Synergymx 96 well microplate reader

Keratinase activity assay was based on the method of Jaouadi et al. [40 (link)], with slight modification. The reaction mixture contained 0.5 mL of 10 g/L of keratin azure (Sigma-Aldrich, St. Louis, MO, USA) in 0.1 M Tris-HCl buffer, pH 7.5, and 0.5 mL of suitably diluted crude enzyme solution. The mixture was incubated at 37 °C for 1 h, with shaking at 220 rpm; after that, the reaction was stopped by placing the assay mixture in ice-cooled water for 10 min. The unutilized substrates were removed by centrifugation at 15,000× g for 10 min, and subsequently filtered (Millipore cellulose filters; 0.45 μm). The azo dye released in the filtrate was determined at 595 nm, using a SYNERGYMx 96 well microplate reader (BioTek Instrument Inc., Winooski, VT, USA). The control was treated at the same condition which contained the enzyme solution and buffer without the substrate. One keratinase unit was defined as the amount of enzyme causing an increase in absorbance of 0.01 per hour under the standard assay condition.
The total protein concentration was estimated by using the Bradford method [41 (link)], with bovine serine albumin as a standard protein. The respective assays were done in triplicate, and the results presented were mean plus standard deviation.

The keratinase activity assay followed the method described by Nnolim et al. (2020b) (link). Briefly, the reaction mixture contained 0.5 ml of a freshly prepared crude enzyme and 0.5 ml of 10 g/L of keratin azure in Tris-HCl (pH 8; 0.1 M). The reaction mixture was incubated at 50°C under a constant shaking condition (220 rpm). After 1 h of incubation, the reaction was stopped by placing the reaction mixture on an ice bath for 10 min. Next, the mixture was centrifuged at 15,000 rpm for 10 min. Finally, an aliquot of the supernatant was used to determine the absorbance at 595 nm using an SYNERGYMx 96 well microplate reader (BioTek, United States).

Assay of keratinase activity using the crude extract was in accordance with the method of Jaouadi et al. [21 (link)], with slight modification as previously described [19 (link)]. Briefly, the reaction mixture contained 0.5 mL of 10 g/L of keratin azure (Sigma-Aldrich, St. Louis, MO, USA) in 0.1 M Tris-HCl buffer, pH 8.0, and 0.5 mL of crude enzyme solution. The mixture was incubated at 60 °C for 1 h, with shaking at 220 rpm; thereafter, the reaction was stopped by placing the assay mixture in ice-cooled water for 10 min. The unutilized substrates were removed by centrifugation at 15,000 rpm for 10 min, and subsequently filtered (Millipore cellulose filters; 0.45 μm, Merck chemicals (Pty) Ltd., Modderfontein, Gauteng, South Africa). The azo dye released in the filtrate was determined at 595 nm, using a SYNERGYMx 96-well microplate reader (BioTek Instrument Inc., Winooski, VT, USA). The control was treated at the same condition which contained the enzyme solution and buffer without the substrate. One unit of keratinase activity (U) was defined as the amount of enzyme causing an increase of 0.1 in absorbance per hour under the standard assay condition.
Total protein concentration in the test sample was quantitated by extrapolating from the standard protein curve constructed using varying concentrations of bovine serum albumin in accordance with the Bradford method [22 (link)].