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Purification and characterization of a bifunctional alginate lyase from Pseudoalteromonas sp. SM0524.

Li JW, Dong S, Song J, Li CB, Chen XL, Xie BB, Zhang YZ - Mar Drugs (2011)

Bottom Line: Aly-SJ02 showed activities toward both polyG (α-l-guluronic acid) and polyM (β-D-mannuronic acid), indicating that it is a bifunctional alginate lyase.Aly-SJ02 had lower K(m) toward polyG than toward polyM and sodium alginate.Thin layer chromatography and ESI-MS analyses showed that aly-SJ02 mainly released dimers and trimers from polyM and alginate, and trimers and tetramers from polyG, which suggests that aly-SJ02 may be a good tool to produce dimers and trimers from alginate.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Jinan 250100, China. sdnyljw@163.com

ABSTRACT
An alginate lyase-producing bacterial strain, Pseudoalteromonas sp. SM0524, was screened from marine rotten kelp. In an optimized condition, the production of alginate lyase from Pseudoalteromonas sp. SM0524 reached 62.6 U/mL, suggesting that strain SM0524 is a good producer of alginate lyases. The bifunctional alginate lyase aly-SJ02 secreted by strain SM0524 was purified. Aly-SJ02 had an apparent molecular mass of 32 kDa. The optimal temperature and pH of aly-SJ02 toward sodium alginate was 50 °C and 8.5, respectively. The half life period of aly-SJ02 was 41 min at 40 °C and 20 min at 50 °C. Aly-SJ02 was most stable at pH 8.0. N-terminal sequence analysis suggested that aly-SJ02 may be an alginate lyase of polysaccharide lyase family 18. Aly-SJ02 showed activities toward both polyG (α-l-guluronic acid) and polyM (β-D-mannuronic acid), indicating that it is a bifunctional alginate lyase. Aly-SJ02 had lower K(m) toward polyG than toward polyM and sodium alginate. Thin layer chromatography and ESI-MS analyses showed that aly-SJ02 mainly released dimers and trimers from polyM and alginate, and trimers and tetramers from polyG, which suggests that aly-SJ02 may be a good tool to produce dimers and trimers from alginate.

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Effects of pH and temperature on the activity and stability of aly-SJ02 toward sodium alginate. (A) Effect of temperature on aly-SJ02 activity. The activity was assayed at pH 8.5. (B) Effect of pH on aly-SJ02 activity. The activity was measured at 50 °C in a broad buffer as described previously [9]. (C) Thermostability of aly-SJ02. The enzyme was incubated at 30 °C, 40 °C and 50 °C for different periods of time, and then the residual activity was assayed at 50 °C. (D) pH stability of aly-SJ02. Residual activities after incubation at various pHs for 20 min were assayed at pH 8.5, 50 °C. The data represent the mean of three experimental repeats with SD ≤ 5%.
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f3-marinedrugs-09-00109: Effects of pH and temperature on the activity and stability of aly-SJ02 toward sodium alginate. (A) Effect of temperature on aly-SJ02 activity. The activity was assayed at pH 8.5. (B) Effect of pH on aly-SJ02 activity. The activity was measured at 50 °C in a broad buffer as described previously [9]. (C) Thermostability of aly-SJ02. The enzyme was incubated at 30 °C, 40 °C and 50 °C for different periods of time, and then the residual activity was assayed at 50 °C. (D) pH stability of aly-SJ02. Residual activities after incubation at various pHs for 20 min were assayed at pH 8.5, 50 °C. The data represent the mean of three experimental repeats with SD ≤ 5%.

Mentions: With sodium alginate as substrate, aly-SJ02 displayed an optimal pH of 8.5 and an optimal temperature of 50 °C (Figure 3A, B). The half life period of aly-SJ02 was 41 min at 40 °C and 20 min at 50 °C, indicating that aly-SJ02 has low thermal stability (Figure 3C). Aly-SJ02 was most stable at pH 8.0 and retained more than 50% activity at pH 7.0–10 after incubation for 20 min (Figure 3D). The effects of metal ions on the activity of aly-SJ02 were analyzed. Most of the investigated metal ions, including Na+, K+, Mg2+, Ca2+, Co2+ Ba2+, Ni2+ and Sr2+, had activating effects on the activity of aly-SJ02. Zn2+ had no effect, and Cu2+ and Sn2+ had slight inhibitory effect on the activity of aly-SJ02. The presence of 1 mM EDTA decreased the enzyme activity to 48.3% of the control (Table 2), suggesting that aly-SJ02 may have metal ions in its structure. Aly-SJ02 showed the highest activity in 0.2 M NaCl, and retained more than 75% activity in 1 M NaCl, indicating its salt-tolerant ability (Figure 4).


Purification and characterization of a bifunctional alginate lyase from Pseudoalteromonas sp. SM0524.

Li JW, Dong S, Song J, Li CB, Chen XL, Xie BB, Zhang YZ - Mar Drugs (2011)

Effects of pH and temperature on the activity and stability of aly-SJ02 toward sodium alginate. (A) Effect of temperature on aly-SJ02 activity. The activity was assayed at pH 8.5. (B) Effect of pH on aly-SJ02 activity. The activity was measured at 50 °C in a broad buffer as described previously [9]. (C) Thermostability of aly-SJ02. The enzyme was incubated at 30 °C, 40 °C and 50 °C for different periods of time, and then the residual activity was assayed at 50 °C. (D) pH stability of aly-SJ02. Residual activities after incubation at various pHs for 20 min were assayed at pH 8.5, 50 °C. The data represent the mean of three experimental repeats with SD ≤ 5%.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3039154&req=5

f3-marinedrugs-09-00109: Effects of pH and temperature on the activity and stability of aly-SJ02 toward sodium alginate. (A) Effect of temperature on aly-SJ02 activity. The activity was assayed at pH 8.5. (B) Effect of pH on aly-SJ02 activity. The activity was measured at 50 °C in a broad buffer as described previously [9]. (C) Thermostability of aly-SJ02. The enzyme was incubated at 30 °C, 40 °C and 50 °C for different periods of time, and then the residual activity was assayed at 50 °C. (D) pH stability of aly-SJ02. Residual activities after incubation at various pHs for 20 min were assayed at pH 8.5, 50 °C. The data represent the mean of three experimental repeats with SD ≤ 5%.
Mentions: With sodium alginate as substrate, aly-SJ02 displayed an optimal pH of 8.5 and an optimal temperature of 50 °C (Figure 3A, B). The half life period of aly-SJ02 was 41 min at 40 °C and 20 min at 50 °C, indicating that aly-SJ02 has low thermal stability (Figure 3C). Aly-SJ02 was most stable at pH 8.0 and retained more than 50% activity at pH 7.0–10 after incubation for 20 min (Figure 3D). The effects of metal ions on the activity of aly-SJ02 were analyzed. Most of the investigated metal ions, including Na+, K+, Mg2+, Ca2+, Co2+ Ba2+, Ni2+ and Sr2+, had activating effects on the activity of aly-SJ02. Zn2+ had no effect, and Cu2+ and Sn2+ had slight inhibitory effect on the activity of aly-SJ02. The presence of 1 mM EDTA decreased the enzyme activity to 48.3% of the control (Table 2), suggesting that aly-SJ02 may have metal ions in its structure. Aly-SJ02 showed the highest activity in 0.2 M NaCl, and retained more than 75% activity in 1 M NaCl, indicating its salt-tolerant ability (Figure 4).

Bottom Line: Aly-SJ02 showed activities toward both polyG (α-l-guluronic acid) and polyM (β-D-mannuronic acid), indicating that it is a bifunctional alginate lyase.Aly-SJ02 had lower K(m) toward polyG than toward polyM and sodium alginate.Thin layer chromatography and ESI-MS analyses showed that aly-SJ02 mainly released dimers and trimers from polyM and alginate, and trimers and tetramers from polyG, which suggests that aly-SJ02 may be a good tool to produce dimers and trimers from alginate.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Jinan 250100, China. sdnyljw@163.com

ABSTRACT
An alginate lyase-producing bacterial strain, Pseudoalteromonas sp. SM0524, was screened from marine rotten kelp. In an optimized condition, the production of alginate lyase from Pseudoalteromonas sp. SM0524 reached 62.6 U/mL, suggesting that strain SM0524 is a good producer of alginate lyases. The bifunctional alginate lyase aly-SJ02 secreted by strain SM0524 was purified. Aly-SJ02 had an apparent molecular mass of 32 kDa. The optimal temperature and pH of aly-SJ02 toward sodium alginate was 50 °C and 8.5, respectively. The half life period of aly-SJ02 was 41 min at 40 °C and 20 min at 50 °C. Aly-SJ02 was most stable at pH 8.0. N-terminal sequence analysis suggested that aly-SJ02 may be an alginate lyase of polysaccharide lyase family 18. Aly-SJ02 showed activities toward both polyG (α-l-guluronic acid) and polyM (β-D-mannuronic acid), indicating that it is a bifunctional alginate lyase. Aly-SJ02 had lower K(m) toward polyG than toward polyM and sodium alginate. Thin layer chromatography and ESI-MS analyses showed that aly-SJ02 mainly released dimers and trimers from polyM and alginate, and trimers and tetramers from polyG, which suggests that aly-SJ02 may be a good tool to produce dimers and trimers from alginate.

Show MeSH
Related in: MedlinePlus