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

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.

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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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Structure Characteristics, Biochemical Properties, and Pharmaceutical Applications of Alginate Lyases

Marine Drugs ◽

10.3390/md19110628 ◽

2021 ◽

Vol 19 (11) ◽

pp. 628

Author(s):

Shu-Kun Gao ◽

Rui Yin ◽

Xiao-Chen Wang ◽

Hui-Ning Jiang ◽

Xiao-Xiao Liu ◽

...

Keyword(s):

Brown Algae ◽

Catalytic Mechanism ◽

Degradation Products ◽

Structural Characteristics ◽

Catalytic Efficiency ◽

Alginate Lyase ◽

Biochemical Properties ◽

Structure And Property ◽

Alginate Oligosaccharides ◽

Alginate Lyases

Alginate, the most abundant polysaccharides of brown algae, consists of various proportions of uronic acid epimers α-L-guluronic acid (G) and β-D-mannuronic acid (M). Alginate oligosaccharides (AOs), the degradation products of alginates, exhibit excellent bioactivities and a great potential for broad applications in pharmaceutical fields. Alginate lyases can degrade alginate to functional AOs with unsaturated bonds or monosaccharides, which can facilitate the biorefinery of brown algae. On account of the increasing applications of AOs and biorefinery of brown algae, there is a scientific need to explore the important aspects of alginate lyase, such as catalytic mechanism, structure, and property. This review covers fundamental aspects and recent developments in basic information, structural characteristics, the structure–substrate specificity or catalytic efficiency relationship, property, molecular modification, and applications. To meet the needs of biorefinery systems of a broad array of biochemical products, alginate lyases with special properties, such as salt-activated, wide pH adaptation range, and cold adaptation are outlined. Withal, various challenges in alginate lyase research are traced out, and future directions, specifically on the molecular biology part of alginate lyases, are delineated to further widen the horizon of these exceptional alginate lyases.

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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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Biochemical Characterization and Elucidation of Action Pattern of a Novel Polysaccharide Lyase 6 Family Alginate Lyase from Marine Bacterium Flammeovirga sp. NJ-04

Marine Drugs ◽

10.3390/md17060323 ◽

2019 ◽

Vol 17 (6) ◽

pp. 323 ◽

Cited By ~ 7

Author(s):

Qian Li ◽

Fu Hu ◽

Benwei Zhu ◽

Yun Sun ◽

Zhong Yao

Keyword(s):

Biochemical Characterization ◽

Alginate Lyase ◽

Ionization Mass ◽

Action Pattern ◽

Polysaccharide Lyase ◽

Glycosidic Bonds ◽

Alginate Oligosaccharides ◽

Degradation Pattern ◽

Alginate Lyases ◽

Layer Chromatography

Alginate lyases have been widely used to prepare alginate oligosaccharides in food, agricultural, and medical industries. Therefore, discovering and characterizing novel alginate lyases with excellent properties has drawn increasing attention. Herein, a novel alginate lyase FsAlyPL6 of Polysaccharide Lyase (PL) 6 family is identified and biochemically characterized from Flammeovirga sp. NJ-04. It shows highest activity at 45 °C and could retain 50% of activity after being incubated at 45 °C for 1 h. The Thin-Layer Chromatography (TLC) and Electrospray Ionization Mass Spectrometry (ESI-MS) analysis indicates that FsAlyPL6 endolytically degrades alginate polysaccharide into oligosaccharides ranging from monosaccharides to pentasaccharides. In addition, the action pattern of the enzyme is also elucidated and the result suggests that FsAlyPL6 could recognize tetrasaccharide as the minimal substrate and cleave the glycosidic bonds between the subsites of −1 and +3. The research provides extended insights into the substrate recognition and degradation pattern of PL6 alginate lyases, which may further expand the application of alginate lyases.

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Molecular identification of a polyM-specific alginate lyase from Pseudomonas sp. strain KS-408 for degradation of glycosidic linkages between two mannuronates or mannuronate and guluronate in alginate

Canadian Journal of Microbiology ◽

10.1139/w11-106 ◽

2011 ◽

Vol 57 (12) ◽

pp. 1032-1041 ◽

Cited By ~ 24

Author(s):

Natania Kam ◽

Yoo Jung Park ◽

Eun Yeol Lee ◽

Hee Sook Kim

Keyword(s):

Signal Peptide ◽

Alginate Lyase ◽

Ionization Mass ◽

Pseudomonas Sp ◽

H Nmr ◽

Alginate Oligosaccharides ◽

Catalytic Active Site ◽

Isoelectric Points ◽

Alginate Lyases ◽

Layer Chromatography

An alginate lyase gene of a newly isolated Pseudomonas sp. strain KS-408 was cloned by using PCR with the specific primers designed from homologous nucleotide sequences. A partial protein sequence of KS-408 alginate lyase was homology-modeled on the basis of the crystal structure of A1-III alginate lyase from Sphingomonas sp. strain A1. The proposed 3-D structure of KS-408 alginate lyase shows that Asn-198, His-199, Arg-246, and Tyr-253 residues are conserved for the catalytic active site. The recombinant KS-408-1F (with signal peptide) and KS-408-2F (without signal peptide) alginate lyases with the (His)6 tag consist of 393 (44.5 kDa) and 372 (42.4 kDa) amino acids with isoelectric points of 8.64 and 8.46, respectively. The purified recombinant KS-408 alginate lyase was very stable when it was incubated at 40 °C for 30 min. Alginate oligosaccharides produced by the KS-408-2F alginate lyase were purified on a Bio-Gel P2 column and analyzed by thin-layer chromatography, fast-protein liquid chromatography, and electrospray ionization mass spectrometry. 1H NMR data showed that the KS-408-2F alginate lyase cleaved the glycosidic linkages between two mannuronates (mannuronate-β(1–4)-mannuronate) or mannuronate and guluronate (mannuronate-β(1–4)-guluronate), indicating that the KS-408 alginate lyase is a polyM-specific lyase.

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Alginate Lyases: Sources, Mechanism of Activity and Potencial Application

SQUALEN Bulletin of Marine and Fisheries Postharvest and Biotechnology ◽

10.15578/squalen.v8i3.39 ◽

2013 ◽

Vol 8 (3) ◽

pp. 105 ◽

Cited By ~ 1

Author(s):

Subaryono Subaryono ◽

Rosmawaty Peranginangin ◽

Maggy Thenawidjaja Suhartono ◽

Fransiska Rungkat Zakaria

Keyword(s):

Rheological Properties ◽

Structure Analysis ◽

Biological Activities ◽

Alginate Lyase ◽

Present Review ◽

Biological Engineering ◽

Alginate Oligosaccharides ◽

Potential Applications ◽

Growth Promoting ◽

Alginate Lyases

Alginate lyases are group of enzymes which catalyze depolymerization of alginate into oligosaccharides. Alginate lyase have been widely used in many applications such as in production of bioactive oligosaccharides, control of polysaccharide rheological properties, and polysaccharide structure analysis. The products of alginate lyase, polysaccharide structure analysis, alginate oligosaccharides (AOS) have many biological activities including act as prebiotics, immune modulator, anticoagulation, antioxidant, anticancer, growth promoting activities, promote production of antibiotics and ethanol. In relation to the importance of alginate lyases, their potential aplications and prospect in development of new bioactive products, we present review of the enzymes, sources, mechanism of activity and potential applications. This paper also discussed some new biological engineering in alginate lyase production.

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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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Efficient Degradation of Alginate And Preparation of Unsaturated Monosaccharides By A Novel Alginate Lyase And Effects of Domain Truncation On Biochemical Characteristics, Degradation Patterns

10.21203/rs.3.rs-832451/v1 ◽

2021 ◽

Author(s):

Qian Li ◽

Shengsheng Cao ◽

Ling Zheng ◽

Benwei Zhu

Keyword(s):

Synergistic Effect ◽

Brown Algae ◽

Catalytic Efficiency ◽

Alginate Lyase ◽

Commercial Application ◽

Action Mode ◽

Domain Truncation ◽

High Conversion Rate ◽

Modular Domain ◽

Alginate Lyases

Abstract BackgroundBrown algae are considered promising crops for the production of sustainable biofuels. However, its commercial application has been limited by lack of efficient methods for converting alginate into fermentable sugars. Recently, exo-type alginate lyases have received extensive attention due to their excellent ability of conversion of alginate into 4-deoxy-L-erythro-5-hexoseulose uronate (DEH), a promising material for bioethanol production and biorefinery systems.ResultsHerein, we cloned and characterized a novel alginate lyase AlyPL17 from Pedobacter hainanensis NJ-02. It possessed outstanding catalytic efficiency towards polymannuronic acid (polyM), polyguluronic acid (polyG) and alginate sodium, with kcat of 39.42 + 1.9 s-1, 32.53 + 0.88 s-1, and 38.30 + 2.12 s-1, respectively. In addition, AlyPL17 adopts a unique hybrid action mode to degrade alginate by the synergistic effect of two domains. Furthermore, the combination of AlyPL17 and AlyPL6 exhibited apparently synergistic effect for the preparation of unsaturated monosaccharides. ConclusionOverall, the results show that AlyPL17 is a PL17 exo-type alginate lyase with high activity and a high conversion rate at low/moderate temperatures, which provides a useful enzymatic tool for the conversion of brown algae into biofuels and enhance our understanding of the function of modular domain of alginate lyase.

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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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Structural insights into the substrate-binding cleft of AlyF reveal the first long-chain alginate-binding mode

Acta Crystallographica Section D Structural Biology ◽

10.1107/s205979832100005x ◽

2021 ◽

Vol 77 (3) ◽

pp. 336-346

Author(s):

Keke Zhang ◽

Tao Liu ◽

Weizhi Liu ◽

Qianqian Lyu

Keyword(s):

Rational Design ◽

Substrate Binding ◽

Alginate Lyase ◽

Titration Calorimetry ◽

Scanning Calorimetry ◽

Alginate Oligosaccharides ◽

Binding Cleft ◽

Alginate Lyases ◽

Loop 2 ◽

Long Chain

The products of alginate degradation, alginate oligosaccharides (AOS), have potential applications in many areas, including functional foods and marine drugs. Enzyme-based approaches using alginate lyases have advantages in the preparation of well defined AOS and have attracted much attention in recent years. However, a lack of structural insight into the whole substrate-binding cleft for most known alginate lyases severely hampers their application in the industrial generation of well defined AOS. To solve this issue, AlyF was co-crystallized with the long alginate oligosaccharide G6 (L-hexaguluronic acid hexasodium salt), which is the longest bound substrate in all solved alginate lyase complex structures. AlyF formed interactions with G6 from subsites −3 to +3 without additional substrate-binding site interactions, suggesting that the substrate-binding cleft of AlyF was fully occupied by six sugars, which was further confirmed by isothermal titration calorimetry and differential scanning calorimetry analyses. More importantly, a combination of structural comparisons and mutagenetic analyses determined that three key loops (loop 1, Lys215–Glu236; loop 2, Gln402–Ile416; loop 3, Arg334–Gly348) mainly function in binding long substrates (degree of polymerization of >4). The potential flexibility of loop 1 and loop 2 might enable the substrate to continue to enter the cleft after binding to subsites +1 to +3; loop 3 stabilizes and orients the substrate at subsites −2 and −3. Taken together, these results provide the first possible alginate lyase–substrate binding profile for long-chain alginates, facilitating the rational design of new enzymes for industrial purposes.

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