Inactivation of a solid-state detergent protease by hydrogen peroxide vapor and humidity.
An experimental study on solid-state stability of a detergent protease (Savinase) is reported. The inactivation kinetics of technical grade enzyme powder was determined as a function of gas phase H(2)O(2) concentration and humidity by employing a quick assay running over few hours instead of several weeks as typical in industry. The results indicated that enzymes adsorbed significant amounts of moisture and H(2)O(2) during exposure. The amount of adsorbed H(2)O(2) did not depend on humidity in the gas stream, which implied that water and H(2)O(2) were not competing for the same adsorption sites. Inactivation of the solid-state enzyme was caused by the mutual effect of increasing hydration and H(2)O(2) (g) concentration. No auto-proteolytic activity or covalently bound aggregate formation was detected. A simple mechanism for solid-state enzyme oxidation was proposed and the kinetic parameters in the resulting rate expression of inactivation were derived. A good agreement between the derived equation and experimental data was obtained. The oxidative alterations on Savinase were investigated by peptide mapping. Molecular mass examination of CNBr-cleaved fragments by MALDI-TOF mass spectroscopy located the oxidation-labile residue. Only one methionine (Met 222) was oxidized, while other residues remained unaffected. The study provides practical information on solid-state stability measurements of biocatalysts in oxidative environments.
Biran
Suzan
S
Department of Chemical and Biochemical Engineering, CHEC Research Centre, Technical University of Denmark, Kgs Lyngby, Denmark.
Jensen
Anker Degn
AD
Kiil
Søren
S
Bach
Poul
P
Simonsen
Ole
O
D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D006813:Humidity; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008715:Methionine / Q000378:metabolism; D010084:Oxidation-Reduction; D010447:Peptide Hydrolases / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D008715:Methionine; D006861:Hydrogen Peroxide; D010447:Peptide Hydrolases