Characterization of Recombinant PolyG-Specific Lyase from a Marine Bacterium, Streptomyces sp. M3

Kim, Hee Sook

doi:10.5352/jls.2010.20.11.1582

Cited by 7 publications

(4 citation statements)

References 27 publications

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“…The molecular mass of purified rNitAly was estimated at ϳ29 kDa under reducing conditions using SDS-PAGE. This value corresponded reasonably well with the calculated mass of rNitAly (29,636 Da) (Fig. 2B, left lane).…”

Section: Resultssupporting

confidence: 86%

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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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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Section: Resultssupporting

confidence: 86%

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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“…These results were similar to reports of M3 lyase and alginate lyase from Streptomyces spp. [44,45] and the alginate lyase from Isoptericola halotolerans CGMCC 5336 [28]. Surprisingly, the activity of AlyDS44 more than doubled in the presence of Mn 2+ and Co 2+ , with increases of 242% and 219% relative activity, respectively.…”

Section: Optimisation Of Ph Temperature and Nacl And The Effect Of Metal Ions On Alginate Lyase Activitymentioning

confidence: 96%

Purification and Characterization of a Novel Alginate Lyase from a Marine Streptomyces Species Isolated from Seaweed

et al. 2021

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Alginate, a natural polysaccharide derived from brown seaweed, is finding multiple applications in biomedicine via its transformation through chemical, physical, and, increasingly, enzymatic processes. In this study a novel alginate lyase, AlyDS44, was purified and characterized from a marine actinobacterium, Streptomyces luridiscabiei, which was isolated from decomposing seaweed. The purified enzyme had a specific activity of 108.6 U/mg, with a molecular weight of 28.6 kDa, and was composed of 260 amino acid residues. AlyDS44 is a bifunctional alginate lyase, active on both polyguluronate and polymannuronate, though it preferentially degrades polyguluronate. The optimal pH of this enzyme is 8.5 and the optimal temperature is 45 °C. It is a salt-tolerant alginate lyase with an optimal activity at 0.6 M NaCl. Metal ions Mn2+, Co2+, and Fe2+ increased the alginate degrading activity, but it was inhibited in the presence of Zn2+ and Cu2+. The highly conserved regions of its amino acid sequences indicated that AlyDS44 belongs to the polysaccharide lyase family 7. The main breakdown products of the enzyme on alginate were disaccharides, trisaccharides, and tetrasaccharides, which demonstrated that this enzyme acted as an endo-type alginate lyase. AlyDS44 is a novel enzyme, with the potential for efficient production of alginate oligosaccharides with low degrees of polymerization.

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“…For protein expression of M3 alginate lyase, the recombinant cells were cultured at 15℃ for 24 h with 1 mM IPTG [10]. The cells were harvested and sonicated in 50 mM potassium phosphate buffer (pH 7.2) containing 300 mM NaCl, 10 mM imidazole, 10% (v/v) glycerol, 0.5% (v/v) Triton X-100, 1 mg/ml lysozyme and 1 mM PMSF by using ultrasonicator Vibra Cell CX400 (Sonics & Materials Inc, USA).…”

Section: Purification Of M3 Alginate Lyasementioning

confidence: 99%

“…Recently, we have cloned and characterized a polyG-specific alginate lyase from a marine bacterium, Streptomyces sp. strain M3 that degrades G-rich region of alginates [10,11]. It releases unsaturated di-, tri-and tetra-saccharides from alginate.…”

Section: Introductionmentioning

confidence: 99%

Enhancing the Alginate Degrading Activity of Streptomyces sp. Strain M3 Alginate Lyase by Mutation

Kim¹

2012

Journal of Life Science

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A polyguluronate-specific lyase from Streptomyces sp. strain M3 has been previously cloned and characterized. In this study, the M3 alginate lyase gene in the pColdI vector was mutated by site-directed mutagenesis and random mutagenesis to enhance the alginate degrading activity. Six mutants were obtained: Ser25Arg, Phe99Leu, Asp142Asn, Val163Ala, Lys191Glu, and Gly194Cys. Phe99Leu and Lys191Glu mutants completely lost their alginate lyase activity, whereas the alginate degrading activity of Gly194Cys mutant increased by nearly 10 fold. The 3-D protein structure of M3 alginate lyase, which was constructed using the Swiss-Model automodeler, was also compared to the crystal structure of another alginate lyase. A mutated glycine residue was positioned between Gly193 and Tyr195 of the C-terminal conserved sequence, YFKAGXYXQ. A phenylalanine residue (at position 99) and a glycine residue (at position 194) mutated in this study were distant from the active site, but the degrading activity was strongly affected by their mutation.

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Characterization of Recombinant PolyG-Specific Lyase from a Marine Bacterium, Streptomyces sp. M3

Cited by 7 publications

References 27 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

Purification and Characterization of a Novel Alginate Lyase from a Marine Streptomyces Species Isolated from Seaweed

Enhancing the Alginate Degrading Activity of Streptomyces sp. Strain M3 Alginate Lyase by Mutation

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