Protease autolysis in heavy-duty liquid detergent formulations: effects of thermodynamic stabilizers and protease inhibitors

Autolysis has been identified as the primary mode of subtilisin activity loss in a heavy-duty liquid detergent (HDLD) formulation containing 10% linear alkylbenzene sulfonate (LAS), 6% alkylether sulfate (AES), and 8% alcohol ethoxylate (AE). Circular dichroism (CD) and Fourier-transform infrared (FTIR) spectra indicate that the protease, a Bacillus lentus subtilisin variant, largely retains its native structure within this formulation. The autolysis reaction is second-order in protease concentration, and a mechanism is proposed wherein both the thermodynamic stability (ΔGunfolding) and Michaelis–Menten catalytic efficiency (kcat/KM) of the enzyme influence the overall autolysis rate. Estimates of ΔGunfolding were calculated from experimental values of the second-order autolysis rate constant using kcat/KM as determined against the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (suc-AAPF-pNA). Sucrose, a known protein stabilizer, was added to the HDLD formulation at a concentration of 0.5 M. The resulting 0.5 kcal/mol increase in ΔGunfolding reduced the second-order autolysis rate constant by 50%. Conversely, addition of the chelating agent EDTA removed bound Ca2+ ions from the subtilisin, destabilizing it by ∼4 kcal/mol; the autolysis rate constant was increased roughly 10,000-fold. Subtilisin inhibitors (borax/propylene glycol) were used to reduce kcat/KM, and concomitant decreases in the autolysis rate were observed. Possible strategies to reduce protease autolysis by thermodynamic stabilization and/or kinetic inhibition are discussed.