Abstract
SummaryMicrobial 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.
Highlights
Livestock industries produce vast amounts of keratin-based waste
Spectral scans indicated an isosbestic point around 415 nm at pH 4–10 (Fig. 1A); measurements at this wavelength are much less sensitive to pH fluctuations compared with 450 nm, eliminating the need for NaOH addition if the reaction is stopped without trichloroacetic acid (TCA) (Fig. 1B)
The Azokeratin procedure implemented to measure microbial keratinolytic activity gave robust and coherent results compared with the popular Keratin Azure method, being more sensitive (3-fold increase)
Summary
Livestock industries produce vast amounts of keratin-based waste (e.g. feathers, bristles, wool, hooves and horns). As each slaughtered pig generates around one kilogram of bristles and hooves, ~22 000 tons of keratin-based by-products are handled yearly in Denmark. Being classified as category-3 waste by European agencies, cautious handling of keratin-based pig by-products is required, as they may pose environmental and sanitary risks (Korniłłowicz-Kowalska and Bohacz, 2011; Verma et al, 2017). There are, several mechanisms to degrade keratinous materials, and controlled microbiological-mediated processes are an alternative for valorization of keratin by-products by conversion into amino acids and short peptides (Korniłłowicz-Kowalska and Bohacz, 2011; Gupta et al, 2013; Kang et al, 2018)
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