Improving Degradation of Polycyclic Aromatic Hydrocarbons by <i>Bacillus atrophaeus</i> Laccase Fused with <i>Vitreoscilla</i> Hemoglobin and a Novel Strong Promoter Replacement
Laccases catalyze a variety of electron-rich substrates by reducing O<sub>2</sub> to H<sub>2</sub>O, with O<sub>2</sub> playing a vital role as the final electron acceptor in the reaction process. In the present study, a laccase gene, <i>lach5</i>, was...
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MDPI AG
2022-07-01
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| author | Luyao Wang Yuzhi Tan Shengwei Sun Liangjie Zhou Guojun Wu Yuting Shao Mengxi Wang Zhihong Xin |
| author_facet | Luyao Wang Yuzhi Tan Shengwei Sun Liangjie Zhou Guojun Wu Yuting Shao Mengxi Wang Zhihong Xin |
| author_sort | Luyao Wang |
| collection | DOAJ |
| description | Laccases catalyze a variety of electron-rich substrates by reducing O<sub>2</sub> to H<sub>2</sub>O, with O<sub>2</sub> playing a vital role as the final electron acceptor in the reaction process. In the present study, a laccase gene, <i>lach5</i>, was identified from <i>Bacillus atrophaeus</i> through sequence-based screening. LacH5 was engineered for modification by fusion expression and promoter replacement. Results showed that the purified enzyme LacH5 exhibited strong oxidative activity towards 2,2’-azinobis(3-ehtylbenzothiazolin-6-sulfnic acid) ammonium salt (ABTS) under optimum pH and temperature conditions (pH 5.0, 60 °C) and displayed remarkable thermostability. The activity of the two fusion enzymes was enhanced significantly from 14.2 U/mg (LacH5) to 22.5 U/mg (LacH5-vgb) and 18.6 U/mg (Vgb-lacH5) toward ABTS after LacH5 fusing with <i>Vitreoscilla</i> hemoglobin (VHb). Three of six tested polycyclic aromatic hydrocarbons (PAHs) were significantly oxidized by two fusion laccases as compared with LacH5. More importantly, the expression level of LacH5 and fusion protein LacH5-vgb was augmented by 3.7-fold and 7.0-fold, respectively, by using a novel strong promoter replacement. The results from the current investigation provide new insights and strategies for improving the activity and expression level of bacterial laccases, and these strategies can be extended to other laccases and multicopper oxidases. |
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| last_indexed | 2024-03-09T11:52:08Z |
| publishDate | 2022-07-01 |
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| spelling | doaj.art-13ef00111efc4adba25f8840d9a4abbf2023-11-30T23:12:55ZengMDPI AGBiology2079-77372022-07-01118112910.3390/biology11081129Improving Degradation of Polycyclic Aromatic Hydrocarbons by <i>Bacillus atrophaeus</i> Laccase Fused with <i>Vitreoscilla</i> Hemoglobin and a Novel Strong Promoter ReplacementLuyao Wang0Yuzhi Tan1Shengwei Sun2Liangjie Zhou3Guojun Wu4Yuting Shao5Mengxi Wang6Zhihong Xin7Key Laboratory of Food Processing and Quality Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, ChinaKey Laboratory of Food Processing and Quality Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, ChinaKey Laboratory of Food Processing and Quality Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, ChinaKey Laboratory of Food Processing and Quality Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, ChinaKey Laboratory of Food Processing and Quality Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, ChinaKey Laboratory of Food Processing and Quality Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, ChinaKey Laboratory of Food Processing and Quality Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, ChinaKey Laboratory of Food Processing and Quality Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, ChinaLaccases catalyze a variety of electron-rich substrates by reducing O<sub>2</sub> to H<sub>2</sub>O, with O<sub>2</sub> playing a vital role as the final electron acceptor in the reaction process. In the present study, a laccase gene, <i>lach5</i>, was identified from <i>Bacillus atrophaeus</i> through sequence-based screening. LacH5 was engineered for modification by fusion expression and promoter replacement. Results showed that the purified enzyme LacH5 exhibited strong oxidative activity towards 2,2’-azinobis(3-ehtylbenzothiazolin-6-sulfnic acid) ammonium salt (ABTS) under optimum pH and temperature conditions (pH 5.0, 60 °C) and displayed remarkable thermostability. The activity of the two fusion enzymes was enhanced significantly from 14.2 U/mg (LacH5) to 22.5 U/mg (LacH5-vgb) and 18.6 U/mg (Vgb-lacH5) toward ABTS after LacH5 fusing with <i>Vitreoscilla</i> hemoglobin (VHb). Three of six tested polycyclic aromatic hydrocarbons (PAHs) were significantly oxidized by two fusion laccases as compared with LacH5. More importantly, the expression level of LacH5 and fusion protein LacH5-vgb was augmented by 3.7-fold and 7.0-fold, respectively, by using a novel strong promoter replacement. The results from the current investigation provide new insights and strategies for improving the activity and expression level of bacterial laccases, and these strategies can be extended to other laccases and multicopper oxidases.https://www.mdpi.com/2079-7737/11/8/1129thermostable laccasePAH biodegradationfusion expressionpromoter screeningstrong promoter replacement |
| spellingShingle | Luyao Wang Yuzhi Tan Shengwei Sun Liangjie Zhou Guojun Wu Yuting Shao Mengxi Wang Zhihong Xin Improving Degradation of Polycyclic Aromatic Hydrocarbons by <i>Bacillus atrophaeus</i> Laccase Fused with <i>Vitreoscilla</i> Hemoglobin and a Novel Strong Promoter Replacement Biology thermostable laccase PAH biodegradation fusion expression promoter screening strong promoter replacement |
| title | Improving Degradation of Polycyclic Aromatic Hydrocarbons by <i>Bacillus atrophaeus</i> Laccase Fused with <i>Vitreoscilla</i> Hemoglobin and a Novel Strong Promoter Replacement |
| title_full | Improving Degradation of Polycyclic Aromatic Hydrocarbons by <i>Bacillus atrophaeus</i> Laccase Fused with <i>Vitreoscilla</i> Hemoglobin and a Novel Strong Promoter Replacement |
| title_fullStr | Improving Degradation of Polycyclic Aromatic Hydrocarbons by <i>Bacillus atrophaeus</i> Laccase Fused with <i>Vitreoscilla</i> Hemoglobin and a Novel Strong Promoter Replacement |
| title_full_unstemmed | Improving Degradation of Polycyclic Aromatic Hydrocarbons by <i>Bacillus atrophaeus</i> Laccase Fused with <i>Vitreoscilla</i> Hemoglobin and a Novel Strong Promoter Replacement |
| title_short | Improving Degradation of Polycyclic Aromatic Hydrocarbons by <i>Bacillus atrophaeus</i> Laccase Fused with <i>Vitreoscilla</i> Hemoglobin and a Novel Strong Promoter Replacement |
| title_sort | improving degradation of polycyclic aromatic hydrocarbons by i bacillus atrophaeus i laccase fused with i vitreoscilla i hemoglobin and a novel strong promoter replacement |
| topic | thermostable laccase PAH biodegradation fusion expression promoter screening strong promoter replacement |
| url | https://www.mdpi.com/2079-7737/11/8/1129 |
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