P. Sears et al., The effect of counterion, water concentration, and stirring on the stability of subtilisin BPN ' in organic solvents, J MOL CAT B, 6(3), 1999, pp. 297-304
The stability of subtilisin BPN' in organic solvents or cosolvent/water mix
tures was studied as a function of the type and concentration of counterion
at the time of freeze-drying, water concentration, and stirring speed/meth
od. It was found that the enzyme is stabilized by high concentrations of co
unterion, at least at very high cosolvent concentrations. The type of count
erion also has a remarkable impact on the enzyme stability; at high concent
rations of DMF (dimethylformamide), multivalent counterions with low solubi
lity in organic solvents are far superior to monovalent, soluble ones. Sodi
um 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 d
isplays 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 e
nzyme particles, had no effect. Explanations for this are discussed. (C) 19
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