PUL were expressed and characterized. The four cooperative alginate lyases provide novel tools for alginate degradation and biological fermentation. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Background: Ovarian cancer treatment is challenged by resistance and off-target effects. Melittin shows promise against cancer but is limited by its instability and harmful cellular interactions. Our study introduces SiO2–alginate–melittin nano-conjugates (SAMNs), incorporating alginate lyase to enhance melittin's release and mitigate alginate drawbacks. Methods: We combined melittin with alginate and mesoporous silica, using alginate lyase to control melittin release. Effects on SKOV3 ovarian cancer cells were evaluated via viability, invasion, migration assays, ROS levels, apoptosis-related proteins, and mitochondrial function tests. Results: SAMNs extended melittin's cell control, reducing proliferation, invasion, and migration compared to free melittin. Alginate lyase facilitated controlled melittin release, decreasing off-target cytotoxicity. The only melittin group showed severe mitochondrial impairment, while the SAMNs and lyase groups had moderated impacts, indicating a dose-dependent effect on mitochondrial health and cell uptake. Conclusions: SAMNs, especially with alginate lyase, offer an effective strategy for ovarian cancer treatment, optimizing melittin delivery while minimizing adverse effects of alginate. This approach enhances the therapeutic potential of melittin in combating ovarian cancer. [ABSTRACT FROM AUTHOR]

Background: Alginate lyase is known to have antibiofilm activity. Toxicity tests for the alginate lyase produced by Streptomyces olivaceus from Serena Kecil Island, North Sulawesi, Indonesia, have never been reported. This research conducted an in vivo toxicity study of the alginate lyase, using male Wistar rats for its development as an oral anti-biofilm agent. Methods: Twenty male Wistar rats (Rattus norvegicus) were divided equally into four groups of: technical control (TC), which was only given food and drink; negative control (NC), given phosphate buffer saline 1x; experimental group (E1), given alginate lyase at a dose of 0.63 g/kg BW, and (E2), given alginate lyase at a dose of 20.85 g/kg BW. The toxicity tests were carried out for 7 days by observing the following parameters: changes in the body weight and behavior; changes in AST and ALT enzyme levels; and evaluation of macroscopic and microscopic damages to the liver. Results: After 7 days of treatment, rats in the NC, E1, and E2 groups experienced insignificant weight losses. They also had normal behavior and did not show signs of toxicity or death. The AST and ALT levels in rats, given alginate lyase (E1 and E2) decreased significantly but were still within the normal range. Alginate lyase also caused an adaptive stress response and degeneration in the liver cells. Conclusions: Alginate lyase at 0.63 g/kg BW and 20.85 g/kg BW were considered safe in the rats and had the prospect of being developed into an anti-biofilm agent. [ABSTRACT FROM AUTHOR]

Alginate lyase can degrade alginate into oligosaccharides through β-elimination for various biological, biorefinery, and agricultural purposes. Here, we report a novel PL7 family exolytic alginate lyase VwAlg7A from marine bacteria Vibrio sp. W13 and achieve the heterologous expression in E. coli BL21 (DE3). VwAlg7A is 348aa with a calculated molecular weight of 36 kDa, containing an alginate lyase 2 domain. VwAlg7A exhibits specificity towards poly-guluronate. The optimal temperature and pH of VwAlg7A are 30 °C and 7.0, respectively. The activity of VwAlg7A can be significantly inhibited by the Ni2+, Zn2+, and NaCl. The Km and Vmax of VwAlg7A are 36.9 mg/ml and 395.6 μM/min, respectively. The ESI and HPAEC-PAD results indicate that VwAlg7A cleaves the sugar bond in an exolytic mode. Based on the molecular docking and mutagenesis results, we further confirmed that R98, H169, and Y303 are important catalytic residues. [ABSTRACT FROM AUTHOR]

ABSTRACTIn order to utilize seaweed waste as a feedstock in biorefinery, it is essential to characterize the microbial communities and their metabolic processes in seaweed decomposition. In this study, the phylogeny and functionality of the prokaryotic and fungal microbiome of Sargassum seaweed waste that has inundated the Barbados coast were assessed for the first time by targeting the 16S rRNA gene and internal transcribed spacer (ITS) region of ribosomal RNA, respectively, using the cultivation-independent Illumina next-generation sequencing approach. The resulting 67 032 and 91 085 high-quality paired-end reads of 16S rRNA gene and ITS were assigned to 734 and 179 amplicon sequence variants (ASVs), respectively. The predominant bacterial genera recorded were Muricauda, Aminobacterium, Mesorhizobium, Marinobacter, Reichenbachiella, Hyphomonas, Simiduia, Aquamicrobium, Oceanicola and Alcanivorax. The major genera of Archaea belonged to Methanosarcina, Methanogenium and Methanosaeta, and that of fungi to Sigmoidea, Tylopilus and Mucor. The metabolic functions of the Sargassum metagenome, analysed via PICRUSt, revealed the predominant metabolic pathways related to the biosynthesis of terpenoids and polyketides, the biosynthesis of other secondary metabolites, the metabolism of cofactors and vitamins, and the xenobiotic biodegradation. Furthermore, the prevalence of diverse alginate lyase-producing bacteria suggests that Sargassum waste is an ideal niche material for the isolation of potent microbial biocatalysts to sustainably depolymerize alginate and thereby produce third-generation biofuels and bioactive alginate oligosaccharides with novel industrial applications.

Alginate lyases are enzymes that hydrolyze alginate polysaccharides. Alginate lyase finds application as a biocatalyst in the production of biochemicals and biofuels and, as an agent in the control of alginate fouling. Furthermore, alginate lyases could be used as potential therapeutic agents for treating P. aeruginosa infections, notably in the treatment of cystic fibrosis by disruption of alginate biofilm and therefore enhancing the efficacy of anti-pseudomonal antibiotics. However, the commercial application of the enzyme is constrained due to high production costs and poor productivity. This issue could be addressed by employing high-yielding strains, optimizing the process parameters and media, and controlling systems. Marine microbes are known to produce alginate lyase for the utilization of seaweed alginate. In this perspective, different sponges and seaweeds were collected from Tuticorin, Gulf of Mannar, Tamilnadu, India, and primarily screened for alginate-degrading bacteria using Gram's Iodine method. Further screening of potential isolates was done by evaluation of alginate lyase activity using DNS assay. The potential marine bacterium was identified as Enterobacter tabaci RAU2C using biochemical tests followed by 16S rRNA sequencing. The initial pH for the production of alginate lyase was optimized. The Plackett–Burman design was used to screen nitrogen and mineral sources for the production of alginate lyase. Central Composite Design was formulated using Design Expert software for five variables to optimise the concentration of media components. Response Surface Methodology was used to estimate optimal concentrations, which were then confirmed by experiments. ANFIS modelling was established using MATLAB to further analyze and predict the composition of variables for enhancing enzyme activity. The optimized media constituents were found to be sodium alginate-5 g/l, peptone-15 g/l, ammonium sulfate-8 g/l, sodium chloride-30 g/l, and dipotassium hydrogen phosphate 4 g/l with an enzyme activity of 12.5 U/ml. Furthermore, the process parameters such as production time, inoculum volume, and age of the inoculum were also optimized for maximizing the alginate lyase production. [ABSTRACT FROM AUTHOR]

An alginate lyase gene aly644 encoding a member of polysaccharide lyase family 6 was obtained from a metagenome of Antarctic macroalgae-associated microbes. The gene was expressed heterologously in Escherichia coli, and the recombinant protein was purified using a Ni-NTA His Tag Kit. With sodium alginate as the substrate, recombinant Aly644 exhibited an optimum reaction temperature of 50°C and an optimum reaction pH of 7.0. The Vmax and Km values of Aly644 toward sodium alginate were 112.36 mg/mL·min and 16.75 mg/mL, respectively. Substrate specificity analysis showed that Aly644 was a bifunctional alginate lyase that hydrolyzed both polyguluronic acid and polymannuronic acid. The hydrolysis products of Aly644 with sodium alginate as the substrate were detected by thin-layer chromatography, and were mainly di- and trisaccharides. The oligosaccharides produced by degradation of sodium alginate by Aly644 inhibited the mycelial growth of the plant pathogens Phytophthora capsici and Fulvia fulva; the 50% maximal effective concentration (EC50) values were 297.45 and 452.89 mg/L, and the 90% maximal effective concentration (EC90) values were 1341.45 and 2693.83 mg/L, respectively. This highlights that Aly644 is a potential candidate enzyme for the industrial production of alginate oligosaccharides with low degree of polymerization. Enzyme-hydrolyzed alginate oligosaccharides could support the development of green agriculture as natural antimicrobial agents. Key points: • An alginate lyase was obtained from a metagenome of Antarctic macroalgae-associated microbes. • Aly644 is a bifunctional alginate lyase with excellent thermostability and pH stability. • The enzymatic hydrolysates of Aly644 directly inhibited Phytophthora capsici and Fulvia fulva. [ABSTRACT FROM AUTHOR]

Alginate lyase has been demonstrated as an efficient tool in the preparation of functional oligosaccharides (AOS) from alginate. The high viscosity resulting from the high concentration of alginate poses a limiting factor affecting enzymatic hydrolysis, particularly in the preparation of the fragments with low degrees of polymerization (DP). Herein, a PL7 family alginate lyase Algt from Microbulbifer thermotolerans DSM 19189 was developed and expressed in Pichia pastoris. The recombinant alginate lyase Algt1 was constructed by adopting the structural domain truncation strategy, and the enzymatic activity towards the alginate was improved from 53.9 U/mg to 212.86 U/mg compared to Algt. Algt1 was stable when incubated at 40 °C for 90 min, remaining with approximately 80.9% of initial activity. The analyses of thin-layer chromatography (TLC), fast protein liquid chromatography (FPLC), and electrospray ionization mass spectrometry (ESI-MS) demonstrated that the DP of the minimum identifiable substrate of Algt1 was five, and the main hydrolysis products were AOS with DP 1–4. Additionally, 1-L the enzymatic hydrolysis system demonstrated that Algt1 exhibited an effective degradation at alginate concentrations of up to 20%, with the resulting products of monosaccharides (14.02%), disaccharides (21.10%), trisaccharides (37.08%), and tetrasaccharides (27.80%). These superior properties of Algt1 make it possible to efficiently generate functional AOS with low DP in industrial processing. [ABSTRACT FROM AUTHOR]

Alginate oligosaccharides prepared by alginate lyases attracted great attention because of their desirable biological activities. However, the hydrolysis products are always a mixture of oligosaccharides with different degrees of polymerization, which increases the production cost because of the following purification procedures. In this study, an alginate lyase, Alg4755, with high product specificity was identified, heterologously expressed, and characterized from Vibrio alginolyticus S10, which was isolated from the intestine of sea cucumber. Alg4755 belonged to the PL7 family with two catalytic domains, which was composed of 583 amino acids. Enzymatic characterization results show that the optimal reaction temperature and pH of Alg4755 were 35 °C and 8.0, respectively. Furthermore, Alg4755 was identified to have high thermal and pH stability. Moreover, the final hydrolysis products of sodium alginate catalyzed by Alg4755 were mainly alginate disaccharides with a small amount of alginate trisaccharides. The results demonstrate that alginate lyase Alg4755 could have a broad application prospect because of its high product specificity and desirable catalytic properties. [ABSTRACT FROM AUTHOR]

The discovery and characterization of new alginate lyases with high activity and stability have been the research topic in utilization of alginate biomass. Herein, we characterized a new Thalassotalea algicola -derived polyM-preferred PL6 family alginate lyase TAPL6 with two-domains (catalytic domain NTD and non-catalytic domain CTD). TAPL6 is a bifunctional enzyme that could endolytically degrade polyM, and could exolytically degrade polyG. It could degrade alginate in a hybrid action mode. The specific activities of polyM, polyG and sodium alginate of TAPL6 were 7174 U/mg, 4140.2 U/mg and 414.3 U/mg, respectively. The optimal temperature and pH for TAPL6 are 25 °C and 8.0, respectively. Deletion of CTD resulted in a loss of 99.8%− 98.2% of TAPL6 activity and a decrease in pH adaptability, but greatly improved the activity and temperature stability above 30 °C. The CTD truncation is more resistant to metal ions than TAPL6, and the promotion effect of Ca2+ on the activity is stronger. Deletion of CTD results in a change in the action mode of TAPL6 to an endo-mode. In addition, the integrity of the linker between NTD and CTD is very important for the soluble expression of recombinant protein, and the complete linker is beneficial to the catalytic activity of NTD. This study provides a new tool enzyme for the production of alginate oligosaccharides, and explores the role of the non-catalytic domain CTD in the PL6 family alginate lyases, providing a rational design basis for the PL6 family alginate lyases to change the enzymatic properties and action mode. [Display omitted] • Biochemical characterization of a new PL6 alginate lyase and its domain truncations. • Function of CTD structural domain in the whole enzyme. • Action mode of TAPL6 and the effect of CTD on product distribution and action mode. [ABSTRACT FROM AUTHOR]

Humans consume alginate in the form of seaweed, food hydrocolloids, and encapsulations, making the digestion of this mannuronic acid (M) and guluronic acid (G) polymer of key interest for human health. To increase knowledge on alginate degradation in the gut, a gene catalog from human feces was mined for potential alginate lyases (ALs). The predicted ALs were present in nine species of the Bacteroidetes phylum, of which two required supplementation of an endo-acting AL, expected to mimic cross-feeding in the gut. However, only a new isolate grew on alginate. Wholegenome sequencing of this alginate-utilizing isolate suggested that it is a new Bacteroides ovatus strain harboring a polysaccharide utilization locus (PUL) containing three ALs of families: PL6, PL17, and PL38. The BoPL6 degraded polyG to oligosaccharides of DP 1-3, and BoPL17 released 4,5-unsaturated monouronate from polyM. BoPL38 degraded both alginates, polyM, polyG, and polyMG, in endo-mode; hence, it was assumed to deliver oligosaccharide substrates for BoPL6 and BoPL17, corresponding well with synergistic action on alginate. BoPL17 and BoPL38 crystal structures, determined at 1.61 and 2.11 Å, respectively, showed (a/a)6-barrel + anti-parallel ß-sheet and (a/a)7-barrel folds, distinctive for these PL families. BoPL17 had a more open active site than the two homologous structures. BoPL38 was very similar to the structure of an uncharacterized PL38, albeit with a different triad of residues possibly interacting with substrate in the presumed active site tunnel. Altogether, the study provides unique functional and structural insights into alginate-degrading lyases of a PUL in a human gut bacterium. [ABSTRACT FROM AUTHOR]

Abstract Background Alginate oligosaccharides (AOs) are the degradation products of alginate, a natural polysaccharide abundant in brown algae. AOs generated by enzymatic hydrolysis have diverse bioactivities and show broad application potentials. AOs production via enzymolysis is now generally with sodium alginate as the raw material, which is chemically extracted from brown algae. In contrast, AOs production by direct degradation of brown algae is more advantageous on account of its cost reduction and is more eco-friendly. However, there have been only a few attempts reported in AOs production from direct degradation of brown algae. Results In this study, an efficient Laminaria japonica-decomposing strain Pseudoalteromonas agarivorans A3 was screened. Based on the secretome and mass spectrum analyses, strain A3 showed the potential as a cell factory for AOs production by secreting alginate lyases to directly degrade L. japonica. By using the L. japonica roots, which are normally discarded in the food industry, as the raw material for both fermentation and enzymatic hydrolysis, AOs were produced by the fermentation broth supernatant of strain A3 after optimization of the alginate lyase production and hydrolysis parameters. The generated AOs mainly ranged from dimers to tetramers, among which trimers and tetramers were predominant. The degradation efficiency of the roots reached 54.58%, the AOs production was 33.11%, and the AOs purity was 85.03%. Conclusion An efficient, cost-effective and green process for AOs production directly from the underutilized L. japonica roots by using strain A3 was set up, which differed from the reported processes in terms of the substrate and strain used for fermentation and the AOs composition. This study provides a promising platform for scalable production of AOs, which may have application potentials in industry and agriculture.

Quorum sensing (QS) system can dynamically control the expression of proteins along with the cell growth. The promoting period of QS system has been little focused on until now. In this study, a self-induced dynamic regulated expression (SIDRE) system was constructed in Escherichia coli. To enable the system suitable for the expression of enzymes, promoter engineering was used to obtain PluxI mutants. To test the SIDRE system, alginate lyase AL493 and esterase Est7 were used as target protein for expression. The enzyme activity of alginate lyase and esterase reached 96.38 % and 106.71 % of the control strains containing the T7 promoter. In high-density fermentation, the activity of alginate lyase expressed by the SIDRE system with PluxI(T-38C) as promoter was 4.34-fold of that expressed by the T7 promoter. Therefore, the PluxI mutants with different promoting periods and/or different strengths show great potential in both laboratory and industrial scale for protein expression.

In order to clarify the effect of the non-catalytic carbohydrate-binding module (CBM) and F5/8 type C domains on the enzymatic properties of AlgL7, an alginate lyase from Microbulbifer sp. ALW1, the full-length enzyme AlgL7 and two truncated enzymes: CD1 (catalytic domain) and CD2 (containing F5/8 type C domain and catalytic domain) were constructed and characterized. The results showed that the truncated enzyme CD2 exhibited higher specific activity, thermostability, Michaelis constant (Km), and maximum reaction velocity (Vmax) compared to the full-length AlgL7, indicating that the CBM domain played an important role in maintaining the substrate affinity of the enzyme, but reduced the catalytic activity, thermostability, and Vmax value the enzyme. Compared to the truncated enzyme CD1, CD2 exhibited higher specific activity, optimal reaction temperature, thermostability, Vmax, and Km, indicating that the F5/8 type C domain contributed to improve the enzymatic activity, optimum reaction temperature, thermostability, and Vmax, but reduced the substrate affinity of the enzyme. Using sodium alginate as the substrate, the specific activity of CD2 was 183.9 U/mg. The optimal reaction temperature and pH were 40 ℃ and 7.0, respectively. The Km and Vmax were 39.80 mg/mL and 2 000 U/mg, respectively. The major enzymatic hydrolysates were disaccharides and trisaccharides. This study promotes the understanding of the structure-activity relationship between the non-catalytic domains and the properties of alginate lyase, and lays a theoretical basis for using the non-catalytic domains to improve the catalytic properties of alginate lysate.

Alginate lyases cleave the 1,4-glycosidic bond of alginate by eliminating sugar molecules from its bond. While earlier reported alginate lyases were primarily single catalytic domains, research on multi-module alginate lyases has been lfiguimited. This study identified VsAly7A, a multi-module alginate lyase present in Vibrio sp. QY108, comprising a “Pro-Asp-Thr(PDT)” fragment and two PL-7 catalytic domains (CD I and CD II). The “PDT” fragment enhances the soluble expression level and increases the thermostability and binding affinity to the substrate. Moreover, CD I exhibited greater catalytic efficiency than CD II. The incorporation of PDT-CD I resulted in an increase in the optimal temperature of VsAly7A, whereas CD II displayed a preference for polyG degradation. The multi-domain structure of VsAly7A provides a new idea for the rational design of alginate lyase, whilst the “PDT” fragment may serve as a fusion tag in the soluble expression of recombinant proteins.

Alginate is derived from brown algae, which can be cultivated in large quantities. It can be broken down by alginate lyase into alginate oligosaccharides (AOSs), which exhibit a higher added value and better bioactivity than alginate. In this study, metagenomic technology was used to screen for genes that code for high-efficiency alginate lyases. The candidate alginate lyase gene alg169 was detected from Psychromonas sp. SP041, the most abundant species among alginate lyase bacteria on selected rotten kelps. The alginate lyase Alg169 was heterologously expressed in Escherichia coli BL21 (DE3), Ni-IDA-purified, and characterized. The optimum temperature and pH of Alg169 were 25 °C and 7.0, respectively. Metal ions including Mn2+, Co2+, Ca2+, Mg2+, Ni2+, and Ba2+ led to significantly increased enzyme activity. Alg169 exhibited a pronounced dependence on Na+, and upon treatment with Mn2+, its activity surged by 687.57%, resulting in the highest observed enzyme activity of 117,081 U/mg. Bioinformatic analysis predicted that Alg169 would be a double-domain lyase with a molecular weight of 65.58 kDa. It is a bifunctional enzyme with substrate specificity to polyguluronic acid (polyG) and polymannuronic acid (polyM). These results suggest that Alg169 is a promising candidate for the efficient manufacturing of AOSs from brown seaweed.

Alginate oligosaccharides (AOS), products of alginate degradation by endotype alginate lyases, possess favorable biological activities and have broad applications. Although many have been reported, alginate lyases with homogeneous AOS products and secretory production by an engineered host are scarce. Herein, the alginate lyase AlyC7 from Vibrio sp. C42 was characterized as a trisaccharide-producing lyase exhibiting high activity and broad substrate specificity. With PelB as the signal peptide and 500 mM glycine as the additive, the extracellular production of AlyC7 in Escherichia coli reached 1122.8 U/mL after 27 h cultivation in Luria-Bertani medium. The yield of trisaccharides from sodium alginate degradation by the produced AlyC7 reached 758.6 mg/g, with a purity of 85.1%. The prepared AOS at 20 μg/mL increased the root length of lettuce, tomato, wheat, and maize by 27.5%, 25.7%, 9.7%, and 11.1%, respectively. This study establishes a robust foundation for the industrial and agricultural applications of AlyC7.

Marine macroalgae are increasingly recognized for their significant biological and economic potential. The key to unlocking this potential lies in the efficient degradation of all carbohydrates from the macroalgae biomass. However, a variety of polysaccharides (alginate, cellulose, fucoidan, and laminarin), are difficult to degrade simultaneously in a short time. In this study, the brown alga Saccharina japonica was found to be rapidly and thoroughly degraded by the marine bacterium Agarivorans albus B2Z047. This strain harbors a broad spectrum of carbohydrate-active enzymes capable of degrading various polysaccharides, making it uniquely equipped to efficiently break down both fresh and dried kelp, achieving a hydrolysis rate of up to 52%. A transcriptomic analysis elucidated the presence of pivotal enzyme genes implicated in the degradation pathways of alginate, cellulose, fucoidan, and laminarin. This discovery highlights the bacterium’s capability for the efficient and comprehensive conversion of kelp biomass, indicating its significant potential in biotechnological applications for macroalgae resource utilization.

Background: Alginate oligosaccharides (AOs) are the degradation products of alginate, a natural polysaccharide abundant in brown algae. AOs generated by enzymatic hydrolysis have diverse bioactivities and show broad application potentials. AOs production via enzymolysis is now generally with sodium alginate as the raw material, which is chemically extracted from brown algae. In contrast, AOs production by direct degradation of brown algae is more advantageous on account of its cost reduction and is more eco-friendly. However, there have been only a few attempts reported in AOs production from direct degradation of brown algae. Results: In this study, an efficient Laminaria japonica-decomposing strain Pseudoalteromonas agarivorans A3 was screened. Based on the secretome and mass spectrum analyses, strain A3 showed the potential as a cell factory for AOs production by secreting alginate lyases to directly degrade L. japonica. By using the L. japonica roots, which are normally discarded in the food industry, as the raw material for both fermentation and enzymatic hydrolysis, AOs were produced by the fermentation broth supernatant of strain A3 after optimization of the alginate lyase production and hydrolysis parameters. The generated AOs mainly ranged from dimers to tetramers, among which trimers and tetramers were predominant. The degradation efficiency of the roots reached 54.58%, the AOs production was 33.11%, and the AOs purity was 85.03%. Conclusion: An efficient, cost-effective and green process for AOs production directly from the underutilized L. japonica roots by using strain A3 was set up, which differed from the reported processes in terms of the substrate and strain used for fermentation and the AOs composition. This study provides a promising platform for scalable production of AOs, which may have application potentials in industry and agriculture. [ABSTRACT FROM AUTHOR]

Alginate oligosaccharides (AOs) prepared through enzymatic reaction by diverse alginate lyases under relatively controllable and moderate conditions possess versatile biological activities. But widely used commercial alginate lyases are still rather rare due to their poor properties (e.g., lower activity, worse thermostability, ion tolerance, etc.). In this work, the alginate lyase Alyw208, derived from Vibrio sp. W2, was expressed in Yarrowia lipolytica of food grade and characterized in order to obtain an enzyme with excellent properties adapted to industrial requirements. Alyw208 classified into the polysaccharide lyase (PL) 7 family showed maximum activity at 35 °C and pH 10.0, indicating its cold-adapted and high-alkaline properties. Furthermore, Alyw208 preserved over 70% of the relative activity within the range of 10–55 °C, with a broader temperature range for the activity compared to other alginate-degrading enzymes with cold adaptation. Recombinant Alyw208 was significantly activated with 1.5 M NaCl to around 2.1 times relative activity. In addition, the endolytic Alyw208 was polyG-preferred, but identified as a bifunctional alginate lyase that could degrade both polyM and polyG effectively, releasing AOs with degrees of polymerization (DPs) of 2–6 and alginate monomers as the final products (that is, DPs 1–6). Alyw208 has been suggested with favorable properties to be a potent candidate for biotechnological and industrial applications. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

There has been a growing demand for efficient disposal of kelp waste. Microbial degradation is a win–win bio-solution for seaweeds waste disposal and acquisition of nutritious byproducts. In this study, the cooperation of an alginate lyase producing bacteria (Paenibacillus sp. QBH) and cellulase producing bacteria (Enterococcus sp. N1) was applied for its high degrading efficiency of kelp waste. Serial single factor experiments revealed that temperature, shaking speed and degradation time were the key factors impacting the alginate lyase and filter paper assay activities upon hydrolyzing kelp waste by N1 + QBH combination. Via response surface method optimization, predicted models for the alginate lyase and filter paper assay activities asserted the highest values could be attained at the temperature 34 °C, shaking speed 180 rpm and degradation time 4.5 days. This prediction was verified by a triplicate experiment giving an average alginate lyase activity of 61.2 U mL−1 and filter paper assay activity of 6.12 U mL−1. Biodegradation of kelp waste by N1 + QBH combination under optimizing conditions remarkably promoted the release of sugars, total phenolic compounds, various minerals and amino acids, generating a nutritious hydrolysate with a total organic matters yield of 56.3 g L−1, total phenolic compounds yield of 18.3 mg g−1 dry weight and total amino acids concentration of 247.3 mg L−1. This study highlights that N1 + QBH combination could be a feasible solution for bioconversion of kelp waste to sustainable production of biostimulant. [ABSTRACT FROM AUTHOR]

Alginate oligosaccharides (AOS) made from the degradation of alginate, to some extent, makes up for the poor solubility and bioavailability of alginate as a macromolecular substance and possess several beneficial biological activities that are absent in alginate. These properties include prebiotic, glycolipid regulatory, immunomodulatory, antimicrobial, antioxidant, anti-tumor, promoting plant growth and other activities. Consequently, AOS has significant potential for use in the agricultural, biomedical, and food industries, and has been the focus of research in the field of marine biological resources. This review comprehensively covers methods (physical, chemical, and enzymatic methods) for the production of AOS from alginate. More importantly, this paper reviews recent advances in the biological activity and potentially industrial and therapeutic applications of AOS, providing a reference for future research and applications of AOS. [ABSTRACT FROM AUTHOR]

Alginates are considered as the major structural material present in the cell-wall of brown seaweeds and are produced by bacterial species like Azotobacter and Pseudomonas. Alginate lyase depolymerizes alginate into alginate oligosaccharides. Alginate lyase (AL) can be classified into two groups such as G block and M block-specific lyase as well as endolytic and exolytic enzymes based on their substrate specificities and mode of action respectively. Oligosaccharides derived from alginate have innumerable biological activities including immune regulation, anticoagulation, anti-oxidant, anti-cancer and health-promoting activities. In the present study, in silico characterization of the different reported alginate lyases was performed. A total of 50 protein sequences of alginate lyase produced by different microorganisms were retrieved from UniProt database and characterized. Multiple sequence alignment was executed using Clustal Omega. ProtParam tool was used to evaluate physicochemical parameters analyses. Phylogenetic tree was constructed by Neighbor-Joining method using MEGA-X. Motif analysis of alginate lyase sequences was carried out by MEME server. 45 structures of alginate lyase were modelled using SWISSMODEL. Multiple sequence alignment validates that sequences are highly variable among various alginate lyases reported in different species. Alginate lyase from different species possesses different molecular weights with amino acid residues ranging from 320 to 1059. The pI ranged between 4.42 and 9.59. The variation within these alginate lyases along with other physicochemical parameters was also examined. The active site for the different reported alginate lyases were predicted through CASTp server. [ABSTRACT FROM AUTHOR]

Alginate lyase (AL) is a polysaccharide-degrading enzyme that can degrade alginate by hydrolyzing glycosidic bonds and produces unsaturated alginate oligosaccharides (AOSs). These AOSs have wide therapeutic and nutraceutical applications. However, to produce alginate oligosaccharides in a cost-effective manner is challenging due to the low availability and high cost of this degrading enzyme. Immobilization of the enzyme facilitates industrial applications owing to its stability, reusability, and cost-effectiveness. This study was focused on the enhancement of the properties of alginate lyase and improvement of the production of AOS. Alginate lyase was immobilized on magnetic nanoparticles (NPs) using glutaraldehyde as the crosslinker. The study showed that the maximum binding achieved between NPs and protein in the enzyme was 71% at a ratio of 1:150 NP:protein. As a result of immobilization, the optimum activity of free enzyme which was obtained at 37 °C and pH 7.4 changed to 45 °C and pH 9. Furthermore, the enzyme was thermostable at 45 °C for 3 h with up to 50% reusability for six consecutive cycles. Storage stability after 15 days showed ~67% relative hydrolysis of alginate. The free alginate lyase (25 IU) showed 76% raw biomass (seaweed) hydrolysis which is higher compared to 63% provided by the immobilized enzyme. As a result of efficient hydrolysis, AOSs with molecular weight profile of 370–1040 kDa were produced and detected using HPLC.

A Gram-stain-negative, rod-shaped bacterium, designated HB171785T, was isolated from soil sample collected from Qishui Bay, Hainan, China. The strain grew optimally at pH 7–8, 37–40 °C and with NaCl 3–4%. The predominant isoprenoid quinone was found to be Q-8 and the major fatty acids were C16:0, C16:1ω7c/C16:1ω6c, C18:1ω7c/C18:1ω6c and C12:0 3OH. The polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The size of the draft genome was 4.32 Mbp with G + C content 49.7%. Phylogenetic analysis of 16S rRNA gene sequence indicated that the closest phylogenetically related species were Neiella marina j221T, “Neiella holothuriorum” 126 and Echinimonas agarilytica KMM 6351T with the similarities of 98.2, 96.0 and 95.0%, respectively. The phylogenetic tree based on 16S rRNA gene and phylogenomic tree based on core genome showed that strain HB171785T clustered together with N. marina j221T, with the highest values of average nucleotide identity (82.9%) and digital DNA-DNA hybridization (25.4%). The combined phylogenetic relatedness, phenotypic and genotypic features supported the conclusion that strain HB171785T represents a novel species of the genus Neiella, for which the name Neiella litorisoli sp. nov. is proposed. The type strain is HB171785T (= MCCC 1K04625T = KCTC 82319T). In addition, Echinimonadaceae fam. nov. in the order Alteromonadales was proposed. [ABSTRACT FROM AUTHOR]

Copyright of Chinese Journal of Bioprocess Engineering is the property of Chinese Journal of Bioprocess Engineering Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Enzymatic alginate oligosaccharides were proved with various biological activities. Developing robust alginate lyase with high production is essential for its industrial application. In this study, a novel alginate lyase gene, AL07, was successfully screened and expressed in Pichia pastoris, and its characteristics were characterized. The secreted alginate lyase has a molecular weight of approximately 40.0 kDa and an activity of 45.4 U/mL. AL07 exhibits superior biological activity at pH 7.0 and 40 °C, with a preference for polyG substrates. Notably, the enzyme exhibits more than 60% relative activity over a pH range of 5.0 to 10.0. AL07 is also independent of ions and exhibits salt tolerance properties. The main degradation product of AL07 is DP2 oligosaccharide. Using AL07-based enzyme cocktail, Laminaria japonica can be degraded within 120 min. Therefore, AL07 has been demonstrated to be an efficient tool for the preparation of alginate oligosaccharides and the degradation of L. japonica. [ABSTRACT FROM AUTHOR]

Seaweeds are considered to be third-generation renewable biomasses, the comprehensive utilization of which has drawn increasing attention in recent years. A novel cold-active alginate lyase (VfAly7) was identified from Vibrio fortis and biochemically characterized for brown seaweed utilization. The alginate lyase gene was high-level expressed in Pichia pastoris, with an enzyme yield of 560 U/mL and a protein content of 9.8 mg/mL by high-cell density fermentation. The recombinant enzyme was most active at 30 °C and pH 7.5, respectively. VfAly7 was a bifunctional alginate lyase with both poly-guluronate and poly-mannuronate hydrolysis activities. On the basis of VfAly7, a bioconversion strategy for the utilization of brown seaweed (Undaria pinnatifida) was developed. The obtained AOSs showed stronger prebiotic activity towards tested probiotics when compared to that of commercial fructooligosaccharides (FOSs), while the obtained protein hydrolysates displayed strong xanthine oxidase inhibitory activity with IC50 of 3.3 mg/mL. This study provided a novel alginate lyase tool as well as a biotransformation route for the utilization of seaweeds. [ABSTRACT FROM AUTHOR]

AbstractAlginate lyase has the potential for the ecofriendly depolymerization of alginate and generation of value-added products important in agriculture, bioenergy, food processing, medical diagnostic and pharmaceutical industries. Substantial research has focused on the cost-effective production of alginate lyase via screening of potent microbial isolates for use as biocatalysts and the optimization of fermentation media with low-cost feedstocks. This study evaluated the alginate lyase-producing efficacy of a novel Pseudomonas sp. LB56 (GenBank accession no. MT176182) isolated from Sargassum seaweed waste. The feedstock for solid-state fermentation (SSF) was decomposed Sargassum biomass amended with cow dung. The production of alginate lyase was maximal using 9.5 g Sargassum, 14 g cow dung and at pH 8 after optimization via a 23–factorial central composite design. Comparative modelling and molecular docking studies were conducted to elucidate the 3D structure of the alginate lyase of this novel isolate, as well as to investigate its catalytic interactions with different polymeric sizes of alginate [polymannuronate (mannuronate disaccharide and mannuronate trisaccharide), polyguluronate (guluronate disaccharide and guluronate trisaccharide) and alginate]. The overall results suggest that biowaste (Sargassum seaweed amended with cow dung) can be used as a feedstock for economical production of alginate lyase. Additionally, the identified active site residues of alginate lyase, which were shared by different polymers of alginate, can be tailored via rational and/or combinatorial approaches for efficient biotransformation of alginate-enriched wastes into biologically active alginate oligomers and third generation biofuels.

Bacterial biofilms generally contribute to chronic infections, including wound infections. Due to the antibiotic resistance mechanisms protecting bacteria living in the biofilm, they are a serious problem in the wound healing process. To accelerate the wound healing process and avoid bacterial infection, it is necessary to select the appropriate dressing material. In this study, the promising therapeutic properties of alginate lyase (AlgL) immobilised on BC membranes for protecting wounds from Pseudomonas aeruginosa infection were investigated. The AlgL was immobilised on never dried BC pellicles via physical adsorption. The maximum adsorption capacity of AlgL was 6.0 mg/g of dry BC, and the equilibrium was reached after 2 h. The adsorption kinetics was studied, and it has been proven that the adsorption was consistent with Langmuir isotherm. In addition, the impact of enzyme immobilisation on bacterial biofilm stability and the effect of simultaneous immobilisation of AlgL and gentamicin on the viability of bacterial cells was investigated. The obtained results showed that the AlgL immobilisation significantly reduced the amount of polysaccharides component of the P. aeruginosa biofilm. Moreover, the biofilm disruption by AlgL immobilised on BC membranes exhibited synergism with the gentamicin, resulting in 86.5% more dead P. aeruginosa PAO-1 cells. [ABSTRACT FROM AUTHOR]

Alginate oligosaccharides (AOS) produced through alginate biodegradation by alginate lyases have received extensive attention for their wide applications in the food, pharmaceutical, and agricultural industries. Thus, alginate lyases with good performance are urgently required for industrial production. In this study, combinational mutagenesis was employed for the alginate lyase from Pseudoalteromonas sp. Alg6B. After combinational mutation of key amino acids (for example, isoleucine and valine) in the catalytic residues, the specific activity of the mutant I62A/A99K/V132S/L157T (1124.1 U/mg) was increased 2.13 fold. The adaptability of the mutant enzyme to temperature (up to 60 °C), pH (4.0–10.0), and chemicals and metal ions was significantly improved. Kinetic analysis showed that the catalytic efficiency of the mutant enzyme was 4.67-fold higher than that of the wild-type enzyme. Our results indicate the feasibility of combinational mutagenesis for the modification of alginate lyases, which shows significant potential for the production of AOS from marine alginate. [Display omitted] • Combinatorial mutagenesis was employed for modification of alginate lyase. • Mutagenesis of isoleucine and valine in catalytic residues enhanced the adaptability. • Catalytic efficiency of the mutant enzyme was increased by 4.67 folds. [ABSTRACT FROM AUTHOR]