A new high-alkaline alginate lyase from a deep-sea bacterium Agarivorans sp.

Extremophiles are expected to represent a source of enzymes having unique functional properties. The hypothetical protein NIS_0185, termed NitAly in this study, was identified as an alginate lyase-homolog protein in the genomic database of ⑀-Proteobacteria Nitratiruptor sp. SB155-2, which was isolated from deep-sea hydrothermal vents at a water depth of 1,000 m. Among the characterized alginate lyases in the polysaccharide lyase family 7 (PL-7), the amino acid sequence of NitAly showed the highest identity (39%) with that of red alga Pyropia yezoensis alginate lyase PyAly. Recombinant NitAly (rNitAly) was successfully expressed in Escherichia coli. Purified rNitAly degraded alginate in an endolytic manner. Among alginate block types, polyM was preferable to polyG and polyMG as a substrate, and its end degradation products were mainly tri-, tetra-, and penta-saccharides. The optimum temperature and pH values were 70°C and around 6, respectively. A high concentration of NaCl (0.8 -1.4 M) was required for maximum activity. In addition, a 50% loss of activity was observed after incubation at 67°C for 30 min. Heat stability was decreased in the presence of 5 mM DTT, and Cys-80 and Cys-232 were identified as the residues responsible for heat stability but not lyase activity. Introducing two cysteines into PyAly based on homology modeling using Pseudomonas aeruginosa alginate lyase PA1167 as the template enhanced its heat stability. Thus, NitAly is a functional alginate lyase, with its unique optimum conditions adapted to its environment. These insights into the heat stability of NitAly could be applied to improve that of other PL-7 alginate lyases.Alginate consists of two uronic acids, ␤-D-mannuronic acid (M) and its C5 epimer ␣-L-guluronic acid (G) (1, 2). These units are linearly linked by a 1-4-glycosidic bond and are arranged in an M-consecutive sequence (M-block), G-consecutive sequence (G-block), and M/G random sequence (MG-block). Only a limited number of organisms produce alginate in nature. As a typical example, brown algae biosynthesize alginate as a cell wall component (3), although some types of mucoid bacteria, such as Pseudomonas spp. and Azotobacter vinelandii, produce alginate as an exopolysaccharide (4).Alginate lyase enzymes catalyze the cleavage of the 1-4-glycosidic bond of alginate via a ␤-elimination mechanism (5). These enzymes are found in organisms that have either alginate-metabolizing systems or alginate biosynthesis systems. In alginate metabolization, endo-type alginate lyase(s) attack alginates and degrade them to oligosaccharides. Then, exo-type alginate lyase(s) release monosaccharides, and 4-deoxy-Lerythro-5-hexoseulose uronic acid is generated by non-enzymatic conversion. 4-Deoxy-L-erythro-5-hexoseulose uronic acid is reduced to 2-keto-3-deoxygluconate by a 4-deoxy-Lerythro-5-hexoseulose uronic acid-specific reductase (6 -8), with 2-keto-3-deoxygluconate possibly being metabolized by the Entner-Doudoroff pathway (9,10

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“…Agarivorans sp. JAM-A1m alginate lyase A1m (16%) (31) and Vibrio sp. alginate lyase A9mT (20%) (25).…”

Section: Resultsmentioning

confidence: 99%

Discovery of a Novel Alginate Lyase from Nitratiruptor sp. SB155-2 Thriving at Deep-sea Hydrothermal Vents and Identification of the Residues Responsible for Its Heat Stability

Inoue

Anraku

Nakagawa

et al. 2016

Journal of Biological Chemistry

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“…[8][9][10][11][12][13][14] Oligomeric alginates obtained from lyase degradation of alginate act as oligosaccharines and regulate physiological processes in plants and microorganisms, such as promoting the growth of Bifidobacterium spp., enhancing the germination and shoot elongation in plants. The oligosaccharides exhibit many physiological activities, such as antitumor, stimulating production of cytokines, regulating the blood lipids and sugars.…”

Section: Functionality and Promising Application Of Alginate Lyasementioning

confidence: 99%

“…13 A number of alginate lyases have been identified, cloned, purified and characterized from various sources, [14][15][16][17][18] including marine and terrestrial bacteria, marine mollusks and algae. They can be classed into 2 groups due to their substrate specificities, 13 molecular weights 19 and action modes.…”

Section: Introductionmentioning

confidence: 99%

Alginate lyase: Review of major sources and classification, properties, structure-function analysis and applications

2015

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A lginate lyases catalyze the degradation of alginate, a complex copolymer of a-L-guluronate and its C5 epimer b-D-mannuronate. The enzymes have been isolated from various kinds of organisms with different substrate specificities, including algae, marine mollusks, marine and terrestrial bacteria, and some viruses and fungi. With the progress of structural biology, many kinds of alginate lyases of different polysaccharide lyases families have been characterized by obtaining crystal structures, and the catalytic mechanism has also been elucidated. Combined with various studies, we summarized the source, classification and properties of the alginate lyases from different polysaccharide lyases families. The relationship between substrate specificity and protein sequence was also investigated.

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“…ALY-1 [29], A1-I from Sphingomonas sp.A1 [30] and A1m from Agarivorans sp. JAM-A1m [31], whose of them preferred polyG as the substrate.…”

Section: Discussionmentioning

confidence: 99%

“…The endo-lytic mode actions of enzyme Alm from Agarivorans sp. JAM-A1m [22] and enzyme AlySY08 from Vibrio sp. SY08 [23] were confirmed in the same method.…”

Section: Reaction Products and Modes Of Actionmentioning

confidence: 99%

Cloning and Characterization of an Alginate Lyase from Marine <em>Vibrio</em>. sp. QD-5

Chao¹,

Wang²,

Wu³

et al. 2017

Preprint

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Abstract:The string of bacteria, Vibrio. sp. QD-5 utilizing alginate, was separated from rotten kelp. The results of genome sequencing showed that the strain QD-5 contained four alginate lyase genes.One of the alginate genes Aly-IV was cloned and linked to the plasmid pET-22b (+). The heterologous expressed alginate lyase aly-IVwas characterized，which possessed the theoretical molecular mass of 62 kDa, and theoretical isoelectric point (pI) of 5.12. -The enzyme aly-IV was purified and the activity reached 1256.78 U/mg, with optimal temperature of 35 o C and pH value of 8.9. Nurtured in the temperature below 25 o C for 30 minutes, the activity was almost stable. The result also suggested that the activity of enzyme was strongly affected by -NaCl whose optimal concentration was 15 mM in a lab environment The TLC and ESI-TOF-MS analysis suggested that the enzyme aly-IV could degrade sodium alginate and polyG in endo-lytic type, producing monomer, dimer and trimmer. So, aly-IV can also be widely applied to make large scale preparation of alginate oligosaccharides with low degree of polymerization (DP).

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A new high-alkaline alginate lyase from a deep-sea bacterium Agarivorans sp.

Cited by 81 publications

References 28 publications

Discovery of a Novel Alginate Lyase from Nitratiruptor sp. SB155-2 Thriving at Deep-sea Hydrothermal Vents and Identification of the Residues Responsible for Its Heat Stability

Discovery of a Novel Alginate Lyase from Nitratiruptor sp. SB155-2 Thriving at Deep-sea Hydrothermal Vents and Identification of the Residues Responsible for Its Heat Stability

Alginate lyase: Review of major sources and classification, properties, structure-function analysis and applications

Cloning and Characterization of an Alginate Lyase from Marine <em>Vibrio</em>. sp. QD-5

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