title: 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.
authors.affiliation: Laboratório de Bioquímica e Microbiologia Aplicada, Instituto de Ciência e Tecnologia de Alimentos, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, 91501-970 Porto Alegre, RS, Brazil.
authors.forename: Daniel Joner
authors.initials: DJ
authors.lastname: Daroit
authors.forename: Voltaire
authors.initials: V
authors.lastname: Sant'anna
authors.forename: Adriano
authors.initials: A
authors.lastname: Brandelli
mesh_terms: D001407:Bacillus / Q000737:chemistry / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D020860:Subtilisin / Q000737:chemistry* / Q000378:metabolism; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D020860:Subtilisin; D008274:Magnesium; D002118:Calcium