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

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.

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

Marine Drugs ◽

10.3390/md18080416 ◽

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.

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

Marine Drugs ◽

10.3390/md18040189 ◽

2020 ◽

Vol 18 (4) ◽

pp. 189 ◽

Cited By ~ 4

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.

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

Marine Drugs ◽

10.3390/md18010025 ◽

2019 ◽

Vol 18 (1) ◽

pp. 25 ◽

Cited By ~ 4

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.

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

Marine Drugs ◽

10.3390/md19030155 ◽

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.

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Cloning, sequence analysis, and expression of gene alyPI encoding an alginate lyase from marine bacterium Pseudoalteromonas sp. CY24

Canadian Journal of Microbiology ◽

10.1139/w09-051 ◽

2009 ◽

Vol 55 (9) ◽

pp. 1113-1118 ◽

Cited By ~ 22

Author(s):

Gaofei Duan ◽

Feng Han ◽

Wengong Yu

Keyword(s):

Amino Acid ◽

Marine Bacterium ◽

Alginate Lyase ◽

Amino Acid Residues ◽

Reading Frame ◽

His Tag ◽

Consensus Region ◽

Encoding Gene ◽

Alginate Lyases ◽

Pcr Techniques

The alginate lyase encoding gene (alyPI) of marine bacterium Pseudoalteromonas sp. CY24 was cloned using a battery of PCR techniques. Gene alyPI was composed of a 1575 bp open reading frame encoding a protein of 57.4 kDa containing 524 amino acid residues with a signal peptide of 23 amino acids. The AlyPI protein was expressed in Escherichia coli with a His-tag sequence fused at the C-terminal end and purified to electrophoretic homogeneity using Ni-sepharose affinity chromatography. AlyPI was most active at 40 °C and pH 7.0 in the presencce of 0.1 mol/L NaCl and stable over a broad range of pH, 6.0–10.6. The presence of Na+, K+, Mn2+, Ca2+, and Fe3+ can enhance the enzyme activity. The alginate lyase consensus region YFKAGXYXQ, regarded as a striking feature at the C termini of several alginate lyase of ~30 kDa, was found in AlyPI, which belongs to the ~60 kDa group. Another nine amino acid consensus region, YXRSELREM, only found in G-specific alginate lyases previously existed in AlyPI, which could degrade sodium alginate, M blocks, and G blocks and appeared to be a broad substrate-specific alginate lyase.

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

Marine Drugs ◽

10.3390/md18040184 ◽

2020 ◽

Vol 18 (4) ◽

pp. 184 ◽

Cited By ~ 2

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.

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Characterization of a Novel PolyM-Preferred Alginate Lyase from Marine Vibrio splendidus OU02

Marine Drugs ◽

10.3390/md16090295 ◽

2018 ◽

Vol 16 (9) ◽

pp. 295 ◽

Cited By ~ 12

Author(s):

Jingjing Zhuang ◽

Keke Zhang ◽

Xiaohua Liu ◽

Weizhi Liu ◽

Qianqian Lyu ◽

...

Keyword(s):

Biological Activities ◽

Alginate Lyase ◽

Potential Candidate ◽

Vibrio Splendidus ◽

Polysaccharide Lyase ◽

Alginate Oligosaccharides ◽

Marine Vibrio ◽

Alginate Oligosaccharide ◽

Alginate Lyases

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, AlyAa and AlyAb, had lyase activities, while AlyAa exhibited polyM preference, and AlyAb was polyG-preferred. In addition, the enzyme activity of AlyAa was much higher than AlyAb at 25 °C. The full-length enzyme of AlyA-OU02 showed polyM preference, which was the same as AlyAa. AlyAa degraded alginate into di-, tri-, and tetra-alginate oligosaccharides, while AlyAb degraded alginate into tri-, tetra-, and penta-alginate oligosaccharides. The degraded products of AlyA-OU02 were similar to AlyAa. 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.

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Characterization of a Novel L-Asparaginase from Mycobacterium gordonae with Acrylamide Mitigation Potential

Foods ◽

10.3390/foods10112819 ◽

2021 ◽

Vol 10 (11) ◽

pp. 2819

Author(s):

Huibing Chi ◽

Meirong Chen ◽

Linshu Jiao ◽

Zhaoxin Lu ◽

Xiaomei Bie ◽

...

Keyword(s):

Food Industry ◽

Specific Activity ◽

Lymphoblastic Leukemia ◽

Data Bank ◽

Clinical Settings ◽

Potential Candidate ◽

Structural Identity ◽

Wide Ph Range ◽

Ph Range

L-asparaginase (E.C.3.5.1.1) is a well-known agent that prevents the formation of acrylamide both in the food industry and against childhood acute lymphoblastic leukemia in clinical settings. The disadvantages of L-asparaginase, which restrict its industrial application, include its narrow range of pH stability and low thermostability. In this study, a novel L-asparaginase from Mycobacterium gordonae (GmASNase) was cloned and expressed in Escherichia coli BL21 (DE3). GmASNase was found to be a tetramer with a monomeric size of 32 kDa, sharing only 32% structural identity with Helicobacter pylori L-asparaginases in the Protein Data Bank database. The purified GmASNase had the highest specific activity of 486.65 IU mg−1 at pH 9.0 and 50 °C. In addition, GmASNase possessed superior properties in terms of stability at a wide pH range of 5.0–11.0 and activity at temperatures below 40 °C. Moreover, GmASNase displayed high substrate specificity towards L-asparagine with Km, kcat, and kcat/Km values of 6.025 mM, 11,864.71 min−1, and 1969.25 mM−1min−1, respectively. To evaluate its ability to mitigate acrylamide, GmASNase was used to treat potato chips prior to frying, where the acrylamide content decreased by 65.09% compared with the untreated control. These results suggest that GmASNase is a potential candidate for applications in the food industry.

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

Journal of Fungi ◽

10.3390/jof7020080 ◽

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.

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