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Enhanced production of a recombinant xylanase (XT6): optimization of production and purification, and scaled-up batch fermentation in a stirred tank bioreactor
The endoxylanase XT6 produced by Geobacillus stearothermophilus is a desirable candidate for industrial applications. In this study, the gene encoding XT6 was cloned using the pET-28a expression vector and expressed in Escherichia coli BL21 (DE3) cells. Recombinant XT6 production was improved by opt...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Nature Publishing Group UK
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10684889/ https://www.ncbi.nlm.nih.gov/pubmed/38017111 http://dx.doi.org/10.1038/s41598-023-48202-5 |
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author | Dhaver, Priyashini Sithole, Tariro Pletschke, Brett Sithole, Bruce Govinden, Roshini |
author_facet | Dhaver, Priyashini Sithole, Tariro Pletschke, Brett Sithole, Bruce Govinden, Roshini |
author_sort | Dhaver, Priyashini |
collection | PubMed |
description | The endoxylanase XT6 produced by Geobacillus stearothermophilus is a desirable candidate for industrial applications. In this study, the gene encoding XT6 was cloned using the pET-28a expression vector and expressed in Escherichia coli BL21 (DE3) cells. Recombinant XT6 production was improved by optimizing cell lysis (sonication, chemical, and enzymatic lysis) and expression conditions. Sonication in a 0.05 M sodium phosphate (pH 6.0) buffer resulted in the highest xylanase activity (16.48 U/ml). Screening and optimization of induction conditions using the Plackett–Burman Design and Box-Behnken Design (BBD) approaches revealed that cell density pre-induction (OD(600 nm)), post-induction incubation time, and IPTG concentration significantly (p < 0.05) influenced the expression levels of XT6 (16.48 U/ml to 40.06 U/ml) representing a 3.60-fold increase. BBD resulted in a further 8.74-fold increase in activity to 144.02 U/ml. Batch fermentation in a 5-l stirred tank bioreactor at 1 vvm aeration boosted recombinant xylanase production levels to 165 U/ml suggesting that heterologous expression of the XT6 enzyme is suitable for scaled-up production. The pure enzyme with a molecular weight of 43 kDa and a 15.69-fold increase in purity was obtained using affinity chromatography and a cobalt column. Future studies will include application of the purified recombinant xylanase to animal feed. |
format | Online Article Text |
id | pubmed-10684889 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-106848892023-11-30 Enhanced production of a recombinant xylanase (XT6): optimization of production and purification, and scaled-up batch fermentation in a stirred tank bioreactor Dhaver, Priyashini Sithole, Tariro Pletschke, Brett Sithole, Bruce Govinden, Roshini Sci Rep Article The endoxylanase XT6 produced by Geobacillus stearothermophilus is a desirable candidate for industrial applications. In this study, the gene encoding XT6 was cloned using the pET-28a expression vector and expressed in Escherichia coli BL21 (DE3) cells. Recombinant XT6 production was improved by optimizing cell lysis (sonication, chemical, and enzymatic lysis) and expression conditions. Sonication in a 0.05 M sodium phosphate (pH 6.0) buffer resulted in the highest xylanase activity (16.48 U/ml). Screening and optimization of induction conditions using the Plackett–Burman Design and Box-Behnken Design (BBD) approaches revealed that cell density pre-induction (OD(600 nm)), post-induction incubation time, and IPTG concentration significantly (p < 0.05) influenced the expression levels of XT6 (16.48 U/ml to 40.06 U/ml) representing a 3.60-fold increase. BBD resulted in a further 8.74-fold increase in activity to 144.02 U/ml. Batch fermentation in a 5-l stirred tank bioreactor at 1 vvm aeration boosted recombinant xylanase production levels to 165 U/ml suggesting that heterologous expression of the XT6 enzyme is suitable for scaled-up production. The pure enzyme with a molecular weight of 43 kDa and a 15.69-fold increase in purity was obtained using affinity chromatography and a cobalt column. Future studies will include application of the purified recombinant xylanase to animal feed. Nature Publishing Group UK 2023-11-28 /pmc/articles/PMC10684889/ /pubmed/38017111 http://dx.doi.org/10.1038/s41598-023-48202-5 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This 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/) . |
spellingShingle | Article Dhaver, Priyashini Sithole, Tariro Pletschke, Brett Sithole, Bruce Govinden, Roshini Enhanced production of a recombinant xylanase (XT6): optimization of production and purification, and scaled-up batch fermentation in a stirred tank bioreactor |
title | Enhanced production of a recombinant xylanase (XT6): optimization of production and purification, and scaled-up batch fermentation in a stirred tank bioreactor |
title_full | Enhanced production of a recombinant xylanase (XT6): optimization of production and purification, and scaled-up batch fermentation in a stirred tank bioreactor |
title_fullStr | Enhanced production of a recombinant xylanase (XT6): optimization of production and purification, and scaled-up batch fermentation in a stirred tank bioreactor |
title_full_unstemmed | Enhanced production of a recombinant xylanase (XT6): optimization of production and purification, and scaled-up batch fermentation in a stirred tank bioreactor |
title_short | Enhanced production of a recombinant xylanase (XT6): optimization of production and purification, and scaled-up batch fermentation in a stirred tank bioreactor |
title_sort | enhanced production of a recombinant xylanase (xt6): optimization of production and purification, and scaled-up batch fermentation in a stirred tank bioreactor |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10684889/ https://www.ncbi.nlm.nih.gov/pubmed/38017111 http://dx.doi.org/10.1038/s41598-023-48202-5 |
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