Screening for physical stability of a Pseudomonas amylase using self-interaction chromatography.

Formulation development is an integral step in the successful commercialization of protein-based products in both the biotechnology and pharmaceutical industries. As the number of these protein formulations increases, so does the need for innovative approaches to characterize physical and chemical product stability. In this study, the osmotic second virial coefficient (B) of a commercial amylase was evaluated by self-interaction chromatography (SIC) as an innovative approach to characterize physical protein stability. B was measured as a function of pH and several common formulation additives (cosolvents), including sodium chloride, sucrose, and sorbitol. Cosolvent- and pH-induced physical stabilization of amylase is discussed in terms of positive shifts in B. Liquid chromatographic measurements of total soluble amylase and enzymatic activity measurements correlated qualitatively with trends in B except near the pI of amylase, where physical stability was minimal.

Surfactant-induced unfolding of cellulase: kinetic studies.

Surfactant-induced unfolding is a significant degradation pathway for detergent enzymes. This study examines the kinetics of surfactant-induced unfolding for endoglucanase III, a detergent cellulase, under conditions of varying pH, temperature, ionic strength, surfactant type, and surfactant concentration. Interactions between protein and surfactant monomer are shown to play a key role in determining the kinetics of the unfolding process. We demonstrate that the unfolding rate can be slowed by (1) modifying protein charge and/or pH conditions to create electrostatic repulsion of ionic surfactants and (2) reducing the amount of monomeric ionic surfactant available for interaction with the enzyme (i.e., by lowering the critical micelle concentration). Additionally, our results illustrate that there is a poor correlation between thermodynamic stability in buffer (DeltaG(unfolding)) and resistance to surfactant-induced unfolding.

Ca2+-surfactant interactions affect enzyme stability in detergent solutions.

Detergent proteases and amylases generally bind Ca(2+) ions. These bound ions enhance enzyme stability, reducing the rates of degradative reactions such as unfolding and proteolysis. Thus, surfactant aggregates, such as micelles, affect protease and amylase stability indirectly, by competing with the enzymes for Ca(2+) ions. Dissociation constants for Ca(2+) interactions with anionic surfactant micelles are in the 10(-3) to 10(-2) M range. These interactions are weak relative to enzyme-Ca(2+) interactions (K(d) of order 10(-6) M). However, surfactant is typically present at much higher concentration than enzyme, and it is the Ca(2+)-micelle equilibrium that largely determines the amount of free Ca(2+) available for binding to enzymes. The problem of surfactant-mediated Ca(2+) removal from enzymes can be avoided by adding calcium to a detergent formulation in an amount such that the concentration of free Ca(2+) is around 10(-5)M.