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. 2017 Jul 5;7:4658. doi: 10.1038/s41598-017-04723-4

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© The Author(s) 2017

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Purification of keratinase from culture medium and keratinase identification. (a) An overview of keratinase purification from the overnight culture medium. Each step is described specifically in Materials and Methods. (b) Elution profile of MtaKer after chromatography using S-Sepharose column. The proteins were eluted with a linear salt gradient. The fraction No. 15 (the major peak) is the contaminating carotenoid pigment that was eluted by 200 mM NaCl. The red box indicates the collected peak (fraction No. 16–19) containing keratinase activates. (c) The proteins from the collected peak were further separated by 4–20% gradient SDS-PAGE to determine the size or molecular weight of unknown proteins. Lane 1: protein markers; lane 2: the collected peak with keratinase activity. (d) The keratinolytic enzyme profile was evaluated by replica-agarose-zymogram analysis on the 1% agarose with keratin powder/casein as substrates. The keratinolytic activity can be visualized as a clear zone (arrowhead) and the corresponding band on SDS-PAGE was subjected to standard proteomic analysis to identify the amino acid sequence of MtaKer.