Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Dhaver, Priyashini, Sithole, Tariro, Pletschke, Brett, Sithole, Bruce, Govinden, Roshini
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2023
Materias:
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
_version_ 1785151505958961152
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
work_keys_str_mv AT dhaverpriyashini enhancedproductionofarecombinantxylanasext6optimizationofproductionandpurificationandscaledupbatchfermentationinastirredtankbioreactor
AT sitholetariro enhancedproductionofarecombinantxylanasext6optimizationofproductionandpurificationandscaledupbatchfermentationinastirredtankbioreactor
AT pletschkebrett enhancedproductionofarecombinantxylanasext6optimizationofproductionandpurificationandscaledupbatchfermentationinastirredtankbioreactor
AT sitholebruce enhancedproductionofarecombinantxylanasext6optimizationofproductionandpurificationandscaledupbatchfermentationinastirredtankbioreactor
AT govindenroshini enhancedproductionofarecombinantxylanasext6optimizationofproductionandpurificationandscaledupbatchfermentationinastirredtankbioreactor