Proteases from Bacillus sp., collectively known as subtilisins, are widely used in laundry and automatic dish detergents to remove proteinaceous stains from surfaces. Commonly, modern detergent powders contain bleaches such as perborate or percarbonate to ensure best wash performance. This poses a challenge for the use of enzymes, because, as all proteins, most enzymes are prone oxidative attack followed by loss of activity [1]. Researchers have three options to address this problem:

• Protein engineer the enzyme to enhance oxidative stability [2]
• Screen for more stable natural and semi-synthetic variants
• Encapsulation and controlled release of the enzyme.

Each of these approaches has been successful in developing proteases that are more stable than the wild type. In this study the latter route was investigated using granulated protease enzyme in conjunction with antioxidants and other materials. As a reporter for the progression of the oxidation of the enzyme one of the three intrinsic methionine residues in subtilisin was used. Enzymatic activity assays coupled with LC/MS peptide maps were employed to quantify the degree and rate of inactivation. Results illustrating the effectiveness of utilizing the combination of methods of analysis to understand the cause of inactivation and identify appropriate solutions will be presented.

[1] Wells, J.A., Estell, D.A., Trends Biochem. Sci., 13, 291-97, 1988.

[2] Estell, D.A., Graycar, T.P., Wells, J.A., J. Biol. Chem., 260, 6518-21, 1985.