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Growth optimization and identification of an ω-transaminase by a novel native PAGE activity staining method in a Bacillus sp. strain BaH isolated from Iranian soil
ω-Transaminases’ (ω-TAs) importance for synthesizing chiral amines led to the development of different methods to quickly identify and characterize new sources of these enzymes. Here we describe the optimization of growth and induction of such an enzyme in a wild type strain of Bacillus sp. strain B...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Springer Berlin Heidelberg
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7988029/ https://www.ncbi.nlm.nih.gov/pubmed/33759017 http://dx.doi.org/10.1186/s13568-021-01207-7 |
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author | Gord Noshahri, Najme Fooladi, Jamshid Engel, Ulrike Muller, Delphine Kugel, Michaela Gorenflo, Pascal Syldatk, Christoph Rudat, Jens |
author_facet | Gord Noshahri, Najme Fooladi, Jamshid Engel, Ulrike Muller, Delphine Kugel, Michaela Gorenflo, Pascal Syldatk, Christoph Rudat, Jens |
author_sort | Gord Noshahri, Najme |
collection | PubMed |
description | ω-Transaminases’ (ω-TAs) importance for synthesizing chiral amines led to the development of different methods to quickly identify and characterize new sources of these enzymes. Here we describe the optimization of growth and induction of such an enzyme in a wild type strain of Bacillus sp. strain BaH (IBRC-M 11337) isolated from Iranian soil in shaking flasks by the response surface methodology (RSM). Optimum conditions were set in a multiplexed bench-top bioreactor system (Sixfors). ω-TA activity of obtained biomass was checked by an innovative efficient colorimetric assay for localizing ω-TAs in crude extracts on acrylamide gel by using ortho-xylylenediamine (OXD) as amino donor. The application of the established OXD assay is thereby expanded from high-throughput activity screenings and colony-based screenings of heterologously expressed mutants to a direct identification of ω-TAs in wild-type strains: This assay can be used to detect the protein band of the respective enzyme in crude extracts of novel isolates by visual inspection of native PAGEs without any upstream protein purification, thus enabling subsequent further investigations of a newly discovered enzyme directly from the crude extract. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13568-021-01207-7. |
format | Online Article Text |
id | pubmed-7988029 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Springer Berlin Heidelberg |
record_format | MEDLINE/PubMed |
spelling | pubmed-79880292021-04-12 Growth optimization and identification of an ω-transaminase by a novel native PAGE activity staining method in a Bacillus sp. strain BaH isolated from Iranian soil Gord Noshahri, Najme Fooladi, Jamshid Engel, Ulrike Muller, Delphine Kugel, Michaela Gorenflo, Pascal Syldatk, Christoph Rudat, Jens AMB Express Original Article ω-Transaminases’ (ω-TAs) importance for synthesizing chiral amines led to the development of different methods to quickly identify and characterize new sources of these enzymes. Here we describe the optimization of growth and induction of such an enzyme in a wild type strain of Bacillus sp. strain BaH (IBRC-M 11337) isolated from Iranian soil in shaking flasks by the response surface methodology (RSM). Optimum conditions were set in a multiplexed bench-top bioreactor system (Sixfors). ω-TA activity of obtained biomass was checked by an innovative efficient colorimetric assay for localizing ω-TAs in crude extracts on acrylamide gel by using ortho-xylylenediamine (OXD) as amino donor. The application of the established OXD assay is thereby expanded from high-throughput activity screenings and colony-based screenings of heterologously expressed mutants to a direct identification of ω-TAs in wild-type strains: This assay can be used to detect the protein band of the respective enzyme in crude extracts of novel isolates by visual inspection of native PAGEs without any upstream protein purification, thus enabling subsequent further investigations of a newly discovered enzyme directly from the crude extract. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13568-021-01207-7. Springer Berlin Heidelberg 2021-03-23 /pmc/articles/PMC7988029/ /pubmed/33759017 http://dx.doi.org/10.1186/s13568-021-01207-7 Text en © The Author(s) 2021 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/. |
spellingShingle | Original Article Gord Noshahri, Najme Fooladi, Jamshid Engel, Ulrike Muller, Delphine Kugel, Michaela Gorenflo, Pascal Syldatk, Christoph Rudat, Jens Growth optimization and identification of an ω-transaminase by a novel native PAGE activity staining method in a Bacillus sp. strain BaH isolated from Iranian soil |
title | Growth optimization and identification of an ω-transaminase by a novel native PAGE activity staining method in a Bacillus sp. strain BaH isolated from Iranian soil |
title_full | Growth optimization and identification of an ω-transaminase by a novel native PAGE activity staining method in a Bacillus sp. strain BaH isolated from Iranian soil |
title_fullStr | Growth optimization and identification of an ω-transaminase by a novel native PAGE activity staining method in a Bacillus sp. strain BaH isolated from Iranian soil |
title_full_unstemmed | Growth optimization and identification of an ω-transaminase by a novel native PAGE activity staining method in a Bacillus sp. strain BaH isolated from Iranian soil |
title_short | Growth optimization and identification of an ω-transaminase by a novel native PAGE activity staining method in a Bacillus sp. strain BaH isolated from Iranian soil |
title_sort | growth optimization and identification of an ω-transaminase by a novel native page activity staining method in a bacillus sp. strain bah isolated from iranian soil |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7988029/ https://www.ncbi.nlm.nih.gov/pubmed/33759017 http://dx.doi.org/10.1186/s13568-021-01207-7 |
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