2.4.2. Thermally induced whey protein gel
Keratinolytic protease activity was measured by reaction of
FolineCiocalteu’s reagent with the trichloroacetic acid (TCA)-
soluble peptides generated during hydrolysis of the substrate
(thermally induced whey protein gel), using Sandhya et al.
(2005) with necessary modification. The substrate and
enzyme reaction was incubated at 70 C for 30 min. One unit
of activity was defined as the amount of enzyme that liberated
1 mg tyrosine min1 under the conditions of the assay. Prior to
assay by this method, crude enzymes extracted from SSF media
were dia-filtered with distilled water using a 10 kDa ultrafiltration
membrane.
2.4.3. Azocasein as substrate
Determination of keratinolytic protease activity using azocasein
as substrate was carried out as previously described by
Boyce et al. (2010) wherein enzyme (250 mL) and substrate
(350 mL of 2.0% (w/v) azocasein in 50 mM NaOH-Glycin buffer
pH 10) were co-incubated at 70 C for 30 min. The reaction was
terminated by the addition of 10% (w/v) trichloroacetic acid