Azo dying of α‐keratin material improves microbial keratinase screening and standardization. Issue 4 (28th February 2020)

• Record Type: Journal Article
• Authors: Gonzalo, Milena; Espersen, Roall; Al‐Soud, Waleed A.; Cristiano Falco, Francesco; Hägglund, Per; Sørensen, Søren J.; Svensson, Birte; Jacquiod, Samuel
• Is Part Of: Microbial biotechnology. Volume 13:Issue 4(2020:Jul.)
• Page Start: 984
• Page End: 996
• Publication Date: 2020-02-28
• Subjects: Microbial biotechnology -- Periodicals; Biotechnology; Microbiology; 660.62
• DOI: 10.1111/1751-7915.13541
• Languages: English
• ISSNs: 1751-7915

Abstract

Summary: 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. Abstract : We introduce 'Azokeratin', an optimized protocol significantly increasing sensitivity for screening of microbial keratinases.