Stability of Enzymes in Granular Enzyme Products for Laundry Detergents

摘要

Enzymes have long been of interest to the detergent industry due to their ability to improve the cleaning efficiency of synthetic detergents, contribute to shortening washing times, and reduce energy and water consumption, provision of environmentally friendlier wash water effluents and fabric care. However, incorporating enzymes in detergent formulations gives rise to numerous practical problems due to their incompatibility with and stability against various detergent components. In powdered detergent formulations, these issues can be partly overcome by physically isolating the enzymes in separate particles. However, enzymes may loose a significant part of their activity over a time period of several weeks. Possible causes of inactivation of enzymes in a granule may be related to the release of hydrogen peroxide from the bleaching chemicals in a moisture-containing atmosphere, humidity, autolysis of enzymes, high local pH in granule, oxygen, defects in granulate structure and the effect of other detergent components. However, the actual mechanism of inactivation is not known yet. It is believed that a combination of the factors mentioned above plays a role in the activity loss, and is the focus of this study. The inactivation kinetics of technical grade enzyme powder was determined in a newly developed experimental setup, which was simple and effective and provided a better control over test conditions and fast sample generation. The method was based on the generation of hydrogen peroxide vapor and humidity by bubbling nitrogen gas through their corresponding solutions. An enzyme column, acting as a plug-flow reactor, was exposed to known concentrations of HO (g) and humidity in a thermally stabilized chamber. Samples were analyzed for adsorptive behavior and residual enzyme activity.Since the moisture is believed to play an important role in the stability of proteins, the monolayer hydration level of Savinase® was experimentally determined and theoretically calculated. Adsorbed moisture was found to have 3 a negative effect on enzyme activity. Below monolayer hydration level, the enzyme stability was significantly conserved, while at multilayer hydration level, especially when samples were exposed to 100% RH, the activity was reduced by 80% in a one week period. Since no auto-proteolytic activity and covalently-bound aggregate formation were detected, humidity possibly induced formation of unfavorable conformational changes, resulting in a decrease in enzyme’s catalytic efficiency. Exposure to HO(g) and humidity also resulted in significant HO adsorption. The amount of adsorbed HO did not depend on humidity in the gas stream, which implied that water and HO were not competing for the same adsorption sites. In addition, the desorption studies revealed that while moisture was adsorbed by physisorption, HO was adsorbed by either chemisorption or possibly involving formation of strong hydrogen bonds. Inactivation of the solid-state enzyme was caused by the mutual effect of hydration and HO (g) concentration. A simple mechanism for solid-state enzyme oxidation was proposed and the kinetic parameters in the resulting rate expression were derived. A good agreement between the derived equation and experimental data was obtained. The enzyme inactivation was found to depend on the square of moisture adsorbed by the enzyme at the corresponding temperature. The inverse of the reaction rate constant was also proportional to the inverse of HO in the system. Activity loss was expected to be caused by the oxidation of the enzyme by HO vapor. 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. Due to its relatively accessible position on the exterior of the enzyme structure, only methionine 222 (Met 222) was oxidized; while other 2 Met residues, buried in the peptide backbone, remained unaffected. Being adjacent to the active site of Savinase®, Met 222 oxidation resulted in conformational and electrostatic shift in the catalytic site, causing a significant reduction of enzyme activity. The findings are in agreement with previously reported HO-induced oxidation studies of Savinase® in solutions. Preliminary formulation studies were conducted and application of the designed setup on stability measurements of commercial granulates was illustrated. Addition of salts resulted in a considerable conservation of enzyme activity. Having an anti-oxidative property, sodium thiosulphate had a better activity-preservation effect compared to sodium carbonate. Due to a possible crack formation on granulate surface and/or deliquescence of sodium thiosulphate at high humidity showed that mixing the antioxidant homogeneously with the enzyme provided better protection than coating the salt as a separate layer. The effect of site-directed mutagenesis on Savinase® stability was illustrated and possible stability enhancing additives for enzyme granulates were proposed. The present study is the first to report the solid-state inactivation kinetics and mechanism of Savinase®, subjected to controlled concentrations of HOvapor and humidity. It provides practical information on solid-state stability measurements of biocatalysts in oxidative environments.