Kinetic Stability Modelling of Keratinolytic Protease P45: Influence of Temperature and Metal Ions

Abstract

The activity and kinetic stability of a keratinolytic subtilisin-like protease from Bacillus sp. P45 was investigated in 100 mM Tris-HCl buffer (pH 8.0; control) and in buffer with addition of Ca(2+) or Mg(2+) (1-10 mM), at different temperatures. Addition of 3 mM Ca(2+) or 4 mM Mg(2+) resulted in a 26% increment on enzyme activity towards azocasein when compared to the control (100%; without added Ca(2+) or Mg(2+)) at 55 °C. Optimal temperature for activity in the control (55 °C) was similar with Mg(2+); however, temperature optimum was increased to 60 °C with 3 mM Ca(2+), displaying an enhancement of 42% in comparison to the control at 55 °C. Stability of protease P45 in control buffer and with Mg(2+) addition was assayed at 40-50 °C, and at 55-62 °C with Ca(2+) addition. Data were fitted to six kinetic inactivation models, and a first-order equation was accepted as the best model to describe the inactivation of protease P45 with and without metal ions. The kinetic and thermodynamic parameters obtained showed the crucial role of calcium ions for enzyme stability. As biocatalyst stability is fundamental for commercial/industrial purposes, the stabilising effect of calcium could be exploited aiming the application of protease P45 in protein hydrolysis.

Kinetics and Thermodynamics of Thermal Inactivation of β-Galactosidase from Aspergillus oryzae

“…Ea can be defined as the energy barrier that molecules need to cross in order to be able to react, and the proportion of molecules able to do that, usually increases with temperature, qualitatively explaining the effect of temperature on rates (33) . Therefore, the higher the Ea values, the higher the energy barrier to be transposed for enzyme inactivation, indicating an increased stability (15). For the thermal inactivation of commercial βgalactosidase from A. oryzae, Ea was 277 kJ mol -1 , which is close to results observed by Ustok and co-workers (17) .…”

“…1) suggests that the reaction happens at one inactivation rate (k-value) in a single step. It has been reported to model heat degradation of several enzymes, including β-galactosidase (15)(16)(17) (1)…”

Potential application spectrum of microbial proteases for clean and green industrial production

“…Keratin in its native state structure is highly stable due to the presence of tightly packed helices and sheets with large number of disulphide bonds and is not easily degraded by common proteolytic enzymes like trypsin, papain and pepsin (Daroit et al 2011). Composition and molecular configuration of keratin, its constituent amino acids, disulphide bonds and cross-linkages are responsible for its hardness and insolubility (Parradoa et al 2014).…”

Queratinasas microbianas: microorganismos, producción y caracterización

“…Cinética de primer orden (ec. 1), esta cinética es empleada generalmente para describir la inactivación térmica y ha sido utilizada por una gran variedad de autores para modelar la inactivación térmica de una gran variedad de enzimas Hasmann et al, 2007;Daroit et al, 2011).…”

“…P 45. En ese caso, la máxima actividad residual a 55 ºC, se observa a una concentración de calcio de 3 mM y de magnesio de 4 mM, respectivamente, la cual disminuye al aumentar la concentración de estos iones (Daroit et al, 2011). por su parte, estudiaron el efecto del incremento de la concentración de sorbitol sobre la estabilidad térmica de una proteasa de B. cereus BG1.…”