The effect of counterion, water concentration, and stirring on the stability of subtilisin BPN′ in organic solvents

Abstract

The stability of subtilisin BPN′ in organic solvents or cosolvent/water mixtures was studied as a function of the type and concentration of counterion at the time of freeze-drying, water concentration, and stirring speed/method. It was found that the enzyme is stabilized by high concentrations of counterion, at least at very high cosolvent concentrations. The type of counterion also has a remarkable impact on the enzyme stability; at high concentrations of DMF (dimethylformamide), multivalent counterions with low solubility in organic solvents are far superior to monovalent, soluble ones. Sodium citrate is the best salt tested in terms of enzyme stability, increasing the half life of the enzyme better than a millionfold over Tris in 99% DMF. The stability of the enzyme was found to have a complex dependence on the amount of water in the DMF. Enzyme lyophilized from the sodium phosphate displays a stability minimum at about 90% DMF, while enzyme lyophilized from Tris becomes increasingly unstable from 30% to 99% DMF, without inflection. Vigorous stirring with a magnetic stir bar, which broke apart the enzyme particles, was found to be extremely deleterious to enzyme stability, while swirling the enzyme with a wrist-action stirrer, which did not grind the enzyme particles, had no effect. Explanations for this are discussed.