Stability and Stabilisation of Doratomyces microsporus Keratinase

Thermal and pH stabilities of a new crude keratinase ( Doratomyces microsporus ) were investigated in the ranges of 20-40°C and pH 4-10, respectively. The stability test was followed by activity measurement on two different substrates: human stratum corneum and haemoglobin. Activity measurement lasted more than 100 h. The effect of calcium ions on enzyme stability was also studied. Crude keratinase was stabilised by crosslinking with glutaraldehyde (GA). The same characteristics were determined for Proteinase K, the commercial enzyme, for comparative purposes. Crude keratinase was most stable at pH 8 in Tris/HCl and borate buffers. The type of buffer used proved to have higher effect on crude keratinase stability than on Proteinase K. Both enzymes were most stable at 20°C. Keratinase stability rapidly decreased at 40°C while Proteinase K showed higher thermal stability. A 1 mM solution of Ca 2+ ions did not significantly influence enzyme stability, but 2.5% GA solution stabilised crude keratinase at 40°C reducing the k d value by about 50%. Crude and crosslinked crude keratinase were used for crude calf skin degradation. A mathematical model, based on Michaelis-Menten kinetics, was developed to describe the crude calf skin degradation in a batch reactor. Validation of the model showed that it could describe the process over a defined range of its conditions.