Cloning, Expression and Characterization of an Alginate Lyase in <i>Bacillus subtilis</i> WB600
The aim of this study was to further broaden the heterologous expression of alginate lyase from <i>Vibrio alginolyticus</i> in a <i>Bacillus subtilis</i> expression vector. A <i>B. subtilis</i> WB600/pP43NMK-<i>alg62</i> strain was constructed. (NH<...
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MDPI AG
2023-02-01
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| Series: | Fermentation |
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| Online Access: | https://www.mdpi.com/2311-5637/9/2/144 |
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| author | Kaixuan Zheng Yaqing Zhu Zhiqiang An Jian Lin Shoushui Shan Hailing Zhang |
| author_facet | Kaixuan Zheng Yaqing Zhu Zhiqiang An Jian Lin Shoushui Shan Hailing Zhang |
| author_sort | Kaixuan Zheng |
| collection | DOAJ |
| description | The aim of this study was to further broaden the heterologous expression of alginate lyase from <i>Vibrio alginolyticus</i> in a <i>Bacillus subtilis</i> expression vector. A <i>B. subtilis</i> WB600/pP43NMK-<i>alg62</i> strain was constructed. (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> precipitation and Ni-affinity chromatography were performed to purify the enzyme. We then characterized the enzyme. Its molecular weight was 57.64 kDa, and it worked optimally at 30 °C with a pH of 8.0. Ca<sup>2+</sup> markedly enhanced the enzymatic activity of Alg62 while Cu<sup>2+</sup> and Ni<sup>2+</sup> inhibited its activity. Alg62 had a wide range of substrate specificity, showing high activity toward sodium alginate and polyG. Following optimization of the fermentation process, the optimal conditions for the recombinant expression of Alg62 were as follows: temperature of 37 °C, pH of 7.0, medium consisting of glycerol 15 g/L, yeast powder 25 g/L and K<sup>+</sup> 1.5 mmol/L. At these optimal conditions, enzyme activity reached 318.21 U/mL, which was 1.54 times higher than the initial enzyme activity. |
| first_indexed | 2024-03-11T08:50:54Z |
| format | Article |
| id | doaj.art-72400803e0e343ada42d369fb03c8e96 |
| institution | Directory Open Access Journal |
| issn | 2311-5637 |
| language | English |
| last_indexed | 2024-03-11T08:50:54Z |
| publishDate | 2023-02-01 |
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| series | Fermentation |
| spelling | doaj.art-72400803e0e343ada42d369fb03c8e962023-11-16T20:26:05ZengMDPI AGFermentation2311-56372023-02-019214410.3390/fermentation9020144Cloning, Expression and Characterization of an Alginate Lyase in <i>Bacillus subtilis</i> WB600Kaixuan Zheng0Yaqing Zhu1Zhiqiang An2Jian Lin3Shoushui Shan4Hailing Zhang5Department of Biological Engineering, College of Life Sciences, Yantai University, Yantai 264005, ChinaDepartment of Biological Engineering, College of Life Sciences, Yantai University, Yantai 264005, ChinaMabPlex International, Ltd., Yantai 264005, ChinaDepartment of Biological Engineering, College of Life Sciences, Yantai University, Yantai 264005, ChinaDepartment of Biological Engineering, College of Life Sciences, Yantai University, Yantai 264005, ChinaDepartment of Biological Engineering, College of Life Sciences, Yantai University, Yantai 264005, ChinaThe aim of this study was to further broaden the heterologous expression of alginate lyase from <i>Vibrio alginolyticus</i> in a <i>Bacillus subtilis</i> expression vector. A <i>B. subtilis</i> WB600/pP43NMK-<i>alg62</i> strain was constructed. (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> precipitation and Ni-affinity chromatography were performed to purify the enzyme. We then characterized the enzyme. Its molecular weight was 57.64 kDa, and it worked optimally at 30 °C with a pH of 8.0. Ca<sup>2+</sup> markedly enhanced the enzymatic activity of Alg62 while Cu<sup>2+</sup> and Ni<sup>2+</sup> inhibited its activity. Alg62 had a wide range of substrate specificity, showing high activity toward sodium alginate and polyG. Following optimization of the fermentation process, the optimal conditions for the recombinant expression of Alg62 were as follows: temperature of 37 °C, pH of 7.0, medium consisting of glycerol 15 g/L, yeast powder 25 g/L and K<sup>+</sup> 1.5 mmol/L. At these optimal conditions, enzyme activity reached 318.21 U/mL, which was 1.54 times higher than the initial enzyme activity.https://www.mdpi.com/2311-5637/9/2/144alginate lyase<i>Bacillus subtilis</i> WB600heterologous expressionalginate oligosaccharidefermentation optimization |
| spellingShingle | Kaixuan Zheng Yaqing Zhu Zhiqiang An Jian Lin Shoushui Shan Hailing Zhang Cloning, Expression and Characterization of an Alginate Lyase in <i>Bacillus subtilis</i> WB600 Fermentation alginate lyase <i>Bacillus subtilis</i> WB600 heterologous expression alginate oligosaccharide fermentation optimization |
| title | Cloning, Expression and Characterization of an Alginate Lyase in <i>Bacillus subtilis</i> WB600 |
| title_full | Cloning, Expression and Characterization of an Alginate Lyase in <i>Bacillus subtilis</i> WB600 |
| title_fullStr | Cloning, Expression and Characterization of an Alginate Lyase in <i>Bacillus subtilis</i> WB600 |
| title_full_unstemmed | Cloning, Expression and Characterization of an Alginate Lyase in <i>Bacillus subtilis</i> WB600 |
| title_short | Cloning, Expression and Characterization of an Alginate Lyase in <i>Bacillus subtilis</i> WB600 |
| title_sort | cloning expression and characterization of an alginate lyase in i bacillus subtilis i wb600 |
| topic | alginate lyase <i>Bacillus subtilis</i> WB600 heterologous expression alginate oligosaccharide fermentation optimization |
| url | https://www.mdpi.com/2311-5637/9/2/144 |
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