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Azo dying of α‐keratin material improves microbial keratinase screening and standardization

Microbial conversion through enzymatic reactions has received a lot of attention as a cost‐effective and environmentally friendly way to recover amino acids and short peptides from keratin materials. However, accurate assessment of microbial keratinase activity is not straightforward, and current av...

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Autores principales: Gonzalo, Milena, Espersen, Roall, Al‐Soud, Waleed A., Cristiano Falco, Francesco, Hägglund, Per, Sørensen, Søren J., Svensson, Birte, Jacquiod, Samuel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264887/
https://www.ncbi.nlm.nih.gov/pubmed/32110845
http://dx.doi.org/10.1111/1751-7915.13541
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author Gonzalo, Milena
Espersen, Roall
Al‐Soud, Waleed A.
Cristiano Falco, Francesco
Hägglund, Per
Sørensen, Søren J.
Svensson, Birte
Jacquiod, Samuel
author_facet Gonzalo, Milena
Espersen, Roall
Al‐Soud, Waleed A.
Cristiano Falco, Francesco
Hägglund, Per
Sørensen, Søren J.
Svensson, Birte
Jacquiod, Samuel
author_sort Gonzalo, Milena
collection PubMed
description Microbial conversion through enzymatic reactions has received a lot of attention as a cost‐effective and environmentally friendly way to recover amino acids and short peptides from keratin materials. However, accurate assessment of microbial keratinase activity is not straightforward, and current available methods lack sensitivity and standardization. Here, we suggest an optimized Azokeratin assay, with substrate generated directly from azo‐dyed raw keratin material. We introduced supernatant filtration in the protocol for optimal stopping of keratinase reactions instead of the widely used trichloroacetic acid (TCA), as it generated biases and impacted the sensitivity. We furthermore suggest a method for standardization of keratinase activity signals using proteinase K, a well‐known keratinase, as a reference enabling reproducibility between studies. Lastly, we evaluated our developed method with several bacterial isolates through benchmarking against a commercial assay (Keratin Azure). Under different setups, the Azokeratin method was more sensitive than commonly used Keratin Azure‐based assays (3‐fold). We argue that this method could be applied with any type of keratin substrate, enabling more robust and sensitive results which can be used for further comparison with other studies, thus representing an important progress within the field of microbial keratin degradation.
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spelling pubmed-72648872020-06-03 Azo dying of α‐keratin material improves microbial keratinase screening and standardization Gonzalo, Milena Espersen, Roall Al‐Soud, Waleed A. Cristiano Falco, Francesco Hägglund, Per Sørensen, Søren J. Svensson, Birte Jacquiod, Samuel Microb Biotechnol Research Articles Microbial conversion through enzymatic reactions has received a lot of attention as a cost‐effective and environmentally friendly way to recover amino acids and short peptides from keratin materials. However, accurate assessment of microbial keratinase activity is not straightforward, and current available methods lack sensitivity and standardization. Here, we suggest an optimized Azokeratin assay, with substrate generated directly from azo‐dyed raw keratin material. We introduced supernatant filtration in the protocol for optimal stopping of keratinase reactions instead of the widely used trichloroacetic acid (TCA), as it generated biases and impacted the sensitivity. We furthermore suggest a method for standardization of keratinase activity signals using proteinase K, a well‐known keratinase, as a reference enabling reproducibility between studies. Lastly, we evaluated our developed method with several bacterial isolates through benchmarking against a commercial assay (Keratin Azure). Under different setups, the Azokeratin method was more sensitive than commonly used Keratin Azure‐based assays (3‐fold). We argue that this method could be applied with any type of keratin substrate, enabling more robust and sensitive results which can be used for further comparison with other studies, thus representing an important progress within the field of microbial keratin degradation. John Wiley and Sons Inc. 2020-02-28 /pmc/articles/PMC7264887/ /pubmed/32110845 http://dx.doi.org/10.1111/1751-7915.13541 Text en © 2020 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology. This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Articles
Gonzalo, Milena
Espersen, Roall
Al‐Soud, Waleed A.
Cristiano Falco, Francesco
Hägglund, Per
Sørensen, Søren J.
Svensson, Birte
Jacquiod, Samuel
Azo dying of α‐keratin material improves microbial keratinase screening and standardization
title Azo dying of α‐keratin material improves microbial keratinase screening and standardization
title_full Azo dying of α‐keratin material improves microbial keratinase screening and standardization
title_fullStr Azo dying of α‐keratin material improves microbial keratinase screening and standardization
title_full_unstemmed Azo dying of α‐keratin material improves microbial keratinase screening and standardization
title_short Azo dying of α‐keratin material improves microbial keratinase screening and standardization
title_sort azo dying of α‐keratin material improves microbial keratinase screening and standardization
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264887/
https://www.ncbi.nlm.nih.gov/pubmed/32110845
http://dx.doi.org/10.1111/1751-7915.13541
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