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Heterologous expression of nattokinase from B. subtilis natto using Pichia pastoris GS115 and assessment of its thrombolytic activity

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...

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Autores principales: Guangbo, Yan, Min, Shu, Wei, Shen, Lixin, Ma, Chao, Zhai, Yaping, Wang, Zunxi, Huang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8353737/
https://www.ncbi.nlm.nih.gov/pubmed/34372833
http://dx.doi.org/10.1186/s12896-021-00708-4
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author Guangbo, Yan
Min, Shu
Wei, Shen
Lixin, Ma
Chao, Zhai
Yaping, Wang
Zunxi, Huang
author_facet Guangbo, Yan
Min, Shu
Wei, Shen
Lixin, Ma
Chao, Zhai
Yaping, Wang
Zunxi, Huang
author_sort Guangbo, Yan
collection PubMed
description 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12896-021-00708-4.
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spelling pubmed-83537372021-08-10 Heterologous expression of nattokinase from B. subtilis natto using Pichia pastoris GS115 and assessment of its thrombolytic activity Guangbo, Yan Min, Shu Wei, Shen Lixin, Ma Chao, Zhai Yaping, Wang Zunxi, Huang BMC Biotechnol Research 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12896-021-00708-4. BioMed Central 2021-08-09 /pmc/articles/PMC8353737/ /pubmed/34372833 http://dx.doi.org/10.1186/s12896-021-00708-4 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Guangbo, Yan
Min, Shu
Wei, Shen
Lixin, Ma
Chao, Zhai
Yaping, Wang
Zunxi, Huang
Heterologous expression of nattokinase from B. subtilis natto using Pichia pastoris GS115 and assessment of its thrombolytic activity
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 Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8353737/
https://www.ncbi.nlm.nih.gov/pubmed/34372833
http://dx.doi.org/10.1186/s12896-021-00708-4
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