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.

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

Marine Drugs ◽

10.3390/md17050308 ◽

2019 ◽

Vol 17 (5) ◽

pp. 308 ◽

Cited By ~ 12

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.

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Characterization of a New Biofunctional, Exolytic Alginate Lyase from Tamlana sp. s12 with High Catalytic Activity and Cold-Adapted Features

Marine Drugs ◽

10.3390/md19040191 ◽

2021 ◽

Vol 19 (4) ◽

pp. 191

Author(s):

Rui Yin ◽

Yan-Jun Yi ◽

Zhuo Chen ◽

Bao-Xun Wang ◽

Xue-Han Li ◽

...

Keyword(s):

Critical Role ◽

Basic Research ◽

Alginate Lyase ◽

Industrial Applications ◽

High Catalytic Activity ◽

Cold Adapted ◽

Acidic Polysaccharide ◽

Alginate Oligosaccharide ◽

Marine Strain ◽

Alginate Lyases

Alginate, a major acidic polysaccharide in brown algae, has attracted great attention as a promising carbon source for biorefinery systems. Alginate lyases, especially exo-type alginate lyase, play a critical role in the biorefinery process. Although a large number of alginate lyases have been characterized, few can efficiently degrade alginate comprised of mannuronate (M) and guluronate (G) at low temperatures by means of an exolytic mode. In this study, the gene of a new exo-alginate lyase—Alys1—with high activity (1350 U/mg) was cloned from a marine strain, Tamlana sp. s12. When sodium alginate was used as a substrate, the recombinant enzyme showed optimal activity at 35 °C and pH 7.0–8.0. Noticeably, recombinant Alys1 was unstable at temperatures above 30 °C and had a low melting temperature of 56.0 °C. SDS and EDTA significantly inhibit its activity. These data indicate that Alys1 is a cold-adapted enzyme. Moreover, the enzyme can depolymerize alginates polyM and polyG, and produce a monosaccharide as the minimal alginate oligosaccharide. Primary substrate preference tests and identification of the final oligosaccharide products demonstrated that Alys1 is a bifunctional alginate lyase and prefers M to G. These properties make Alys1 a valuable candidate in both basic research and industrial applications.

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

Marine Drugs ◽

10.3390/md18080388 ◽

2020 ◽

Vol 18 (8) ◽

pp. 388

Author(s):

Hai-Xiang Zhou ◽

Shan-Shan Xu ◽

Xue-Jing Yin ◽

Feng-Long Wang ◽

Yang Li

Keyword(s):

Biological Activities ◽

Alginate Lyase ◽

Industrial Applications ◽

Cold Adapted ◽

Polysaccharide Lyase ◽

Alginate Oligosaccharides ◽

Optimal Reaction Temperature ◽

High Level ◽

Optimal Reaction

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.

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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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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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Introducing a Thermo-Alkali-Stable, Metallic Ion-Tolerant Laccase Purified From White Rot Fungus Trametes hirsuta

Frontiers in Microbiology ◽

10.3389/fmicb.2021.670163 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jing Si ◽

Hongfei Ma ◽

Yongjia Cao ◽

Baokai Cui ◽

Yucheng Dai

Keyword(s):

Specific Activity ◽

Endocrine Disrupting Chemicals ◽

Environmental Pollutants ◽

Fold Increase ◽

Industrial Applications ◽

White Rot ◽

White Rot Fungus ◽

Metallic Ions ◽

Trametes Hirsuta ◽

Ph Range

This study introduces a valuable laccase, designated ThLacc-S, purified from white rot fungus Trametes hirsuta. ThLacc-S is a monomeric protein in nature with a molecular weight of 57.0 kDa and can efficiently metabolize endocrine disrupting chemicals. The enzyme was successfully purified to homogeneity via three consecutive steps consisting of salt precipitation and column chromatography, resulting in a 20.76-fold increase in purity and 46.79% yield, with specific activity of 22.111 U/mg protein. ThLacc-S was deciphered as a novel member of the laccase family and is a rare metalloenzyme that contains cysteine, serine, histidine, and tyrosine residues in its catalytic site, and follows Michaelis-Menten kinetic behavior with a Km and a kcat/Km of 87.466 μM and 1.479 s–1μM–1, respectively. ThLacc-S exerted excellent thermo-alkali stability, since it was markedly active after a 2-h incubation at temperatures ranging from 20 to 70°C and retained more than 50% of its activity after incubation for 72 h in a broad pH range of 5.0–10.0. Enzymatic activities of ThLacc-S were enhanced and preserved when exposed to metallic ions, surfactants, and organic solvents, rendering this novel enzyme of interest as a green catalyst for versatile biotechnological and industrial applications that require these singularities of laccases, particularly biodegradation and bioremediation of environmental pollutants.

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Production and Characterization of Esterase in Lantinus tigrinus for Degradation of Polystyrene

Polish Journal of Microbiology ◽

10.33073/pjm-2013-015 ◽

2013 ◽

Vol 62 (1) ◽

pp. 101-108 ◽

Cited By ~ 6

Author(s):

LUBNA TAHIR ◽

MUHAMMAD ISHTIAQ ALI ◽

MUHAMMAD ZIA ◽

NAIMA ATIQ ◽

FARIHA HASAN ◽

...

Keyword(s):

Acidic Medium ◽

Specific Activity ◽

Mineral Salt Medium ◽

Tween 80 ◽

Synthetic Polymers ◽

Ftir Analysis ◽

Polystyrene Film ◽

Biological Degradation ◽

Ph Range

Polystyrene is considered stable to biological degradation. Lantinus tigrinus isolated from wood sample produced esterase in growth medium under normal conditions. However, acidic medium, 37 degrees C temperature, presence of tween 80; and urea and yeast extract in mineral salt medium enhance the production of esterase and specific activity. Purified esterase was active at broad pH range and 45 degrees C. FTIR analysis confirmed that esterase produced by Lantinus tigrinus effectively degraded polystyrene film and broke macromolecules down to non-toxic molecules. This study concludes that the presence of Lantinus tigrinus at dumping sites can be exploited for waste management containing high molecular weight synthetic polymers.

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Characterization of a Robust and pH-Stable Tannase from Mangrove-Derived Yeast Rhodosporidium diobovatum Q95

Marine Drugs ◽

10.3390/md18110546 ◽

2020 ◽

Vol 18 (11) ◽

pp. 546

Author(s):

Jie Pan ◽

Ni-Na Wang ◽

Xue-Jing Yin ◽

Xiao-Ling Liang ◽

Zhi-Peng Wang

Keyword(s):

Gallic Acid ◽

Tannic Acid ◽

Specific Activity ◽

Maximum Activity ◽

Sds Page ◽

Rhodosporidium Diobovatum ◽

Bacteria And Fungi ◽

Ph Range ◽

First Time

Tannase plays a crucial role in many fields, such as the pharmaceutical industry, beverage processing, and brewing. Although many tannases derived from bacteria and fungi have been thoroughly studied, those with good pH stabilities are still less reported. In this work, a mangrove-derived yeast strain Rhodosporidium diobovatum Q95, capable of efficiently degrading tannin, was screened to induce tannase, which exhibited an activity of up to 26.4 U/mL after 48 h cultivation in the presence of 15 g/L tannic acid. The tannase coding gene TANRD was cloned and expressed in Yarrowia lipolytica. The activity of recombinant tannase (named TanRd) was as high as 27.3 U/mL. TanRd was purified by chromatography and analysed by SDS-PAGE, showing a molecular weight of 75.1 kDa. The specific activity of TanRd towards tannic acid was 676.4 U/mg. Its highest activity was obtained at 40 °C, with more than 70% of the activity observed at 25–60 °C. Furthermore, it possessed at least 60% of the activity in a broad pH range of 2.5–6.5. Notably, TanRd was excellently stable at a pH range from 3.0 to 8.0; over 65% of its maximum activity remained after incubation. Besides, the broad substrate specificity of TanRd to esters of gallic acid has attracted wide attention. In view of the above, tannase resources were developed from mangrove-derived yeasts for the first time in this study. This tannase can become a promising material in tannin biodegradation and gallic acid production.

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