Heterologous expression of nattokinase from B. subtilis natto using Pichia pastoris GS115 and assessment of its thrombolytic activity
Abstract Background Nattokinase is a fibrinolytic enzyme that has huge market value as a nutritional supplement for health promotion. In order to increase nattokinase yields, fermentation conditions, strains, cultivation media, and feeding strategies have been optimized. Nattokinase has been express...
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| Format: | Article |
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BMC
2021-08-01
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| Series: | BMC Biotechnology |
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| Online Access: | https://doi.org/10.1186/s12896-021-00708-4 |
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| author | Yan Guangbo Shu Min Shen Wei Ma Lixin Zhai Chao Wang Yaping Huang Zunxi |
| author_facet | Yan Guangbo Shu Min Shen Wei Ma Lixin Zhai Chao Wang Yaping Huang Zunxi |
| author_sort | Yan Guangbo |
| collection | DOAJ |
| description | Abstract Background Nattokinase is a fibrinolytic enzyme that has huge market value as a nutritional supplement for health promotion. In order to increase nattokinase yields, fermentation conditions, strains, cultivation media, and feeding strategies have been optimized. Nattokinase has been expressed using several heterologous expression systems. Pichia pastoris heterologous expression system was the alternative. Results This report aimed to express high levels of nattokinase from B. subtilis natto (NK-Bs) using a Pichia pastoris heterologous expression system and assess its fibrinolytic activity in vivo. Multicopy expression strains bearing 1–7 copies of the aprN gene were constructed. The expression level of the target protein reached a maximum at five copies of the target gene. However, multicopy expression strains were not stable in shake-flask or high-density fermentation, causing significant differences in the yield of the target protein among batches. Therefore, P. pastoris bearing a single copy of aprN was used in shake-flask and high-density fermentation. Target protein yield was 320 mg/L in shake-flask fermentation and approximately 9.5 g/L in high-density fermentation. The recombinant nattokinase showed high thermo- and pH-stability. The present study also demonstrated that recombinant NK-Bs had obvious thrombolytic activity. Conclusions This study suggests that the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of nattokinase. |
| first_indexed | 2024-12-16T11:35:17Z |
| format | Article |
| id | doaj.art-851f02633e1d4b999430463ac2ca7f83 |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-12-16T11:35:17Z |
| publishDate | 2021-08-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-851f02633e1d4b999430463ac2ca7f832022-12-21T22:33:08ZengBMCBMC Biotechnology1472-67502021-08-0121111210.1186/s12896-021-00708-4Heterologous expression of nattokinase from B. subtilis natto using Pichia pastoris GS115 and assessment of its thrombolytic activityYan Guangbo0Shu Min1Shen Wei2Ma Lixin3Zhai Chao4Wang Yaping5Huang Zunxi6State Key Laboratory of Biocatalysis and Enzyme, Engineering Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty of Hubei University, Hubei UniversityState Key Laboratory of Biocatalysis and Enzyme, Engineering Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty of Hubei University, Hubei UniversityState Key Laboratory of Biocatalysis and Enzyme, Engineering Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty of Hubei University, Hubei UniversityState Key Laboratory of Biocatalysis and Enzyme, Engineering Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty of Hubei University, Hubei UniversityState Key Laboratory of Biocatalysis and Enzyme, Engineering Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty of Hubei University, Hubei UniversityState Key Laboratory of Biocatalysis and Enzyme, Engineering Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty of Hubei University, Hubei UniversitySchool of Life Sciences, Yunnan Normal UniversityAbstract Background Nattokinase is a fibrinolytic enzyme that has huge market value as a nutritional supplement for health promotion. In order to increase nattokinase yields, fermentation conditions, strains, cultivation media, and feeding strategies have been optimized. Nattokinase has been expressed using several heterologous expression systems. Pichia pastoris heterologous expression system was the alternative. Results This report aimed to express high levels of nattokinase from B. subtilis natto (NK-Bs) using a Pichia pastoris heterologous expression system and assess its fibrinolytic activity in vivo. Multicopy expression strains bearing 1–7 copies of the aprN gene were constructed. The expression level of the target protein reached a maximum at five copies of the target gene. However, multicopy expression strains were not stable in shake-flask or high-density fermentation, causing significant differences in the yield of the target protein among batches. Therefore, P. pastoris bearing a single copy of aprN was used in shake-flask and high-density fermentation. Target protein yield was 320 mg/L in shake-flask fermentation and approximately 9.5 g/L in high-density fermentation. The recombinant nattokinase showed high thermo- and pH-stability. The present study also demonstrated that recombinant NK-Bs had obvious thrombolytic activity. Conclusions This study suggests that the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of nattokinase.https://doi.org/10.1186/s12896-021-00708-4Nattokinase; High-density fermentation; Multi-copy strains; Pichia pastoris |
| spellingShingle | Yan Guangbo Shu Min Shen Wei Ma Lixin Zhai Chao Wang Yaping Huang Zunxi Heterologous expression of nattokinase from B. subtilis natto using Pichia pastoris GS115 and assessment of its thrombolytic activity BMC Biotechnology Nattokinase; High-density fermentation; Multi-copy strains; Pichia pastoris |
| title | Heterologous expression of nattokinase from B. subtilis natto using Pichia pastoris GS115 and assessment of its thrombolytic activity |
| title_full | Heterologous expression of nattokinase from B. subtilis natto using Pichia pastoris GS115 and assessment of its thrombolytic activity |
| title_fullStr | Heterologous expression of nattokinase from B. subtilis natto using Pichia pastoris GS115 and assessment of its thrombolytic activity |
| title_full_unstemmed | Heterologous expression of nattokinase from B. subtilis natto using Pichia pastoris GS115 and assessment of its thrombolytic activity |
| title_short | Heterologous expression of nattokinase from B. subtilis natto using Pichia pastoris GS115 and assessment of its thrombolytic activity |
| title_sort | heterologous expression of nattokinase from b subtilis natto using pichia pastoris gs115 and assessment of its thrombolytic activity |
| topic | Nattokinase; High-density fermentation; Multi-copy strains; Pichia pastoris |
| url | https://doi.org/10.1186/s12896-021-00708-4 |
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