Alginate Lyase Research Articles

Exolytic products of alginate by the immobilized alginate lyase confer antioxidant and antiapoptotic bioactivities in human umbilical vein endothelial cells

Alginate is a natural polysaccharide resource abundant in brown algae and it can be cleaved into alginate oligosaccharides by alginate lyase. Alginate lyases and the bioactive alginate oligosaccharides have been applied in diverse fields such as pharmaceutical therapy and nutraceutical supplementation. Immobilized enzymes greatly facilitate their industrial application owing to their reusability, stability, and tunability. In this study, magnetic Fe3O4 nanoparticles were synthesized and used to immobilize an exolytic alginate lyase AlgL17 that was characterized previously. The immobilized AlgL17 demonstrated enhanced thermal and pH tolerance, extended storage stability, and moderate reusability. The mass spectrum indicated the specific activity of the immobilized AlgL17 to release alginate oligosaccharides (AOS) from alginate polysaccharide. The produced AOS exhibited their antioxidant and antiapoptotic activities in H2O2-stressed human umbilical vein endothelial cells by upregulation of reactive oxygen species scavenging activities and attenuation of the caspase-mediated apoptosis pathway.

Characterization of a New Intracellular Alginate Lyase with Metal Ions-Tolerant and pH-Stable Properties.

Alginate lyases play an important role in alginate oligosaccharides (AOS) preparation and brown seaweed processing. Many extracellular alginate lyases have been characterized to develop efficient degradation tools needed for industrial applications. However, few studies focusing on intracellular alginate lyases have been conducted. In this work, a novel intracellular alkaline alginate lyase Alyw202 from Vibrio sp. W2 was cloned, expressed and characterized. Secretory expression was performed in a food-grade host, Yarrowia lipolytica. Recombinant Alyw202 with a molecular weight of approximately 38.3 kDa exhibited the highest activity at 45 °C and more than 60% of the activity in a broad pH range of 3.0 to 10.0. Furthermore, Alyw202 showed remarkable metal ion-tolerance, NaCl independence and the capacity of degrading alginate into oligosaccharides of DP2-DP4. Due to the unique pH-stable and high salt-tolerant properties, Alyw202 has potential applications in the food and pharmaceutical industries.

Characterization of a New Bifunctional and Cold-Adapted Polysaccharide Lyase (PL) Family 7 Alginate Lyase from Flavobacterium sp.

Alginate oligosaccharides produced by enzymatic degradation show versatile physiological functions and biological activities. In this study, a new alginate lyase encoding gene alyS02 from Flavobacterium sp. S02 was recombinantly expressed at a high level in Yarrowia lipolytica, with the highest extracellular activity in the supernatant reaching 36.8 ± 2.1 U/mL. AlyS02 was classified in the polysaccharide lyase (PL) family 7. The optimal reaction temperature and pH of this enzyme were 30 °C and 7.6, respectively, indicating that AlyS02 is a cold-adapted enzyme. Interestingly, AlyS02 contained more than 90% enzyme activity at 25 °C, higher than other cold-adapted enzymes. Moreover, AlyS02 is a bifunctional alginate lyase that degrades both polyG and polyM, producing di- and trisaccharides from alginate. These findings suggest that AlyS02 would be a potent tool for the industrial applications.

Isolation, identification, and whole genome sequence analysis of the alginate-degrading bacterium Cobetia sp. cqz5-12

Alginate-degrading bacteria or alginate lyases can be used to oligomerize alginate. In this study, an alginate-degrading bacterium with high alginolytic activity was successfully screened by using Sargassum fusiforme sludge. When the strain was grown on a plate containing sodium alginate, the transparent ring diameter (D) was 2.2 cm and the ratio (D/d) of transparent ring diameter to colony diameter (d) was 8.8. After 36 h in culture at a temperature of 28 °C shaken at 150 r/min, the enzymatic activity of the fermentation supernatant reached 160 U/mL, and the enzymatic activity of the bacterial precipitate harvested was 2,645 U/mL. The strain was named Cobetia sp. cqz5-12. Its genome is circular in shape, 4,209,007 bp in size, with a 62.36% GC content. It contains 3,498 predicted coding genes, 72 tRNA genes, and 21 rRNA genes. The functional annotations for the coding genes demonstrated that there were 181 coding genes in the genome related to carbohydrate transport and metabolism and 699 coding genes with unknown functions. Three putative coding genes, alg2107, alg2108 and alg2112, related to alginate degradation were identified by analyzing the carbohydrate active enzyme (CAZy) database. Moreover, proteins Alg2107 and Alg2112 were successfully expressed and exhibited alginate lyase activity.

Unveiling the CAZyme repertoire of a sponge-derived Pseudoalteromonas sp. strain for the degradation of marine polysaccharides

Despite their well‐known importance to the holobiont homeostasis, carbohydrate‐active enzymes (CAZymes) from the sponge microbiome have to date been largely understudied from an industrial perspective. Enzymes that degrade marine polysaccharides (MPs) are receiving increasing attention as alternatives in the biorefinery sector and for use in the generation of high‐value‐added hydrolysis products. Screening of the agarolytic Pseudoalteromonas sp. strain PA2MD11, isolated from the Brazilian sponge Plakina cyanorosea, on selective media indicated that the strain degrades agarose, κ‐carrageenan and sodium alginate. Subsequently in silico analysis of the strain's genome indicated that around 3 % of the total genome encoded for CAZymes. These consisted mostly of glycoside hydrolases (GH) and glycosyltransferases (GT). Four agarase genes were also present, three from the family GH50 and one from the family GH16, with sequences from other free‐living and psychrophile Pseudoalteromonas genomes as its closest relatives. These agarases are structurally related to exo‐(GH50) and endo‐β‐agarases (GH16), and appear to encode for a complete agarolytic pathway. The two alginate lyases were classified as polysaccharide lyases (PL) from the families 6 and 17, with chondroitin‐ and heparin‐like domains, respectively, and shared common homologues sequences with an estuarine Pseudoalteromonas lipolytica strain. Data on the cloning and heterologous expression of these genes will be presented. To our knowledge, this is the first time that a “genome mining” based approach has been undertaken to identify these groups of MPs‐degrading CAZymes from cultivable members of the sponge microbiome.

Unveiling epidemiology and treatment strategies for efficient management of lung infection in cystic fibrosis

Cystic Fibrosis (CF) is a genetic disorder which results in abnormal Cystic Fibrosis Transmembrane Regulator (CFTR) protein. CFTR protein actively transports chloride and bicarbonate out of cells which produce mucus in epithelium of various organs. However, when there is an abnormality in this protein then ion and water transport across airway surface is affected and form viscous mucus due to dehydration. This disease affects lung, pancreases, intestine and hepatobiliary system. Infection of lungs is main reason of high morbidity and mortality. Patients with CF are more prone to bacterial infection of the respiratory tract. Mainly four bacterial pathogens are found in the respiratory tract of CF patient viz. Staphylococcus aureus, Haemophilus influenzae, Burkholderia cepacia, Pseudomonas aeruginosa. Mucoid P. aeruginosa is the most dangerous pathogen among them due to ability to form biofilm. There are many conventional and new treatment options available for CF patients. New therapies are used to correct the CFTR protein and antibiotic treatment is used to prevent or early infection or suppression of chronic infection. However, when P. aeruginosa turn into mucoid form, formation of extracellular polysaccharide biofilm starts with alginate as major constituent. The antibiotics fail to reach the target as EPS of the biofilm shield the microbe from antibiotics action. Alginate lyase, an alginate degrading has the ability to break the alginate and subsequently increases the efficacy of antibiotics therapy. The co-administration of alginate lyase and Dnase along with antibiotics has proved to be very efficient in the management of patients with CF.

Expression and Characterization of an Alginate Lyase and Its Thermostable Mutant in Pichia pastoris.

Alginate is one of the most abundant polysaccharides in algae. Alginate lyase degrades alginate through a β-elimination mechanism to produce alginate oligosaccharides with special bioactivities. Improving enzyme activity and thermal stability can promote the application of alginate lyase in the industrial preparation of alginate oligosaccharides. In this study, the recombinant alginate lyase cAlyM and its thermostable mutant 102C300C were expressed and characterized in Pichia pastoris. The specific activities of cAlyM and 102C300C were 277.1 U/mg and 249.6 U/mg, respectively. Both enzymes showed maximal activity at 50 °C and pH 8.0 and polyG preference. The half-life values of 102C300C at 45 °C and 50 °C were 2.6 times and 11.7 times the values of cAlyM, respectively. The degradation products of 102C300C with a lower degree of polymerization contained more guluronate. The oligosaccharides with a polymerization degree of 2–4 were the final hydrolytic products. Therefore, 102C300C is potentially valuable in the production of alginate oligosaccharides with specific M/G ratio and molecular weights.

Characterization of different alginate lyases for dissolving Pseudomonas aeruginosa biofilms

Aggregates of Pseudomonas aeruginosa form a protective barrier against antibiotics and the immune system. These barriers, known as biofilms, are associated with several infectious diseases. One of the main components of these biofilms is alginate, a homo- and hetero-polysaccharide that consists of β-D-mannuronate (M) and α-L-guluronate (G) units. Alginate lyases degrade this sugar and have been proposed as biotherapeutic agents to dissolve P. aeruginosa biofilms. However, there are contradictory reports in the literature regarding the efficacy of alginate lyases against biofilms and their synergistic effect with antibiotics. We found that most positive reports used a commercial crude extract from Flavobacterium multivorum as the alginate lyase source. By using anion exchange chromatography coupled to nano LC MS/MS, we identified two distinct enzymes in this extract, one has both polyM and polyG (polyM/G) degradation activities and it is similar in sequence to a broad-spectrum alginate lyase from Flavobacterium sp. S20 (Alg2A). The other enzyme has only polyG activity and it is similar in sequence to AlyA1 from Zobellia galactanivorans. By characterizing both of these enzymes together with three recombinant alginate lyases (a polyM, a polyG and a polyM/G), we showed that only enzymes with polyM/G activity such as Alg2A and A1-II’ (alginate lyase from Sphingomonas sp.) are effective in dissolving biofilms. Furthermore, both activities are required to have a synergistic effect with antibiotics.

Recent progress on engineering microbial alginate lyases towards their versatile role in biotechnological applications.

Biomass feedstock is an efficient and harmless source of energy. There are various sources of feedstock, such as plant, microbial, macro, and microalgae, and agricultural waste. The major component in biomass feedstock material is a polysaccharide, such as cellulose, cellobiose, starch, and alginate. Alginate is mainly found in macroalgae as one of the significant polysaccharide components. It is made up of β-d-mannuronate (M) and α-l-guluronate (G) blocks. Alginate lyase is an enzyme that degrades alginate by breaking the glycosidic linkage between the poly M and G blocks to liberate oligosaccharides. Several organisms, including bacteria, fungi, viruses, and algae can produce alginate lyases. The species of bacteria, such as Bacillus, Vibrio, Pseudomonas, and Microbulbifer, are some of the important sources of alginate lyases. They are industrially essential enzymes used in food, biofuel, and biomedical industries. There are various assays available to determine the alginate lyase activity qualitatively as well as quantitatively. Qualitatively, different dyes like Gram's iodine, cetyl pyridinium chloride, and rutanium red can be used to visualize the zone formed due to the alginate lyase activity. DNS assay, UV absorption, and the Somogyi-Nelson method help to determine the alginate lyase activity quantitatively. Since the alginate lyase production in the native organisms is relatively lower, the genes encoding alginate lyases are heterologously cloned and expressed in E. coli to maximize the production and to characterize the enzyme. Different chromatographic techniques like size exclusion, affinity, gel permeation, and ion-exchange chromatography are used to purify the protein. In this paper, the source of alginate and alginate lyases, the mechanism of action of the enzyme, the engineering approaches to enhance the enzyme production, its purification strategy, and the potential applications of alginate lyases has been discussed.

Functional Characterization of a Novel Oligoalginate Lyase of Stenotrophomonas maltophilia KJ-2 Using Site-Specific Mutation Reveals Bifunctional Mode of Action, Possessing Both Endolytic and Exolytic Degradation Activity Toward Alginate in Seaweed Biomass

A novel oligoalginate lyase from the marine bacterium, Stenotrophomonas maltophilia KJ-2, completely depolymerizes alginate to monomers with generation of oligomer intermediates. In order to analyze whether KJ-2 oligoalginate lyase has endolytic and exolytic alginate lyase activities, five mutants were developed and characterized using homology modeling based on the crystal structure of Alg17c, an exolytic oligoalginate lyase from Saccharophagus degradans 2-40. The important residues of Tyr238, Arg241, Arg418 and Glu644, which were predicted to be located within hydrogen bonding distance of the non-reducing end of alginate during exolytic catalysis, were mutated to Phe, Ala, Ala and Ala, respectively. Exolytic activities of Arg241Ala, Glu644Ala and Arg241Ala_Glu644Ala mutants decreased compared to the wild type, indicating that Arg241 and Glu644 are key residues for exolytic catalysis. Interestingly, these mutants produced oligomers as the main product due to inherent endolytic degradation activity. These mutational characterization results showed that KJ-2 oligoalginate lyase possesses catalytic domains for both inherent endolytic and exolytic activities.

Enzyme-Assisted Fucoidan Extraction from Brown Macroalgae Fucus distichus subsp. evanescens and Saccharina latissima.

Fucoidans from brown macroalgae (brown seaweeds) have different structures and many interesting bioactivities. Fucoidans are classically extracted from brown seaweeds by hot acidic extraction. Here, we report a new targeted enzyme-assisted methodology for fucoidan extraction from brown seaweeds. This enzyme-assisted extraction protocol involves a one-step combined use of a commercial cellulase preparation (Cellic®CTec2) and an alginate lyase from Sphingomonas sp. (SALy), reaction at pH 6.0, 40 °C, removal of non-fucoidan polysaccharides by Ca2+ precipitation, and ethanol-precipitation of crude fucoidan. The workability of this method is demonstrated for fucoidan extraction from Fucus distichus subsp. evanescens (basionym Fucus evanescens) and Saccharina latissima as compared with mild acidic extraction. The crude fucoidans resulting directly from the enzyme-assisted method contained considerable amounts of low molecular weight alginate, but this residual alginate was effectively removed by an additional ion-exchange chromatographic step to yield pure fucoidans (as confirmed by 1H NMR). The fucoidan yields that were obtained by the enzymatic method were comparable to the chemically extracted yields for both F. evanescens and S. latissima, but the molecular sizes of the fucoidans were significantly larger with enzyme-assisted extraction. The molecular weight distribution of the fucoidan fractions was 400 to 800 kDa for F. evanescens and 300 to 800 kDa for S. latissima, whereas the molecular weights of the corresponding chemically extracted fucoidans from these seaweeds were 10–100 kDa and 50–100 kDa, respectively. Enzyme-assisted extraction represents a new gentle strategy for fucoidan extraction and it provides new opportunities for obtaining high yields of native fucoidan structures from brown macroalgae.

Biochemical characteristics and molecular mechanism of an exo-type alginate lyase VxAly7D and its use for the preparation of unsaturated monosaccharides

BackgroundAs the most abundant polysaccharide in brown algae, alginate has become a promising economical material for bioethanol production. Recently, exo-type alginate lyases have received extensive attention because the unsaturated monosaccharides produced by their degradation of alginate can be easily converted into 4-deoxy-l-erythro-5-hexoseulose uronate (DEH), a promising material for bioethanol production and biorefinery systems.ResultsIn this study, we cloned and characterized an exo-type polysaccharide lyase family 7 (PL7) alginate lyase VxAly7D from the marine bacterium Vibrio xiamenensis QY104. Recombinant VxAly7D was most active at 30 °C and exhibited 21%, 46% and 90% of its highest activity at 0, 10 and 20 °C, respectively. Compared with other exo-type alginate lyases, recombinant VxAly7D was shown to be a bifunctional alginate lyase with higher specific activity towards sodium alginate, polyG and polyM (462.4 ± 0.64, 357.37 ± 0.53 and 441.94 ± 2.46 U/mg, respectively). A total of 13 μg recombinant VxAly7D could convert 3 mg sodium alginate to unsaturated monosaccharides in 1 min with a yield of 37.6%, and the yield reached 95% in 1 h. In addition, the three-dimensional structure of VxAly7D was modelled using the crystal structure of AlyA5 from Zobellia galactanivorans DsijT as the template. The action mode and the end products of the W295A mutant revealed that Trp295 is a key amino acid residue responsible for the exolytic action mode of VxAly7D.ConclusionOverall, our results show that VxAly7D is a PL7 exo-type alginate lyase with high activity and a high conversion rate at low/moderate temperatures, which provides a useful enzymatic tool for the development of biofuel production from brown algae and enriches the understanding of the structure and functional relationships of polysaccharide lyases.

Disruption of the extracellular polymeric network of Pseudomonas aeruginosa biofilms by alginate lyase enhances pathogen eradication by antibiotics

BackgroundPseudomonas aeruginosa forms antibiotic-resistant biofilms that are responsible for the treatment failure or relapses of the bacterial infections in the lungs of patients with cystic fibrosis (CF). The alginate lyases that target extracellular polysaccharide alginate of P. aeruginosa biofilms are promising therapeutic candidates for treatment of P. aeruginosa biofilm infections. MethodsImmunofluorescent staining and thin layer chromatography were used to demonstrate the alginolytic activity of the alginate lyase enzyme (AlyP1400) purified from a marine Pseudoalteromonas bacterium. Anti-biofilm activities of AlyP1400 were tested alone or in combination with antibiotics on the biofilms of a mucoid Pseudomonas aeruginosa clinical isolate CF27 that were cultivated in 96-well plates and a flow cell. ResultsWe showed that AlyP1400 facilitated antibiotic activities to eliminate CF27 biofilms. The combination of AlyP1400 with antibiotics reduced the biofilm biomass and boosted bactericidal activity of antibiotics. Importantly, we demonstrated that the enzymatic activity of AlyP1400 was required for its biofilm disruption activity and its synergy with antibiotics to eradicate biofilm cells. ConclusionThis work shed new light on the potential mechanisms of the therapeutic activity for the combinational use of alginate lyase and antibiotics to treat P. aeruginosa infections in CF lungs or other P. aeruginosa biofilm-related infections.

Effects of a Newly Developed Enzyme-Assisted Extraction Method on the Biological Activities of Fucoidans in Ocular Cells.

Fucoidans from brown seaweeds are promising substances as potential drugs against age-related macular degeneration (AMD). The heterogeneity of fucoidans requires intensive research in order to find suitable species and extraction methods. Ten different fucoidan samples extracted enzymatically from Laminaria digitata (LD), Saccharina latissima (SL) and Fucus distichus subsp. evanescens (FE) were tested for toxicity, oxidative stress protection and VEGF (vascular endothelial growth factor) inhibition. For this study crude fucoidans were extracted from seaweeds using different enzymes and SL fucoidans were further separated into three fractions (SL_F1-F3) by ion-exchange chromatography (IEX). Fucoidan composition was analyzed by high performance anion exchange chromatography (HPAEC) after acid hydrolysis. The crude extracts contained alginate, while two of the fractionated SL fucoidans SL_F2 and SL_F3 were highly pure. Cell viability was assessed with an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay in OMM-1 and ARPE-19. Protective effects were investigated after 24 h of stress insult in OMM-1 and ARPE-19. Secreted VEGF was analyzed via ELISA (enzyme-linked immunosorbent assay) in ARPE-19 cells. Fucoidans showed no toxic effects. In OMM-1 SL_F2 and several FE fucoidans were protective. LD_SiAT2 (Cellic®CTec2 + Sigma-Aldrich alginate lyase), FE_SiAT3 (Cellic® CTec3 + Sigma-Aldrich alginate lyase), SL_F2 and SL_F3 inhibited VEGF with the latter two as the most effective. We could show that enzyme treated fucoidans in general and the fractionated SL fucoidans SL_F2 and SL_F3 are very promising for beneficial AMD relevant biological activities.

Hydrolyzing Laminaria japonica with a combination of microbial alginate lyase and cellulase

A novel seaweed-hydrolyzing strain designated as Pseudoalteromonas sp. Alg6B was isolated from the surface of brown seaweed (Laminaria japonica). The activity of crude alginate lyase produced by Alg6B was 54.5 U/ml and the main products of hydrolyzing alginate were disaccharide and tetrasaccharide. The hydrolysis rate of seaweed reached up to 97% after combining 3% (v/v) Alg6B and 0.2% (w/v) solid cellulase. In kelp hydrolysate, the contents of nutrients are much more than raw seaweed. Alg6B grows quickly and has the ability of producing alginate oligosaccharides with low molecular weight (MW) (≤2 kDa). Furthermore, this study demonstrates that a combination of microbial alginate lyase and cellulase could almost hydrolyze seaweed completely. This research indicated that Alg6B could provide a feasible pathway to produce alginate oligosaccharides (AOS), and the synergistic effect of alginate lyase and cellulase on seaweed bioconversion can potentially pave the way to the sustainable production of seaweed fertilizer.

Optimization of protoplast isolation from the gametophytes of brown alga Undaria pinnatifida using response surface methodology

The microscopic gametophytic phase of the commercial brown alga, Undaria pinnatifida, can be used for several applications, including the production of bioactive compounds, aquaculture and as germplasm bank. Therefore, gametophytes are good candidates for cellular biotechnology techniques, many of which rely on protoplasts (“naked” living plant cells). This study reports on the optimization of protoplast yield from male and female gametophytes of U. pinnatifida using different mixtures of commercial enzymes and chelation pre-treatment. Key conditions for achieving the highest protoplast yield, such as enzyme combinations, chelation pre-treatment, growth, temperature, incubation time, pH and osmolarity, were investigated. Protoplast isolation conditions were modelled by using response surface methodology (RSM) via Box-Behnken design (BBD) and subsequently experimentally verified in its predictability of protoplast production. The enzyme composition with 1% cellulase RS, 2 U mL−1 alginate lyase and 1% driselase with chelation pre-treatment, at 2481–2591 mOsm L−1 H2O and adjusted to pH 5.8–6.1, produced the highest protoplast yields of 3.12 ± 0.51 × 106 protoplasts g−1 fresh weight for male gametophyte and 2.11 ± 0.08 × 106 for female gametophyte when incubated at 20 °C for 4–7 h using cultures at mid or early exponential phase, respectively. These conditions also gave high amounts of protoplasts from other strains of Korea. Our results show the effectiveness of commercial enzymes combined with chelation pre-treatment in protoplast isolation and RSM with BBD is a useful method for rapidly producing the higher yields of protoplasts from brown alga.

Biochemical characterization and degradation pattern analysis of a novel PL-6 alginate lyase from Streptomyces coelicolor A3(2)

Alginate is the main component of brown algae which contributes to a huge biomass. The alginate oligosaccharides (AOs) have been widely used in food, cosmetic and pharmaceutical industries due to their various physiological activities. In this study, we expressed and characterized a novel PL-6 alginate lyase, named OUC-ScCD6. The results indicated that OUC-ScCD6 showed highest activity at 50 °C and pH 9.0. OUC-ScCD6 prefers to degrade poly M blocks and could digest poly G blocks as well. Endolytic action mode towards polysaccharides contributes to the creation of AOs with the degrees of polymerization 2–6. Degradation towards saturated oligosaccharides showed that saturated trisaccharides (M3 and G3) were minimum identifiable substrates. Furthermore, OUC-ScCD6 shows an even-numbered glycosidic bonds preference from non-reducing end which provided clearer insights into the substrate recognition and action mode of PL-6 family alginate lyases.

Biochemical and computational study of an alginate lyase produced by Pseudomonas aeruginosa strain S21.

Objective(s):Alginates play a key role in mucoid Pseudomonas aeruginosa colonization, biofilm formation, and driving out of cationic antibiotics. P. aeruginosa alginate lyase (AlgL) is a periplasmic enzyme that is necessary for alginate synthesis and secretion. It also has a role in depolymerization of alginates. Using AlgLs in cystic fibrosis patients along with antibiotics enhances bacterial killing and host healing. In this study, we investigated the different biochemical properties of a newly isolated AlgL from P. aeruginosa S21 to complete the databank of AlgLsMaterials and Methods:The enzyme was extracted from the periplasmic space of the bacteria by the heat shock method. Using the TBA method, the enzyme activity and biochemical properties were assessed. The mutability of P. aeruginosa S21 AlgL to increase its thermal stability was investigated. The most favorable mutations were studied computationally. The molecular dynamics simulation (MDS) package GROMACS was used for determining the effect of S34R mutation on enzyme’s thermal stability.Results:Data showed that this enzyme has the best activity at 37 °C and pH 7.5 and it can degrade mannuronate blocks, guluronate blocks, and sodium alginate. After 7 hr at 80 °C, 45% of the enzyme activity was retained. This enzyme needed 15 min to completely degrade accessible sodium alginate. Tris buffer, pH 8.5 and Britton-Robinson buffer, pH 7.0 were the preferable buffers for the enzyme activity. MDS of native and mutated enzymes showed desirable results.Conclusion: P. aeruginosa S21 AlgL can be used in medical and industrial applications to degrade alginates.

Cloning, Secretory Expression and Characterization of a Unique pH-Stable and Cold-Adapted Alginate Lyase.

Cold-adapted alginate lyases have unique advantages for alginate oligosaccharide (AOS) preparation and brown seaweed processing. Robust and cold-adapted alginate lyases are urgently needed for industrial applications. In this study, a cold-adapted alginate lyase-producing strain Vibrio sp. W2 was screened. Then, the gene ALYW201 was cloned from Vibrio sp. W2 and expressed in a food-grade host, Yarrowia lipolytica. The secreted Alyw201 showed the activity of 64.2 U/mL, with a molecular weight of approximate 38.0 kDa, and a specific activity of 876.4 U/mg. Alyw201 performed the highest activity at 30 °C, and more than 80% activity at 25–40 °C. Furthermore, more than 70% of the activity was obtained in a broad pH range of 5.0–10.0. Alyw201 was also NaCl-independent and salt-tolerant. The degraded product was that of the oligosaccharides of DP (Degree of polymerization) 2–6. Due to its robustness and its unique pH-stable property, Alyw201 can be an efficient tool for industrial production.

Purification and Characterization of a Novel Endolytic Alginate Lyase from Microbulbifer sp. SH-1 and Its Agricultural Application.

Alginate, an important acidic polysaccharide in marine multicellular algae, has attracted attention as a promising biomass resource for the production of medical and agricultural chemicals. Alginate lyase is critical for saccharification and utilization of alginate. Discovering appropriate and efficient enzymes for depolymerizing alginate into fermentable fractions plays a vital role in alginate commercial exploitation. Herein, a unique alginate lyase, AlgSH7, belonging to polysaccharide lyase 7 family is purified and characterized from an alginate-utilizing bacterium Microbulbifer sp. SH-1. The purified AlgSH7 shows a specific activity of 12,908.26 U/mg, and its molecular weight is approximately 66.4 kDa. The optimal temperature and pH of AlgSH7 are 40 °C and pH 9.0, respectively. The enzyme exhibits stability at temperatures below 30 °C and within an extensive pH range of 5.0–9.0. Metal ions including Na+, K+, Al3+, and Fe3+ considerably enhance the activity of the enzyme. AlgSH7 displays a preference for poly-mannuronic acid (polyM) and a very low activity towards poly-guluronic acid (polyG). TLC and ESI-MS analysis indicated that the enzymatic hydrolysates mainly include disaccharides, trisaccharides, and tetrasaccharides. Noteworthy, the alginate oligosaccharides (AOS) prepared by AlgSH7 have an eliciting activity against chilling stress in Chinese flowering cabbage (Brassica parachinensis L.). These results suggest that AlgSH7 has a great potential to design an effective process for the production of alginate oligomers for agricultural applications.