An optimised keratin azure assay of keratinase activity

Keratin, in the form of coarse sheep wool, has been identified as an undervalued natural resource, which with the appropriate tools (e.g. a keratinase biocatalyst) can be repurposed for various textile and industrial biotechnology applications. For these purposes, we describe a novel method for identifying keratinase activity through the use of a-keratin azure (KA), an anthraquinone dyed substrate. An optimised colourimetric method monitored the keratinase activity of Proteinase K (PK), which degrades the KA substrate and releases soluble products that are observed at 595 nm. In addition to optimising the method, the azure dye standard, Remazol Brilliant Blue R (RBBR), was used to calibrate the assay and provide information on the kinetics of the keratinase-catalysed reaction. The optimised method was also used to investigate different reaction quenching/work up conditions and centrifugation was found to be the most effective method for removing unreacted KA substrate. This assay was then applied to investigate the effect of reduction of the keratin disulfide bond on keratinase-catalysed degradation. This method will enable identification of keratinases ideally suited for application in the valorisation of the a-keratin found in natural wool fibres. The dataset relates to the upcoming publication 'An improved keratin azure assay of keratinase activity' (in submission).