Search Results (19)

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. Full article

Alginate oligosaccharides (AOS) and their derivatives become popular due to their favorable biological activity, and the key to producing functional AOS is to find efficient alginate lyases. This study showed one alginate lyase TsAly7A found in Thalassomonas sp. LD5, which was predicted to have excellent industrial properties. Bioinformatics analysis and enzymatic properties of recombinant TsAly7A (rTsAly7A) were investigated. TsAly7A belonged to the fifth subfamily of polysaccharide lyase family 7 (PL7). The optimal temperature and pH of rTsAly7A was 30 °C and 9.1 in Glycine-NaOH buffer, respectively. The pH stability of rTsAly7A under alkaline conditions was pretty good and it can remain at above 90% of the initial activity at pH 8.9 in Glycine-NaOH buffer for 12 h. In the presence of 100 mM NaCl, rTsAly7A showed the highest activity, while in the absence of NaCl, 50% of the highest activity was observed. The rTsAly7A was an endo-type alginate lyase, and its end-products of alginate degradation were unsaturated oligosaccharides (degree of polymerization 2–6). Collectively, the rTsAly7A may be a good industrial production tool for producing AOS with high degree of polymerization. Full article

Most reports on signal peptides focus on their ability to affect the normal folding of proteins, thereby affecting their secreted expression, while few studies on its effects on enzymatic properties were published. Therefore, biochemical characterization and comparison of alginate lyase rALYI1/rALYI1-1 (rALYI1: without signal peptides; rALYI1-1:with signal peptides) were conducted in our study, and the results showed that the signal peptide affected the biochemical properties, especially in temperature and pH. rALYI1 (32.15 kDa) belonging to polysaccharide lyase family 7 was cloned from sea-cucumber-gut bacterium Tamlana sp. I1. The optimum temperature of both rALYI1 and rALYI1-1 was 40 °C, but the former had a wider optimum temperature range and better thermal stability. The optimum pH of rALYI1 and rALYI1-1 were 7.6 and 8.6, respectively. The former was more stable and acid resistant. Noticeably, rALYI1 was a salt-activated enzyme and displayed remarkable salt tolerance. Alginate, an essential polysaccharide in algae and Pseudomonas aeruginosa biofilms, is composed of α-L-guluronate and β-D-mannuronate. It is also found in our study that rALYI1 is also effective in removing mature biofilms compared with controls. In conclusion, the signal peptide affects several biochemical properties of the enzyme, and alginate lyase rALYI1 may be an effective method for inhibiting biofilm formation of Pseudomonas aeruginosa. Full article

Alginate lyases with unique biochemical properties have irreplaceable value in food and biotechnology industries. Herein, the first new hybrid action mode Thalassotalea algicola-derived alginate lyase gene (TAPL7A) with both psychrophilic and cold-tolerance was cloned and expressed heterologously in E. coli. With the highest sequence identity (43%) to the exolytic alginate lyase AlyA5 obtained from Zobellia galactanivorans, TAPL7A was identified as a new polysaccharide lyases family 7 (PL7) alginate lyase. TAPL7A has broad substrate tolerance with specific activities of 4186.1 U/mg, 2494.8 U/mg, 2314.9 U/mg for polyM, polyG, and sodium alginate, respectively. Biochemical characterization of TAPL7A showed optimal activity at 15 °C, pH 8.0. Interestingly, TAPL7A exhibits both extreme psychrophilic and cold tolerance, which other cold-adapted alginate lyase do not possess. In a wide range of 5–30 °C, the activity can reach 80–100%, and the residual activity of more than 70% can still be maintained after 1 h of incubation. Product analysis showed that TAPL7A adopts a hybrid endo/exo-mode on all three substrates. FPLC and ESI-MS confirmed that the final products of TAPL7A are oligosaccharides with degrees of polymerization (Dps) of 1–2. This study provides excellent alginate lyase candidates for low-temperature environmental applications in food, agriculture, medicine and other industries. Full article

Carbohydrate-active enzymes (CAZymes) are an important characteristic of bacteria in marine systems. We herein describe the CAZymes of Paenibacillus algicola HB172198^T, a novel type species isolated from brown algae in Qishui Bay, Hainan, China. The genome of strain HB172198^T is a 4,475,055 bp circular chromosome with an average GC content of 51.2%. Analysis of the nucleotide sequences of the predicted genes shows that strain HB172198^T encodes 191 CAZymes. Abundant putative enzymes involved in the degradation of polysaccharides were identified, such as alginate lyase, agarase, carrageenase, xanthanase, xylanase, amylases, cellulase, chitinase, fucosidase and glucanase. Four of the putative polysaccharide lyases from families 7, 15 and 38 were involved in alginate degradation. The alginate lyases of strain HB172198^T exhibited the maximum activity 152 U/mL at 50 °C and pH 8.0, and were relatively stable at pH 7.0 and temperatures lower than 40 °C. The average degree of polymerization (DP) of the sodium alginate oligosaccharide (AOS) degraded by the partially purified alginate lyases remained around 14.2, and the thin layer chromatography (TCL) analysis indicated that it contained DP2-DP8 oligosaccharides. The complete genome sequence of P. algicola HB172198^T will enrich our knowledge of the mechanism of polysaccharide lyase production and provide insights into its potential applications in the degradation of polysaccharides such as alginate. Full article

Alginate lyase has received extensive attention as an important tool for oligosaccharide preparation, pharmaceutical production, and energy biotransformation. Noncatalytic module carbohydrate-binding modules (CBM) have a major impact on the function of alginate lyases. Although the effects of two different families of CBMs on enzyme characteristics have been reported, the effect of two combined CBM32s on enzyme function has not been elucidated. Herein, we cloned and expressed a new multimodular alginate lyase, VxAly7C, from Vibrioxiamenensis QY104, consisting of two CBM32s at N-terminus and a polysaccharide lyase family 7 (PL7) at C-terminus. To explore the function of CBM32s in VxAly7C, full-length (VxAly7C-FL) and two truncated mutants, VxAly7C-TM1 (with the first CBM32 deleted) and VxAly7C-TM2 (with both CBM32s deleted), were characterized. The catalytic efficiency of recombinant VxAly7C-TM2 was 1.82 and 4.25 times higher than that of VxAly7C-TM1 and VxAly7C-FL, respectively, indicating that CBM32s had an antagonistic effect. However, CBM32s improved the temperature stability, the adaptability in an alkaline environment, and the preference for polyG. Moreover, CBM32s contributed to the production of tri- and tetrasaccharides, significantly affecting the end-product distribution. This study advances the understanding of module function and provides a reference for broader enzymatic applications and further enzymatic improvement and assembly. Full article

Alginate is the main component of brown algae, which is an important primary production in marine ecosystems and represents a huge marine biomass. The efficient utilization of alginate depends on alginate lyases to catalyze the degradation, and remains to be further explored. In this study, 354 strains were isolated from the gut of adult abalones, which mainly feed on brown algae. Among them, 100 alginate-degrading strains were gained and the majority belonged to the Gammaproteobacteria, followed by the Bacteroidetes and Alphaproteobacteria. A marine bacterium, Agarivorans sp. B2Z047, had the strongest degradation ability of alginate with the largest degradation circle and the highest enzyme activity. The optimal alginate lyase production medium of strain B2Z047 was determined as 1.1% sodium alginate, 0.3% yeast extract, 1% NaCl, and 0.1% MgSO₄ in artificial seawater (pH 7.0). Cells of strain B2Z047 were Gram-stain-negative, aerobic, motile by flagella, short rod-shaped, and approximately 0.7–0.9 µm width and 1.2–1.9 µm length. The optimal growth conditions were determined to be at 30 °C, pH 7.0–8.0, and in 3% (w/v) NaCl. A total of 12 potential alginate lyase genes were identified through whole genome sequencing and prediction, which belonged to polysaccharide lyase family 6, 7, 17, and 38 (PL6, PL7, PL17, and PL38, respectively). Furthermore, the degradation products of nine alginate lyases were detected, among which Aly38A was the first alginate lyase belonging to the PL38 family that has been found to degrade alginate. The combination of alginate lyases functioning in the alginate-degrading process was further demonstrated by the growth curve and alginate lyase production of strain B2Z047 cultivated with or without sodium alginate, as well as the content changes of total sugar and reducing sugar and the transcript levels of alginate lyase genes. A simplified model was proposed to explain the alginate utilization process of Agarivorans sp. B2Z047. Full article

Alginate, a natural polysaccharide derived from brown seaweed, is finding multiple applications in biomedicine via its transformation through chemical, physical, and, increasingly, enzymatic processes. In this study a novel alginate lyase, AlyDS44, was purified and characterized from a marine actinobacterium, Streptomyces luridiscabiei, which was isolated from decomposing seaweed. The purified enzyme had a specific activity of 108.6 U/mg, with a molecular weight of 28.6 kDa, and was composed of 260 amino acid residues. AlyDS44 is a bifunctional alginate lyase, active on both polyguluronate and polymannuronate, though it preferentially degrades polyguluronate. The optimal pH of this enzyme is 8.5 and the optimal temperature is 45 °C. It is a salt-tolerant alginate lyase with an optimal activity at 0.6 M NaCl. Metal ions Mn²⁺, Co²⁺, and Fe²⁺ increased the alginate degrading activity, but it was inhibited in the presence of Zn²⁺ and Cu²⁺. The highly conserved regions of its amino acid sequences indicated that AlyDS44 belongs to the polysaccharide lyase family 7. The main breakdown products of the enzyme on alginate were disaccharides, trisaccharides, and tetrasaccharides, which demonstrated that this enzyme acted as an endo-type alginate lyase. AlyDS44 is a novel enzyme, with the potential for efficient production of alginate oligosaccharides with low degrees of polymerization. Full article

We carried out a detailed investigation of PL7 alginate lyases across the Zobellia genus. The main findings were obtained using the methods of comparative genomics and spatial structure modeling, as well as a phylogenomic approach. Initially, in order to elucidate the alginolytic potential of Zobellia, we calculated the content of polysaccharide lyase (PL) genes in each genome. The genus-specific PLs were PL1, PL6, PL7 (the most abundant), PL14, PL17, and PL40. We revealed that PL7 belongs to subfamilies 3, 5, and 6. They may be involved in local and horizontal gene transfer and gene duplication processes. Most likely, an individual evolution of PL7 genes promotes the genetic variability of the Alginate Utilization System across Zobellia. Apparently, the PL7 alginate lyases may acquire a sub-functionalization due to diversification between in-paralogs. Full article

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. Full article

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. Full article

Alginate extracted from widely cultured brown seaweed can be hydrolyzed by alginate lyase to produce alginate oligosaccharides (AOS) with intriguing biological activities. Herein, a novel alginate lyase Aly1281 was cloned from marine bacterium Pseudoalteromonas carrageenovora ASY5 isolated from mangrove soil and found to belong to polysaccharide lyase family 7. Aly1281 exhibited maximum activity at pH 8.0 and 50 °C and have broad substrate specificity for polyguluronate and polymannuronate. Compared with other alginate lyases, Aly1281 exhibited high degradation specificity and mainly produced di-alginate oligosaccharides which displayed good antioxidant function to reduce ferric and scavenge radicals such as hydroxyl, ABTS⁺ and DPPH. Moreover, the catalytic activity and kinetic performance of Aly1281 were highly improved with the addition of salt, demonstrating a salt-activation property. A putative conformational structural feature of Aly1281 was found by MD simulation analysis for understanding the salt-activation effect. Full article

Alginate lyases degrade alginate into oligosaccharides, of which the biological activities have vital roles in various fields. Some alginate lyases contain one or more carbohydrate-binding modules (CBMs), which assist the function of the catalytic modules. However, the precise function of CBMs in alginate lyases has yet to be fully elucidated. We have identified a new multi-domain alginate lyase, TsAly7B, in the marine bacterium Thalassomonas sp. LD5. This novel lyase contains an N-terminal CBM9, an internal CBM32, and a C-terminal polysaccharide lyase family 7 (PL7) catalytic module. To investigate the specific function of each of these CBMs, we expressed and characterized the full-length TsAly7B and three truncated mutants: TM1 (CBM32-PL7), TM2 (CBM9-PL7), and TM3 (PL7 catalytic module). CBM9 and CBM32 could enhance the degradation of alginate. Notably, the specific activity of TM2 was 7.6-fold higher than that of TM3. CBM32 enhanced the resistance of the catalytic module to high temperatures. In addition, a combination of CBM9 and CBM32 showed enhanced thermostability when incubated at 80 °C for 1 h. This is the first report that finds CBM9 can significantly improve the ability of enzyme degradation. Our findings provide new insight into the interrelationships of tandem CBMs and alginate lyases and other polysaccharide-degrading enzymes, which may inspire CBM fusion strategies. Full article

Alginate oligosaccharides (AOS) show versatile bioactivities. Although various alginate lyases have been characterized, enzymes with special characteristics are still rare. In this study, a polysaccharide lyase family 7 (PL7) alginate lyase-encoding gene, aly08, was cloned from the marine bacterium Vibrio sp. SY01 and expressed in Escherichia coli. The purified alginate lyase Aly08, with a molecular weight of 35 kDa, showed a specific activity of 841 U/mg at its optimal pH (pH 8.35) and temperature (45 °C). Aly08 showed good pH-stability, as it remained more than 80% of its initial activity in a wide pH range (4.0–10.0). Aly08 was also a thermo-tolerant enzyme that recovered 70.8% of its initial activity following heat shock treatment for 5 min. This study also demonstrated that Aly08 is a polyG-preferred enzyme. Furthermore, Aly08 degraded alginates into disaccharides and trisaccharides in an endo-manner. Its thermo-tolerance and pH-stable properties make Aly08 a good candidate for further applications. Full article

Alginate lyases are enzymes that degrade alginate into oligosaccharides which possess a variety of biological activities. Discovering and characterizing novel alginate lyases has great significance for industrial and medical applications. In this study, we reported a novel alginate lyase, AlyA-OU02, derived from the marine Vibrio splendidus OU02. The BLASTP searches showed that AlyA-OU02 belonged to polysaccharide lyase family 7 (PL7) and contained two consecutive PL7 domains, which was rare among the alginate lyases in PL7 family. Both the two domains, AlyA^a and AlyA^b, had lyase activities, while AlyA^a exhibited polyM preference, and AlyA^b was polyG-preferred. In addition, the enzyme activity of AlyA^a was much higher than AlyA^b at 25 °C. The full-length enzyme of AlyA-OU02 showed polyM preference, which was the same as AlyA^a. AlyA^a degraded alginate into di-, tri-, and tetra-alginate oligosaccharides, while AlyA^b degraded alginate into tri-, tetra-, and penta-alginate oligosaccharides. The degraded products of AlyA-OU02 were similar to AlyA^a. Our work provided a potential candidate in the application of alginate oligosaccharide production and the characterization of the two domains might provide insights into the use of alginate of this organism. Full article