Optimized Recombinant Expression and Characterization of Collagenase in <i>Bacillus subtilis</i> WB600
Background: The collagenase encoding gene <i>col</i> was cloned into a pP43NMK vector and amplified in <i>Escherichia coli</i> JM109 cells. The shuttle vector pP43NMK was used to sub-clone the <i>col</i> gene to obtain the vector pP43NMK-<i>col</i> for...
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MDPI AG
2022-09-01
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| Online Access: | https://www.mdpi.com/2311-5637/8/9/449 |
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| author | Yaqing Zhu Linlin Wang Kaixuan Zheng Ping Liu Wenkang Li Jian Lin Wenjing Liu Shoushui Shan Liqin Sun Hailing Zhang |
| author_facet | Yaqing Zhu Linlin Wang Kaixuan Zheng Ping Liu Wenkang Li Jian Lin Wenjing Liu Shoushui Shan Liqin Sun Hailing Zhang |
| author_sort | Yaqing Zhu |
| collection | DOAJ |
| description | Background: The collagenase encoding gene <i>col</i> was cloned into a pP43NMK vector and amplified in <i>Escherichia coli</i> JM109 cells. The shuttle vector pP43NMK was used to sub-clone the <i>col</i> gene to obtain the vector pP43NMK-<i>col</i> for the expression of collagenase in <i>Bacillus subtilis</i> WB600. The enzyme was characterized and the composition of the expression medium and culture conditions were optimized. Methods: The expressed recombinant enzyme was purified by ammonium sulfate, ultrafiltration, and through a nickel column. The purified collagenase had an activity of 9405.54 U/mg. Results: The recombinant enzyme exhibited optimal activity at pH 9.0 and 50 °C. Catalytic efficiency of the recombinant collagenase was inhibited by Fe<sup>3+</sup> and Cu<sup>2+</sup>, but stimulated by Co<sup>2+</sup>, Ca<sup>2+</sup>, Zn<sup>2+</sup>, and Mg<sup>2+</sup>. The optimal conditions for its growth were at pH 7.0 and 35 °C, using 15 g/L of fructose and 36 g/L of yeast powder and peptone mixture (2:1) at 260 rpm with 11% inoculation. The maximal extracellular activity of the recombinant collagenase reached 2746.7 U/mL after optimization of culture conditions, which was 2.4-fold higher than that before optimization. Conclusions: This study is a first attempt to recombinantly express collagenase in <i>B. subtilis</i> WB600 and optimize its expression conditions, its production conditions, and possible scale-up. |
| first_indexed | 2024-03-10T00:04:43Z |
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| language | English |
| last_indexed | 2024-03-10T00:04:43Z |
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| series | Fermentation |
| spelling | doaj.art-d20fbff643aa46d4b5c095b7596922592023-11-23T16:10:53ZengMDPI AGFermentation2311-56372022-09-018944910.3390/fermentation8090449Optimized Recombinant Expression and Characterization of Collagenase in <i>Bacillus subtilis</i> WB600Yaqing Zhu0Linlin Wang1Kaixuan Zheng2Ping Liu3Wenkang Li4Jian Lin5Wenjing Liu6Shoushui Shan7Liqin Sun8Hailing Zhang9Department of Biological Engineering, College of Life Sciences, Yantai University, Yantai 264005, ChinaDepartment of Food Engineering, Shandong Business Institute, Yantai 264670, ChinaDepartment of Biological Engineering, College of Life Sciences, Yantai University, Yantai 264005, ChinaCentral Innovation Biotechnology (Shandong) Co., 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, ChinaSchool of Medicine, Yunnan University, Kunming 650091, 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, ChinaBackground: The collagenase encoding gene <i>col</i> was cloned into a pP43NMK vector and amplified in <i>Escherichia coli</i> JM109 cells. The shuttle vector pP43NMK was used to sub-clone the <i>col</i> gene to obtain the vector pP43NMK-<i>col</i> for the expression of collagenase in <i>Bacillus subtilis</i> WB600. The enzyme was characterized and the composition of the expression medium and culture conditions were optimized. Methods: The expressed recombinant enzyme was purified by ammonium sulfate, ultrafiltration, and through a nickel column. The purified collagenase had an activity of 9405.54 U/mg. Results: The recombinant enzyme exhibited optimal activity at pH 9.0 and 50 °C. Catalytic efficiency of the recombinant collagenase was inhibited by Fe<sup>3+</sup> and Cu<sup>2+</sup>, but stimulated by Co<sup>2+</sup>, Ca<sup>2+</sup>, Zn<sup>2+</sup>, and Mg<sup>2+</sup>. The optimal conditions for its growth were at pH 7.0 and 35 °C, using 15 g/L of fructose and 36 g/L of yeast powder and peptone mixture (2:1) at 260 rpm with 11% inoculation. The maximal extracellular activity of the recombinant collagenase reached 2746.7 U/mL after optimization of culture conditions, which was 2.4-fold higher than that before optimization. Conclusions: This study is a first attempt to recombinantly express collagenase in <i>B. subtilis</i> WB600 and optimize its expression conditions, its production conditions, and possible scale-up.https://www.mdpi.com/2311-5637/8/9/449collagenase<i>Bacillus subtilis</i> WB600protein purificationcharacterizationrecombinant protein expression |
| spellingShingle | Yaqing Zhu Linlin Wang Kaixuan Zheng Ping Liu Wenkang Li Jian Lin Wenjing Liu Shoushui Shan Liqin Sun Hailing Zhang Optimized Recombinant Expression and Characterization of Collagenase in <i>Bacillus subtilis</i> WB600 Fermentation collagenase <i>Bacillus subtilis</i> WB600 protein purification characterization recombinant protein expression |
| title | Optimized Recombinant Expression and Characterization of Collagenase in <i>Bacillus subtilis</i> WB600 |
| title_full | Optimized Recombinant Expression and Characterization of Collagenase in <i>Bacillus subtilis</i> WB600 |
| title_fullStr | Optimized Recombinant Expression and Characterization of Collagenase in <i>Bacillus subtilis</i> WB600 |
| title_full_unstemmed | Optimized Recombinant Expression and Characterization of Collagenase in <i>Bacillus subtilis</i> WB600 |
| title_short | Optimized Recombinant Expression and Characterization of Collagenase in <i>Bacillus subtilis</i> WB600 |
| title_sort | optimized recombinant expression and characterization of collagenase in i bacillus subtilis i wb600 |
| topic | collagenase <i>Bacillus subtilis</i> WB600 protein purification characterization recombinant protein expression |
| url | https://www.mdpi.com/2311-5637/8/9/449 |
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