Protease enzymes in detergent formulations facilitate protein stain removal, allowing greater cleaning at a lower wash temperature. But incorporating enzymes into detergent formulations has numerous practical problems, such as the influence of surfactants on enzymatic hydrolysis and the aggregation, oxidation, denaturation and autolytic degradation of the enzymes. A greater understanding of the effect of surfactants on enzymatic degradation of proteins may lead to development of detergents with greater enzyme-surfactant cleaning synergy.

In this work we study the factors that affect fluorescently-labelled bovine serum albumin (FBSA) bulk proteolysis by subtilisin Carlsberg. We study systematically the effect of the molecular structure and concentration of non-ionic and ionic surfactants on the kinetics of subtilisin-Carlsberg-catalyzed bulk proteolysis of bovine serum albumin. To measure bulk proteolysis kinetics we use flouresecence spectroscopy with a fluorescently-labelled protein substrate. The studied surfactants have tails consisting of a twelve-carbon alkyl chain and head groups of varying composition. The surfactants are nonionic tetraethylene glycol momododecyl ether (C12E4), anionic sodium dodecyl sulfate (SDS), and cationic dodecyltrimethylammonium bromide (DTAB). To explain the kinetic results, conformational studies of the interaction of these surfactants with BSA and subtilisin Carlsberg are reported, using static fluorescence and circular dichroism spectroscopy.

We find that the rate of bulk proteolysis decreases in the presence of increasing ionic surfactant concentration, and is less affected by the nonionic surfactant C12E4. We observe the change in the bulk proteolysis rate using the parameter enzyme specificity, kcat/KM, measured as the Michaelis-Menten catalytic turnover number over the Michaelis constant. Below the critical micellar concentration (CMC), the enzyme specificity decreases 20% for C12E4 at 0.9CMC, 40% for SDS at 0.6CMC, and almost 80% for DTAB at 0.4CMC. Above the CMC, the enzyme specificity decreases 50% for C12E4 at 4.4CMC, almost 90% for SDS at 1.5CMC, and approximately 80% for DTAB at 2.1CMC. In addition to the decrease observed in the enzymatic activity, studies of the conformation of the protein substrate and the enzyme in the presence of surfactant suggest that the decreases in enzyme specificity are also due to conformational changes in FBSA which affect enzyme-substrate interactions.