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

Marine Drugs ◽  
2020 ◽  
Vol 18 (4) ◽  
pp. 184 ◽  
Author(s):  
Jin Yang ◽  
Dandan Cui ◽  
Diwen Chen ◽  
Wenkang Chen ◽  
Shuo Ma ◽  
...  
Keyword(s):  
Specific Activity ◽  
Chilling Stress ◽  
Alginate Lyase ◽  
Vital Role ◽  
Acidic Polysaccharide ◽  
Mannuronic Acid ◽  
Guluronic Acid ◽  
Alginate Oligosaccharides ◽  
Agricultural Applications ◽  
Ph Range

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.

scholarly journals Characterization of an Alkaline Alginate Lyase with pH-Stable and Thermo-Tolerance Property

Marine Drugs ◽  
2019 ◽  
Vol 17 (5) ◽  
pp. 308 ◽  
Author(s):  
Yanan Wang ◽  
Xuehong Chen ◽  
Xiaolin Bi ◽  
Yining Ren ◽  
Qi Han ◽  
...  
Keyword(s):  
Marine Bacterium ◽  
Specific Activity ◽  
Alginate Lyase ◽  
Initial Activity ◽  
Alginate Oligosaccharides ◽  
Wide Ph Range ◽  
Encoding Gene ◽  
Ph Range ◽  
Alginate Lyases

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.

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

Marine Drugs ◽  
2020 ◽  
Vol 18 (8) ◽  
pp. 416
Author(s):  
Yan Ma ◽  
Jie Li ◽  
Xin-Yue Zhang ◽  
Hao-Dong Ni ◽  
Feng-Biao Wang ◽  
...  
Keyword(s):  
Metal Ion ◽  
Brown Seaweed ◽  
Alginate Lyase ◽  
Industrial Applications ◽  
Alginate Oligosaccharides ◽  
Potential Applications ◽  
Pharmaceutical Industries ◽  
Ph Range ◽  
Alginate Lyases

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.

scholarly journals Characterization of Three New Azotobacter vinelandii Alginate Lyases, One of Which Is Involved in Cyst Germination

2009 ◽  
Vol 191 (15) ◽  
pp. 4845-4853 ◽  
Author(s):  
Martin Gimmestad ◽  
Helga Ertesvåg ◽  
Tonje Marita Bjerkan Heggeset ◽  
Olav Aarstad ◽  
Britt Iren Glærum Svanem ◽  
...  
Keyword(s):  
Azotobacter Vinelandii ◽  
Alginate Lyase ◽  
Growth Conditions ◽  
Type I ◽  
Wild Type ◽  
Mannuronic Acid ◽  
Guluronic Acid ◽  
Polysaccharide Lyases ◽  
Alginate Lyases

ABSTRACT Alginates are polysaccharides composed of 1-4-linked β-d-mannuronic acid and α-l-guluronic acid. The polymer can be degraded by alginate lyases, which cleave the polysaccharide using a β-elimination reaction. Two such lyases have previously been identified in the soil bacterium Azotobacter vinelandii, as follows: the periplasmic AlgL and the secreted bifunctional mannuronan C-5 epimerase and alginate lyase AlgE7. In this work, we describe the properties of three new lyases from this bacterium, AlyA1, AlyA2, and AlyA3, all of which belong to the PL7 family of polysaccharide lyases. One of the enzymes, AlyA3, also contains a C-terminal module similar to those of proteins secreted by a type I secretion system, and its activity is stimulated by Ca2+. All three enzymes preferably cleave the bond between guluronic acid and mannuronic acid, resulting in a guluronic acid residue at the new reducing end, but AlyA3 also degrades the other three possible bonds in alginate. Strains containing interrupted versions of alyA1, alyA3, and algE7 were constructed, and their phenotypes were analyzed. Genetically pure alyA2 mutants were not obtained, suggesting that this gene product may be important for the bacterium during vegetative growth. After centrifugation, cultures from the algE7 mutants form a large pellet containing alginate, indicating that AlgE7 is involved in the release of alginate from the cells. Upon encountering adverse growth conditions, A. vinelandii will form a resting stage called cyst. Alginate is a necessary part of the protective cyst coat, and we show here that strains lacking alyA3 germinate poorly compared to wild-type cells.

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

Marine Drugs ◽  
2020 ◽  
Vol 18 (4) ◽  
pp. 189 ◽  
Author(s):  
Zhi-Peng Wang ◽  
Min Cao ◽  
Bing Li ◽  
Xiao-Feng Ji ◽  
Xin-Yue Zhang ◽  
...  
Keyword(s):  
Specific Activity ◽  
Brown Seaweed ◽  
Alginate Lyase ◽  
Industrial Applications ◽  
Degree Of Polymerization ◽  
Cold Adapted ◽  
Alginate Oligosaccharide ◽  
Ph Range ◽  
Alginate Lyases

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.

scholarly journals Role of the Pseudomonas fluorescens Alginate Lyase (AlgL) in Clearing the Periplasm of Alginates Not Exported to the Extracellular Environment

2005 ◽  
Vol 187 (24) ◽  
pp. 8375-8384 ◽  
Author(s):  
Karianne Bakkevig ◽  
Håvard Sletta ◽  
Martin Gimmestad ◽  
Randi Aune ◽  
Helga Ertesvåg ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Alginate Lyase ◽  
Wild Type ◽  
Brown Seaweeds ◽  
Mannuronic Acid ◽  
Guluronic Acid ◽  
Genes Encoding ◽  
In The Wild ◽  
Extracellular Environment

ABSTRACT Alginate is an industrially widely used polysaccharide produced by brown seaweeds and as an exopolysaccharide by bacteria belonging to the genera Pseudomonas and Azotobacter. The polymer is composed of the two sugar monomers mannuronic acid and guluronic acid (G), and in all these bacteria the genes encoding 12 of the proteins essential for synthesis of the polymer are clustered in the genome. Interestingly, 1 of the 12 proteins is an alginate lyase (AlgL), which is able to degrade the polymer down to short oligouronides. The reason why this lyase is associated with the biosynthetic complex is not clear, but in this paper we show that the complete lack of AlgL activity in Pseudomonas fluorescens in the presence of high levels of alginate synthesis is toxic to the cells. This toxicity increased with the level of alginate synthesis. Furthermore, alginate synthesis became reduced in the absence of AlgL, and the polymers contained much less G residues than in the wild-type polymer. To explain these results and other data previously reported in the literature, we propose that the main biological function of AlgL is to degrade alginates that fail to become exported out of the cell and thereby become stranded in the periplasmic space. At high levels of alginate synthesis in the absence of AlgL, such stranded polymers may accumulate in the periplasm to such an extent that the integrity of the cell is lost, leading to the observed toxic effects.

scholarly journals Expression and Characterization of a Cold-Adapted Alginate Lyase with Exo/Endo-Type Activity from a Novel Marine Bacterium Alteromonas portus HB161718T

Marine Drugs ◽  
2021 ◽  
Vol 19 (3) ◽  
pp. 155
Author(s):  
Huiqin Huang ◽  
Shuang Li ◽  
Shixiang Bao ◽  
Kunlian Mo ◽  
Dongmei Sun ◽  
...  
Keyword(s):  
Marine Bacterium ◽  
Reducing Power ◽  
Alginate Lyase ◽  
Maximum Activity ◽  
Ionization Mass ◽  
Cold Adapted ◽  
Guluronic Acid ◽  
Type Activity ◽  
Alginate Oligosaccharides ◽  
Alginate Lyases

The alginate lyases have unique advantages in the preparation of alginate oligosaccharides and processing of brown algae. Herein, a gene alg2951 encoding a PL7 family alginate lyase with exo/endo-type activity was cloned from a novel marine bacterium Alteromonas portus HB161718T and then expressed in Escherichia coli. The recombinant Alg2951 in the culture supernatant reached the activity of 63.6 U/mL, with a molecular weight of approximate 60 kDa. Alg2951 exhibited the maximum activity at 25 °C and pH 8.0, was relatively stable at temperatures lower than 30 °C, and showed a special preference to poly-guluronic acid (polyG) as well. Both NaCl and KCl had the most promotion effect on the enzyme activity of Alg2951 at 0.2 M, increasing by 21.6 and 19.1 times, respectively. The TCL (Thin Layer Chromatography) and ESI-MS (Electrospray Ionization Mass Spectrometry) analyses suggested that Alg2951 could catalyze the hydrolysis of sodium alginate to produce monosaccharides and trisaccharides. Furthermore, the enzymatic hydrolysates displayed good antioxidant activity by assays of the scavenging abilities towards radicals (hydroxyl and ABTS+) and the reducing power. Due to its cold-adapted and dual exo/endo-type properties, Alg2951 can be a potential enzymatic tool for industrial production.

scholarly journals Biochemical Properties and Substrate Specificities of a Recombinantly Produced Azotobacter vinelandiiAlginate Lyase

1998 ◽  
Vol 180 (15) ◽  
pp. 3779-3784 ◽  
Author(s):  
Helga Ertesvåg ◽  
Frode Erlien ◽  
Gudmund Skjåk-Bræk ◽  
Bernd H. A. Rehm ◽  
Svein Valla
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Azotobacter Vinelandii ◽  
Divalent Cations ◽  
Alginate Lyase ◽  
Biochemical Properties ◽  
Mature Protein ◽  
Substrate Specificities ◽  
Mannuronic Acid ◽  
Guluronic Acid

ABSTRACT Alginate is a polysaccharide composed of β-d-mannuronic acid (M) and α-l-guluronic acid (G). An Azotobacter vinelandii alginate lyase gene,algL, was cloned, sequenced, and expressed inEscherichia coli. The deduced molecular mass of the corresponding protein is 41.4 kDa, but a signal peptide is cleaved off, leaving a mature protein of 39 kDa. Sixty-three percent of the amino acids in this mature protein are identical to those in AlgL fromPseudomonas aeruginosa. AlgL was partially purified, and the activity was found to be optimal at a pH of 8.1 to 8.4 and at 0.35 M NaCl. Divalent cations are not necessary for activity. The pI of the enzyme is 5.1. When an alginate rich in mannuronic acid was used as the substrate, the Km was found to be 4.6 × 10−4 M (sugar residues). AlgL was found to cleave M-M and M-G bonds but not G-M or G-G bonds. Bonds involving acetylated residues were also cleaved, but this activity may be sensitive to the extent of acetylation.

scholarly journals IMMUNOCYTOCHEMICAL LOCALIZATION OF THE EXTRACELLULAR POLYSACCHARIDE ALGINIC ACID IN THE BROWN SEAWEED, FUCUS DISTICHUS

1972 ◽  
Vol 20 (5) ◽  
pp. 358-367 ◽  
Author(s):  
VALERIE VREELAND
Keyword(s):  
Brown Seaweed ◽  
Extracellular Polysaccharide ◽  
Degree Of Polymerization ◽  
Extracellular Polysaccharides ◽  
Acidic Polysaccharide ◽  
Antibody Staining ◽  
Mannuronic Acid ◽  
Guluronic Acid ◽  
Fucus Distichus ◽  
Acid Ratio

Fluorescent and peroxidase-labeled antibody techniques were employed to localize alginate, which is one component in the complex extracellular acidic polysaccharide situation of the brown seaweed, Fucus distichus. The alginate antigen was extracted and characterized by a variety of methods. It had a low degree of polymerization and a mannuronic acid to guluronic acid ratio of 2.2. No contaminants were detected. The specificity of rabbit antisera was tested by immunodiffusion. Antisera reacted with alginate and fractions of alginate but not with other brown algal polysaccharides. When cryostat sections of Fucus proved unsuitable, 1-µ sections embedded in glycol methacrylate were used for indirect fluorescent or peroxidase antibody staining. Highly sulfated extracellular polysaccharides and intracellular phenolic compounds were implicated in nonspecific staining. Texture artifacts were caused by solubility of alginates and other polysaccharides from embedded sections. Alginate was localized in variable patterns in all cell walls and some matrix regions. The applicability of the methods is discussed.

scholarly journals The Pseudomonas fluorescens AlgG Protein, but Not Its Mannuronan C-5-Epimerase Activity, Is Needed for Alginate Polymer Formation

2003 ◽  
Vol 185 (12) ◽  
pp. 3515-3523 ◽  
Author(s):  
Martin Gimmestad ◽  
Håvard Sletta ◽  
Helga Ertesvåg ◽  
Karianne Bakkevig ◽  
Sumita Jain ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Azotobacter Vinelandii ◽  
Sequence Similarity ◽  
Alginate Lyase ◽  
Wild Type ◽  
Mannuronic Acid ◽  
Guluronic Acid ◽  
Pure Cell ◽  
Pure Cell Line

ABSTRACT Bacterial alginates are produced as 1-4-linked β-d-mannuronan, followed by epimerization of some of the mannuronic acid residues to α-l-guluronic acid. Here we report the isolation of four different epimerization-defective point mutants of the periplasmic Pseudomonas fluorescens mannuronan C-5-epimerase AlgG. All mutations affected amino acids conserved among AlgG-epimerases and were clustered in a part of the enzyme also sharing some sequence similarity to a group of secreted epimerases previously reported in Azotobacter vinelandii. An algG-deletion mutant was constructed and found to produce predominantly a dimer containing a 4-deoxy-l- erythro-hex-4-enepyranosyluronate residue at the nonreducing end and a mannuronic acid residue at the reducing end. The production of this dimer is the result of the activity of an alginate lyase, AlgL, whose in vivo activity is much more limited in the presence of AlgG. A strain expressing both an epimerase-defective (point mutation) and a wild-type epimerase was constructed and shown to produce two types of alginate molecules: one class being pure mannuronan and the other having the wild-type content of guluronic acid residues. This formation of two distinct classes of polymers in a genetically pure cell line can be explained by assuming that AlgG is part of a periplasmic protein complex.

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