Discovery and characterization of a novel poly-mannuronate preferred alginate lyase: The first member of a new polysaccharide lyase family.

Alginate is one of the most important marine colloidal polysaccharides, and its oligosaccharides have been proven to possess diverse biological functions. Alginate lyases could specifically degrade alginate and therefore serve as desirable tools for the research and development of alginate. In this report, a novel catalytic domain, which demonstrated no significant sequence similarity with all previously defined functional domains, was verified to exhibit a random endo-acting lyase activity to alginate. The action pattern analysis revealed that the heterologously expressed protein, named Aly44A, preferred to degrade polyM. Its minimum substrates and the minimum products were identified as unsaturated alginate trisaccharides and disaccharides, respectively. Based on the sequence novelty of Aly44A and its homologs, a new polysaccharide lyase family (PL44) was proposed. The discovery of the novel enzyme and polysaccharide lyase family provided a new entrance for the gene-mining and acquiring of alginate lyases, and would facilitate to the utilization of alginate and its oligosaccharides.

A chemo-enzymatic method for preparation of saturated oligosaccharides from alginate and other uronic acid-containing polysaccharides.

Oligosaccharides from uronic acid-containing polysaccharides can be produced either by chemical or enzymatic degradation. The benefit of using enzymes, called lyases, is their high specificity for various glycosidic linkages. Lyases cleave the polysaccharide chain by an ß-elimination reaction, yielding oligosaccharides with an unsaturated sugar (4-deoxy-l-erythro-hex-4-enepyranosyluronate) at the non-reducing end. In this work we have systematically studied acid degradation of unsaturated uronic acid oligosaccharides. Based on these findings, a method for preparing saturated oligosaccharides by enzymatic degradation of uronic acid-containing polysaccharides was developed. This results in oligosaccharides with a pre-defined distribution and proportion of sugar residues compared to the products of chemical degradation, while maintaining the chemical structure of the non-reducing end. The described method was demonstrated for generating saturated oligosaccharides of alginate, heparin and polygalacturonic acid. In the case of alginate, the ratio of hydrolysis rate of Δ-G and Δ-M linkages to that of G-G and M-M linkages, respectively, was found to be approximately 65 and 43, at pH* 3.4, 90 °C. Finally, this method has been demonstrated to be superior in the production of α-l-guluronate oligosaccharides with a lower content of ß-d-mannuronate residues compared to what can be achieved using chemical depolymerization alone.

Cloning, expression and characterization of novel hyaluronan lyases Vhylzx1 and Vhylzx2 from Vibrio sp. ZG1.

Hyaluronidases are a class of enzymes that can degrade hyaluronic acid and have a wide range of applications in the medical field. In this study, the marine bacterium Vibrio sp. ZG1, which can degrade HA, was isolated, leading to the discovery of two novel hyaluronan lyases, Vhylzx1 and Vhylzx2, through genome sequencing and bioinformatic analysis. These lyases belong to the polysaccharide lyase-8 family. Vhylzx1 and Vhylzx2 specifically degrade HA, with highest activity at 35 °C, pH 5.7 and 50 °C, pH 7.1. Vhylzx1 and Vhylzx2 are endo-type enzymes that can fully degrade HA into unsaturated disaccharides. Sequence homology assessment and site-directed mutagenesis revealed that the catalytic residues of Vhylzx1 are Asn231, His281, and Tyr290, and that the catalytic residues of Vhylzx2 are Asn227, His277, and Tyr286. Moreover, this study used consensus sequences to enhance the specific activity of Vhylzx2 mutants. Notably, the mutants V564I, N742D, L619F, and D658G increases the specific activity by 2.4, 2.2, 1.3, and 1.2-fold. These characteristics are useful for further basic research and applications, and have a promising application in the preparation of biologically active hyaluronic acid oligosaccharides.

Cloning and characterization of a hyaluronate lyase EsHyl8 from Escherichia sp. A99.

Hyaluronidase, an enzyme that degrades hyaluronic acid (HA), is utilized in clinical settings to facilitate drug diffusion, manage extravasation, and address injection-related complications linked to HA-based fillers. In this study, a novel hyaluronate lyase EsHyl8 was cloned, expressed, and characterized from Escherichia sp. A99 of human intestinal origin. This lyase belongs to polysaccharide lyase (PL) family 8, and showed specific activity towards HA. EsHyl8 exhibited optimal degradation at 40 °C and pH 6.0. EsHyl8 exhibited a high activity of 376.32 U/mg among hyaluronidases of human gut microorganisms. EsHyl8 was stable at 37 °C and remained about 70 % of activity after incubation at 37 °C for 24 h, demonstrating excellent thermostability. The activity of EsHyl8 was inhibited by Zn2+, Cu2+, Fe3+, and SDS. EsHyl8 was an endo-type enzyme whose end-product was unsaturated disaccharide. This study enhances our understanding of hyaluronidases from human gut microorganisms.

The structure of a pectin-active family 1 polysaccharide lyase from the marine bacterium Pseudoalteromonas fuliginea.

Pseudoalteromonas fuliginea sp. PS47 is a recently identified marine bacterium that has extensive enzymatic machinery to metabolize polysaccharides, including a locus that targets pectin-like substrates. This locus contains a gene (locus tag EU509_03255) that encodes a pectin-degrading lyase, called PfPL1, that belongs to polysaccharide lyase family 1 (PL1). The 2.2 Šresolution X-ray crystal structure of PfPL1 reveals the compact parallel ß-helix fold of the PL1 family. The back side of the core parallel ß-helix opposite to the active site is a meandering set of five α-helices joined by lengthy loops. A comparison of the active site with those of other PL1 enzymes suggests a catalytic mechanism that is independent of metal ions, such as Ca2+, but that substrate recognition may require metal ions. Overall, this work provides the first structural insight into a pectinase of marine origin and the first structure of a PL1 enzyme in subfamily 2.

Impact of dietary Laminaria digitata with alginate lyase or carbohydrase mixture on nutrient digestibility and gut health of weaned piglets.

Laminaria digitata is a brown seaweed rich in prebiotic polysaccharides, mainly laminarin, but its alginate-rich cell wall could compromise nutrient access. Carbohydrase supplementation, such as individual alginate lyase and carbohydrases mixture (Rovabio® Excel AP), could enhance nutrient digestibility and prebiotic potential. This study aimed to evaluate the effect of these enzymes on nutrient digestibility and gut health of weaned piglets fed with 10% L. digitata. Diets did not affect growth performance (P > 0.05). The majority of the feed fractions had similar digestibility across all diets, but the supplementation of alginate lyase increased hemicellulose digestibility by 3.3% compared to the control group (P = 0.047). Additionally, we observed that algal zinc was more readily available compared to the control group, even without enzymatic supplementation (P < 0.001). However, the increased digestibility of some minerals, such as potassium, raises concerns about potential mineral imbalance. Seaweed groups had a higher abundance of beneficial bacteria in colon contents, such as Prevotella, Oscillospira and Catenisphaera. Furthermore, the addition of alginate lyase led to a lower pH in the colon (P < 0.001) and caecum (P < 0.001) of piglets, which is possibly a result of released fermentable laminarin, and is consistent with the higher proportion of butyric acid found in these intestinal compartments. L. digitata is a putative supplement to enhance piglet gut health due to its prebiotic polysaccharides. Alginate lyase supplementation further improves nutrient digestibility and prebiotic potential. These results suggest the potential use of L. digitata and these enzymatic supplements in commercial piglet-feeding practices.

The two-step strategy for enhancing the specific activity and thermostability of alginate lyase AlyG2 with mechanism for improved thermostability.

To overcome the trade-off challenge encountered in the engineering of alginate lyase AlyG2 from Seonamhaeicola algicola Gy8T and to expand its potential industrial applications, we devised a two-step strategy encompassing activity enhancement followed by thermal stability engineering. To enhance the specific activity of efficient AlyG2, we strategically substituted residues with bulky steric hindrance proximal to the active pocket with glycine or alanine. This led to the generation of three promising positive mutants, with particular emphasis on the T91S mutant, exhibiting a 1.91-fold specific activity compared to the wild type. To mitigate the poor thermal stability of T91S, mutants with negative ΔΔG values in the thermal flexibility region were screened out. Notably, the S72Ya mutant not only displayed 17.96 % further increase in specific activity but also exhibited improved stability compared to T91S, manifesting as a remarkable 30.97 % increase in relative activity following a 1-hour incubation at 42 °C. Furthermore, enhanced kinetic stability was observed. To gain deeper insights into the mechanism underlying the enhanced thermostability of the S72Ya mutant, we conducted molecular dynamics simulations, principal component analysis (PCA), dynamic cross-correlation map (DCCM), and free energy landscape (FEL) analysis. The results unveiled a reduction in the flexibility of the surface loop, a stronger correlation dynamic and a narrower motion subspace in S72Ya system, along with the formation of more stable hydrogen bonds. Collectively, our findings suggest amino acids substitutions resulting in smaller side chains proximate to the active site can positively impact enzyme activity, while reducing the flexibility of surface loops emerges as a pivotal factor in conferring thermal stability. These insights offer valuable guidance and a framework for the engineering of other enzyme types.

Discovery of a class of glycosaminoglycan lyases with ultrabroad substrate spectrum and their substrate structure preferences.

Glycosaminoglycan (GAG) lyases are often strictly substrate specific, and it is especially difficult to simultaneously degrade GAGs with different types of glycosidic bonds. Herein, we found a new class of GAG lyases (GAGases) from different bacteria. These GAGases belong to polysaccharide lyase 35 family and share quite low homology with the identified GAG lyases. The most surprising thing is that GAGases can not only degrade three types of GAGs: hyaluronan, chondroitin sulfate, and heparan sulfate but also even one of them can also degrade alginate. Further investigation of structural preferences revealed that GAGases selectively act on GAG domains composed of non/6-O-/N-sulfated hexosamines and d-glucoronic acids as well as on alginate domains composed of d-mannuronic acids. In addition, GAG lyases were once speculated to have evolved from alginate lyases, but no transitional enzymes have been found. The discovery of GAGases not only broadens the category of GAG lyases, provides new enzymatic tools for the structural and functional studies of GAGs with specific structures, but also provides candidates for the evolution of GAG lyases.

Unveiling novel insights into Bacillus velezensis 16B pectin lyase for improved fruit juice processing.

Microbial pectinolytic enzymes are important in various industries, particularly food processing. This study focuses on uncovering insights into a novel pectin lyase, BvPelB, from Bacillus velezensis 16B, with the aim of enhancing fruit juice processing. The study examines the structural and functional characteristics of pectinolytic enzyme, underscoring the critical nature of substrate specificity and enzymatic reaction mechanisms. BvPelB was successfully expressed and purified, exhibiting robust activity under alkaline conditions and thermal stability. Structural analysis revealed similarities with other pectin lyases, despite limited sequence identity. Biochemical characterization showed BvPelB's preference for highly methylated pectins and its endo-acting mode of cleavage. Treatment with BvPelB significantly increased juice yield and clarity without generating excessive methanol, making it a promising candidate for fruit juice processing. Overall, this study provides valuable insights into the enzymatic properties of BvPelB and its potential industrial applications in improving fruit juice processing efficiency and quality.

A "Pro-Asp-Thr" Amino Acid Repeat from Vibrio sp. QY108 Alginate Lyase Exhibits Alginate-Binding Capacity and Enhanced Soluble Expression and Thermostability.

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.

Effect of enzymatic modification on the structure and rheological properties of diluted alkali-soluble pectin fraction rich in RG-I.

This study focuses on pectin covalently linked in cell walls from two sources, apples and carrots, that was extracted using diluted alkali, and it describes changes in the rheological properties of diluted alkali-soluble pectin (DASP) due to enzymatic treatment. Given DASP's richness of rhamnogalacturonan I (RG-I), RG-I acetyl esterase (RGAE), rhamnogalacturonan endolyase (RGL), and arabinofuranosidase (ABF) were employed in various combinations for targeted degradation of RG-I pectin chains. Enzymatic degradations were followed by structural studies of pectin molecules using atomic force microscopy (AFM) as well as measurements of rheological and spectral properties. AFM imaging revealed a significant increase in the length of branched molecules after incubation with ABF, suggesting that arabinose side chains limit RG-I aggregation. Structural modifications were confirmed by changes in the intensity of bands in the pectin fingerprint and anomeric region on Fourier transform infrared spectra. ABF treatment led to a decrease in the stability of pectic gels, while the simultaneous use of ABF, RGAE, and RGL enzymes did not increase the degree of aggregation compared to the control sample. These findings suggest that the association of pectin chains within the DASP fraction may rely significantly on intermolecular interactions. Two mechanisms are proposed, which involve side chains as short-range attachment points or an extended linear homogalacturonan conformation favoring inter-chain interactions over self-association.

Enhancing Acid Resistance of Aspergillus niger Pectin Lyase through Surface Charge Design for Improved Application in Juice Clarification.

Pectin lyases (PNLs) can enhance juice clarity and flavor by degrading pectin in highly esterified fruits, but their inadequate acid resistance leads to rapid activity loss in juice. This study aimed to improve the acid resistance of Aspergillus niger PNL pelA through surface charge design. A modification platform was established by fusing pelA with a protein tag and expressing the fusion enzyme in Escherichia coli. Four single-point mutants were identified to increase the surface charge using computational tools. Moreover, the combined mutant M6 (S514D/S538E) exhibited 99.8% residual activity at pH 3.0. The M6 gene was then integrated into the A. niger genome using a multigene integration system to obtain the recombinant PNL AM6. Notably, AM6 improved the light transmittance of orange juice to 45.3%, which was 8.39 times higher than that of pelA. In conclusion, AM6 demonstrated the best-reported acid resistance, making it a promising candidate for industrial juice clarification.

Biochemical Characterization of a Novel Thermostable Ulvan Lyase from Tamlana fucoidanivorans CW2-9.

Ulvan is a complex sulfated polysaccharide extracted from Ulva, and ulvan lyases can degrade ulvan through a ß-elimination mechanism to obtain oligosaccharides. In this study, a new ulvan lyase, EPL15085, which belongs to the polysaccharide lyase (PL) 28 family from Tamlana fucoidanivorans CW2-9, was characterized in detail. The optimal pH and salinity are 9.0 and 0.4 M NaCl, respectively. The Km and Vmax of recombinant EPL15085 toward ulvan are 0.80 mg·mL-1 and 11.22 µmol·min -1 mg-1·mL-1, respectively. Unexpectedly, it is very resistant to high temperatures. After treatment at 100 °C, EPL15085 maintained its ability to degrade ulvan. Molecular dynamics simulation analysis and site-directed mutagenesis analysis indicated that the strong rigidity of the disulfide bond between Cys74-Cys102 in the N-terminus is related to its thermostability. In addition, oligosaccharides with disaccharides and tetrasaccharides were the end products of EPL15085. Based on molecular docking and site-directed mutagenesis analysis, Tyr177 and Leu134 are considered to be the crucial residues for enzyme activity. In conclusion, our study identified a new PL28 family of ulvan lyases, EPL15085, with excellent heat resistance that can expand the database of ulvan lyases and provide the possibility to make full use of ulvan.

High-Level Extracellular Production of a Trisaccharide-Producing Alginate Lyase AlyC7 in Escherichia coli and Its Agricultural Application.

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.

Rational design of PspAlgL to improve its thermostability and anti-biofilm activity against Pseudomonas aeruginosa.

Pseudomonas aeruginosa biofilm enhances tolerance to antimicrobials and immune system defenses. Alginate is an important component of biofilm and a virulence factor of P. aeruginosa. The degradation of alginate by alginate lyases has come to serve as an adjunctive therapeutic strategy against P. aeruginosa biofilm, but poor stability of the enzyme limited this application. Thus, PspAlgL, an alginate lyase, can degrade acetylated alginate but has poor thermostability. The 3D structure of PspAlgL was predicted, and the thermostability of PspAlgL was rationally designed by GRAPE strategy, resulting in two variants with better stability. These variants, PspAlgLS270F/E311P and PspAlgLG291S/E311P, effectively degraded the alginate in biofilm. In addition, compared with PspAlgL, these variants were more efficient in inhibiting biofilm formation and degrading the established biofilm of P. aeruginosa PAO1, and they were also able to destroy the biofilm attached to catheters and to increase the sensitivity of P. aeruginosa to the antibiotic amikacin. This study provides one potential anti-biofilm agent for P. aeruginosa infection.

Efficacious bioconversion of alginate/cellulose to value-added oligosaccharides by alginate-degrading GH5 endoglucanase from Trichoderma asperellum.

Enzymatic degradation of lignocellulosic biomass provides an eco-friendly approach to produce value-added macromolecules, e.g., bioactive polysaccharides. A novel acidophilic GH5 ß-1,4-endoglucanase (termed TaCel5) from Trichoderma asperellum ND-1 was efficiently expressed in Komagataella phaffii (∼1.5-fold increase, 38.42 U/mL). TaCel5 displayed both endoglucanase (486.3 U/mg) and alginate lyase (359.5 U/mg) enzyme activities. It had optimal pH 3.0 and strong pH stability (exceed 86 % activity retained over pH range 3.0-5.0). 80 % activity (both endoglucanase and alginate lyase) was retained in the presence of 15 % ethanol or 3.42 M NaCl. Analysis of action mode revealed that hydrolytic activity of TaCel5 required at least three glucose (cellotriose) residues, yielding mainly cellobiose. Glu241 and Glu352 are essential catalytic residues, while Asp106, Asp277 and Asp317 play auxiliary roles in cellulose degradation. TaCel5 displayed high hydrolysis efficiency for glucan and alginate substrates. ESI-MS analysis indicated that the enzymatic hydrolysates of alginate mainly contained disaccharides and heptasaccharides. This is the first detailed report of a bifunctional GH5 endoglucanase/alginate lyase enzyme from T. asperellum. Thus TaCel5 has strong potential in food and feed industries as a catalyst for bioconversion of cellulose- and alginate-containing waste materials into value-added products oligosaccharides, which was of great benefit both for the economy and environment.

Construction and characterization of a novel fusion alginate lyase with endolytic and exolytic cleavage activity for industrial preparation of alginate oligosaccharides.

Alginate lyases with high activity and good thermostability are lacking for the preparation of alginate oligosaccharides (AOS) with various biological activities. We constructed a fusion alginate lyase with both endo-and exo-activities. AlyRm6A-Zu7 was successfully constructed by connecting the highly thermostable AlyRm6A to a new exotype lyase, AlyZu7. The fusion enzyme exhibited high catalytic activity and thermostability. It transformed sodium alginate into oligosaccharides with degrees of polymerization (DP) of 2-4 while producing 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). The maximum reducing sugar, AOS, and DP1 + DEH yields were 75 %, 45 %, and 40 %, respectively. Molecular docking confirmed the formation of a stable complex between the substrate and AlyRm6A-Zu7. Protein interactions increased the thermostability of AlyZu7. This work provides new insights into the industrial formation of AOS and monosaccharide DEH using thermally stable fusion enzymes, which has a positive effect in the fields of functional oligosaccharide production and biofuel formation.

In vitro effects of alginate lyase SG4 + produced by Paenibacillus lautus alone and combined with antibiotics on biofilm formation by mucoid Pseudomonas aeruginosa.

Alginate is a major extra polymeric substance in the biofilm formed by mucoid Pseudomonas aeruginosa. It is the main proven perpetrator of lung infections in patients suffering from cystic fibrosis. Alginate lyases are very important in the treatment of cystic fibrosis. This study evaluated the role of standalone and in conjugation, effect of alginate lyase of SG4 + isolated from Paenibacillus lautus in enhancing in vitro bactericidal activity of gentamicin and amikacin on mucoid P. aeruginosa. Using Response Surface Methodology (RSM) alginate lyase SG4 + production was optimized in shake flask and there 8.49-fold enhancement in enzyme production. In fermenter, maximum growth (10.15 mg/ml) and alginate lyase (1.46 International Units) production, 1.71-fold was increased using Central Composite Design (CCD). Further, fermentation time was reduced from 48 to 20 h. To the best of our knowledge this is the first report in which CCD was used for fermenter studies to optimize alginate lyase production. The Km and Vmax of purified enzyme were found to be 2.7 mg/ml and 0.84 mol/ml-min, respectively. The half-life (t 1/2) of purified alginate lyase SG4 + at 37 °C was 180 min. Alginate lyase SG4 + in combination with gentamicin and amikacin eradiated 48.4- 52.3% and 58- 64.6%, alginate biofilm formed by P. aeruginosa strains, respectively. The study proves that alginate lyase SG4 + has excellent exopolysaccharide disintegrating ability and may be useful in development of potent therapeutic agent to treat P. aeruginosa biofilms.

Rhamnogalacturonan lyase 1 (RGL1), as a suppressor of E3 ubiquitin ligase Arabidopsis thaliana ring zinc finger 1 (AtRZF1), is involved in dehydration response to mediate proline synthesis and pectin rhamnogalacturonan-I composition.

Proline metabolism plays a crucial role in both environmental stress responses and plant growth. However, the specific mechanism by which proline contributes to abiotic stress processes remains to be elucidated. In this study, we utilized atrzf1 (Arabidopsis thaliana ring zinc finger 1) as a parental line for T-DNA tagging mutagenesis and identified a suppressor mutant of atrzf1, designated proline content alterative 31 (pca31). The pca31 mutant suppressed the insensitivity of atrzf1 to dehydration stress during early seedling growth. Using Thermal Asymmetric Interlaced-PCR, we found that the T-DNA of pca31 was inserted into the promoter region of the At2g22620 gene, which encodes the cell wall enzyme rhamnogalacturonan lyase 1 (RGL1). Enzymatic assays indicated that RGL1 exhibited rhamnogalacturonan lyase activity, influencing cell wall pectin composition. The decrease in RGL1 gene expression suppressed the transcriptomic perturbation of the atrzf1 mutant. Silencing of the RGL1 gene in atrzf1 resulted in a sensitive phenotype similar to pca31 under osmotic stress conditions. Treatment with mannitol, salt, hydrogen peroxide, and abscisic acid induced RGL1 expression. Furthermore, we uncovered that RGL1 plays a role in modulating root growth and vascular tissue development. Molecular, physiological, and genetic experiments revealed that the positive modulation of RGL1 during abiotic stress was linked to the AtRZF1 pathway. Taken together, these findings establish that pca31 acts as a suppressor of atrzf1 in abiotic stress responses through proline and cell wall metabolisms.

Expression and Characterization of an Efficient Alginate Lyase from Psychromonas sp. SP041 through Metagenomics Analysis of Rotten Kelp.

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.