scholarly journals Specificities and Synergistic Actions of Novel PL8 and PL7 Alginate Lyases from the Marine Fungus Paradendryphiella salina

2021 ◽  
Vol 7 (2) ◽  
pp. 80
Author(s):  
Bo Pilgaard ◽  
Marlene Vuillemin ◽  
Jesper Holck ◽  
Casper Wilkens ◽  
Anne S. Meyer
Keyword(s):  
Alginate Lyase ◽  
Marine Fungus ◽  
Brown Macroalgae ◽  
Polysaccharide Lyases ◽  
Alginate Lyases ◽  
Encoding Genes ◽  
Substrate Affinities ◽  
Alginate Degradation ◽  
Insight Into

Alginate is an anionic polysaccharide abundantly present in the cell walls of brown macroalgae. The enzymatic depolymerization is performed solely by alginate lyases (EC 4.2.2.x), categorized as polysaccharide lyases (PLs) belonging to 12 different PL families. Until now, the vast majority of the alginate lyases have been found in bacteria. We report here the first extensive characterization of four alginate lyases from a marine fungus, the ascomycete Paradendryphiella salina, a known saprophyte of seaweeds. We have identified four polysaccharide lyase encoding genes bioinformatically in P. salina, one PL8 (PsMan8A), and three PL7 alginate lyases (PsAlg7A, -B, and -C). PsMan8A was demonstrated to exert exo-action on polymannuronic acid, and no action on alginate, indicating that this enzyme is most likely an exo-acting polymannuronic acid specific lyase. This enzyme is the first alginate lyase assigned to PL8 and polymannuronic acid thus represents a new substrate specificity in this family. The PL7 lyases (PsAlg7A, -B, and -C) were found to be endo-acting alginate lyases with different activity optima, substrate affinities, and product profiles. PsAlg7A and PsMan8A showed a clear synergistic action for the complete depolymerization of polyM at pH 5. PsAlg7A depolymerized polyM to mainly DP5 and DP3 oligomers and PsMan8A to dimers and monosaccharides. PsAlg7B and PsAlg7C showed substrate affinities towards both polyM and polyG at pH 8, depolymerizing both substrates to DP9-DP2 oligomers. The findings elucidate how P. salina accomplishes alginate depolymerization and provide insight into an efficient synergistic cooperation that may provide a new foundation for enzyme selection for alginate degradation in seaweed bioprocessing.

scholarly journals Structural and molecular basis for the substrate positioning mechanism of a new PL7 subfamily alginate lyase from the arctic

2020 ◽  
Vol 295 (48) ◽  
pp. 16380-16392 ◽  
Author(s):  
Fei Xu ◽  
Xiu-Lan Chen ◽  
Xiao-Hui Sun ◽  
Fang Dong ◽  
Chun-Yang Li ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Catalytic Properties ◽  
Alginate Lyase ◽  
The Arctic ◽  
Amino Acid Residues ◽  
Biochemical Analyses ◽  
Alginate Lyases ◽  
First Time ◽  
Alginate Degradation

Alginate lyases play important roles in alginate degradation in the ocean. Although a large number of alginate lyases have been characterized, little is yet known about those in extremely cold polar environments, which may have unique mechanisms for environmental adaptation and for alginate degradation. Here, we report the characterization of a novel PL7 alginate lyase AlyC3 from Psychromonas sp. C-3 isolated from the Arctic brown alga Laminaria, including its phylogenetic classification, catalytic properties, and structure. We propose the establishment of a new PM-specific subfamily of PL7 (subfamily 6) represented by AlyC3 based on phylogenetic analysis and enzymatic properties. Structural and biochemical analyses showed that AlyC3 is a dimer, representing the first dimeric endo-alginate lyase structure. AlyC3 is activated by NaCl and adopts a novel salt-activated mechanism; that is, salinity adjusts the enzymatic activity by affecting its aggregation states. We further solved the structure of an inactive mutant H127A/Y244A in complex with a dimannuronate molecule and proposed the catalytic process of AlyC3 based on structural and biochemical analyses. We show that Arg82 and Tyr190 at the two ends of the catalytic canyon help the positioning of the repeated units of the substrate and that His127, Tyr244, Arg78, and Gln125 mediate the catalytic reaction. Our study uncovers, for the first time, the amino acid residues for alginate positioning in an alginate lyase and demonstrates that such residues involved in alginate positioning are conserved in other alginate lyases. This study provides a better understanding of the mechanisms of alginate degradation by alginate lyases.

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 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 Functional Characterization of Carbohydrate-Binding Modules in a New Alginate Lyase, TsAly7B, from Thalassomonas sp. LD5

Marine Drugs ◽  
2019 ◽  
Vol 18 (1) ◽  
pp. 25 ◽  
Author(s):  
Zhelun Zhang ◽  
Luyao Tang ◽  
Mengmeng Bao ◽  
Zhigang Liu ◽  
Wengong Yu ◽  
...  
Keyword(s):  
Specific Activity ◽  
Biological Activities ◽  
Functional Characterization ◽  
Alginate Lyase ◽  
Carbohydrate Binding ◽  
Enzyme Degradation ◽  
Carbohydrate Binding Modules ◽  
Catalytic Module ◽  
Alginate Lyases

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.

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 Insights into alginate degradation through the characterization of a thermophilic exolytic alginate lyase

2021 ◽  
Author(s):  
Magnus Ø. Arntzen ◽  
Bjørn Pedersen ◽  
Leesa J. Klau ◽  
Runar Stokke ◽  
Maren Oftebro ◽  
...  
Keyword(s):  
Nmr Assignment ◽  
Alginate Lyase ◽  
Product Formation ◽  
Site Directed Mutagenesis ◽  
Fermentable Sugars ◽  
Reaction Products ◽  
Processing Technologies ◽  
Future Assessment ◽  
Alginate Lyases ◽  
Alginate Degradation

Enzymatic depolymerization of seaweed polysaccharides is gaining interest for the production of functional oligosaccharides and fermentable sugars. Herein, we describe a thermostable alginate lyase that belongs to Polysaccharide Lyase family 17 (PL17) and was derived from an Arctic Mid-Ocean Ridge (AMOR) metagenomics dataset. This enzyme, AMOR_PL17A, is a thermostable exolytic oligoalginate lyase (EC 4.2.2.26), which can degrade alginate, poly β-d-mannuronate and poly α-l-guluronate within a broad range of pH, temperature and salinity conditions. Site-directed mutagenesis showed that tyrosine Y251, previously suggested to act as catalytic acid, indeed is essential for catalysis; whereas mutation of tyrosine Y446, previously proposed to act as catalytic base, did not affect enzyme activity. The observed reaction products are protonated and deprotonated forms of the 4,5-unsaturated uronic acid monomer, Δ, two hydrates of DEH (4-deoxy-l-erythro-5-hexulosuronate), which are formed after ring opening and, finally, two epimers of a 5-membered hemiketal called 4-deoxy-d-manno-hexulofuranosidonate (DHF) formed through intramolecular cyclisation of hydrated DEH. The detection and NMR assignment of these hemiketals refine our current understanding of alginate degradation. Importance The potential markets for seaweed-derived products and seaweed processing technologies are growing, yet commercial enzyme-cocktails for complete conversion of seaweed to fermentable sugars are not available. Such an enzyme-cocktail would require the catalytic properties of a variety of different enzymes, where fucoidanases, laminarinases and cellulases together with endo- and exo-acting alginate lyases would be the key enzymes. Here we present an exo-acting alginate lyase that efficiently produces monomeric sugars from alginate. Since it is only the second characterized exo-acting alginate lyase capable of degrading alginate at industrially relevant higher temperatures of 60 °C, this enzyme may be of great biotechnological and industrial interest. In addition, in-depth NMR-based structural elucidation reveal previously undescribed rearrangement products of the unsaturated monomeric sugars generated from exo-acting lyases. The insight provided by the NMR assignment of these products facilitates future assessment of product formation by alginate lyases.

scholarly journals Biochemical characteristics and synergistic effect of two novel alginate lyases from Photobacterium sp. FC615

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Danrong Lu ◽  
Qingdong Zhang ◽  
Shumin Wang ◽  
Jingwen Guan ◽  
Runmiao Jiao ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Synergistic Effect ◽  
Alginate Lyase ◽  
Enzymatic Digestion ◽  
Biofuel Production ◽  
Molar Ratio ◽  
Brown Macroalgae ◽  
Alginate Oligosaccharides ◽  
Novel Method ◽  
Alginate Lyases

Abstract Background Macroalgae and microalgae, as feedstocks for third-generation biofuel, possess competitive strengths in terms of cost, technology and economics. The most important compound in brown macroalgae is alginate, and the synergistic effect of endolytic and exolytic alginate lyases plays a crucial role in the saccharification process of transforming alginate into biofuel. However, there are few studies on the synergistic effect of endolytic and exolytic alginate lyases, especially those from the same bacterial strain. Results In this study, the endolytic alginate lyase AlyPB1 and exolytic alginate lyase AlyPB2 were identified from the marine bacterium Photobacterium sp. FC615. These two enzymes showed quite different and novel enzymatic properties whereas behaved a strong synergistic effect on the saccharification of alginate. Compared to that when AlyPB2 was used alone, the conversion rate of alginate polysaccharides to unsaturated monosaccharides when AlyPB1 and AlyPB2 acted on alginate together was dramatically increased approximately sevenfold. Furthermore, we found that AlyPB1 and AlyPB2 acted the synergistic effect basing on the complementarity of their substrate degradation patterns, particularly due to their M-/G-preference and substrate-size dependence. In addition, a novel method for sequencing alginate oligosaccharides was developed for the first time by combining the 1H NMR spectroscopy and the enzymatic digestion with the exo-lyase AlyPB2, and this method is much simpler than traditional methods based on one- and two-dimensional NMR spectroscopy. Using this strategy, the sequences of the final tetrasaccharide and pentasaccharide product fractions produced by AlyPB1 were easily determined: the tetrasaccharide fractions contained two structures, ΔGMM and ΔMMM, at a molar ratio of 1:3.2, and the pentasaccharide fractions contained four structures, ΔMMMM, ΔMGMM, ΔGMMM, and ΔGGMM, at a molar ratio of ~ 1:1.5:3.5:5.25. Conclusions The identification of these two novel alginate lyases provides not only excellent candidate tool-type enzymes for oligosaccharide preparation but also a good model for studying the synergistic digestion and saccharification of alginate in biofuel production. The novel method for oligosaccharide sequencing described in this study will offer a very useful approach for structural and functional studies on alginate.

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 Characterization of Alginate Lyase from Pseudomonas syringae pv. syringae

2000 ◽  
Vol 182 (21) ◽  
pp. 6268-6271 ◽  
Author(s):  
Lori A. Preston ◽  
T. Y. Wong ◽  
Carol L. Bender ◽  
Neal L. Schiller
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Syringae ◽  
Alginate Lyase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Gene Encoding ◽  
Tryptophan Residues ◽  
Lyase Activity ◽  
Alginate Lyases

ABSTRACT The gene encoding alginate lyase (algL) inPseudomonas syringae pv. syringae was cloned, sequenced, and overexpressed in Escherichia coli. Alginate lyase activity was optimal when the pH was 7.0 and when assays were conducted at 42°C in the presence of 0.2 M NaCl. In substrate specificity studies, AlgL from P. syringae showed a preference for deacetylated polymannuronic acid. Sequence alignment with other alginate lyases revealed conserved regions within AlgL likely to be important for the structure and/or function of the enzyme. Site-directed mutagenesis of histidine and tryptophan residues at positions 204 and 207, respectively, indicated that these amino acids are critical for lyase activity.

scholarly journals Bacterial alginate metabolism: an important pathway for bioconversion of brown algae

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Lanzeng Zhang ◽  
Xue Li ◽  
Xiyue Zhang ◽  
Yingjie Li ◽  
Lushan Wang
Keyword(s):  
Metabolic Pathways ◽  
Cell Walls ◽  
Direct Conversion ◽  
Significant Progress ◽  
Brown Macroalgae ◽  
Sustainable Solutions ◽  
Important Pathway ◽  
Alternative Feedstock ◽  
Alginate Lyases ◽  
Alginate Degradation

AbstractBrown macroalgae have attracted great attention as an alternative feedstock for biorefining. Although direct conversion of ethanol from alginates (major components of brown macroalgae cell walls) is not amenable for industrial production, significant progress has been made not only on enzymes involved in alginate degradation, but also on metabolic pathways for biorefining at the laboratory level. In this article, we summarise recent advances on four aspects: alginate, alginate lyases, different alginate-degrading systems, and application of alginate lyases and associated pathways. This knowledge will likely inspire sustainable solutions for further application of both alginate lyases and their associated pathways.

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