On the Salt-Induced Activation of Lyophilized Enzymes in Organic Solvents:  Effect of Salt Kosmotropicity on Enzyme Activity

Abstract

The dramatic activation of enzymes in nonaqueous media upon co-lyophilization with simple inorganic salts has been investigated as a function of the Jones-Dole B coefficient, a thermodynamic parameter for characterizing the salt's affinity for water and its chaotropic (water-structure breaking) or kosmotropic (waterstructure making) character. In general, the water content, active-site content, and transesterification activity of freeze-dried subtilisin Carlsberg preparations containing >96% w/w salt increased with increasing kosmotropicity of the activating salt. Degrees of activation relative to the salt-free enzyme ranged from 33fold for chaotropic sodium iodide to 2480-fold for kosmotropic sodium acetate. Exceptions to the general trend can be explained by the mechanical properties and freezing characteristics of the salts undergoing lyophilization. The profound activating effect can thus be attributed in part to the stabilizing (salting-out) effect of kosmotropic salts and the phenomenon of preferential hydration.

Quaternary Diffusion Coefficients in a Protein−Polymer−Salt−Water System Determined by Rayleigh Interferometry

“…Our results are shown in Figure 3. From the error bar comparison along the C 3 coordinate, we observe that (D 21 ) V and (D 21 ) V (12) are equal to each other within the experimental error. We therefore conclude that the addition of salt has negligible effect on both lysozyme-PEG2000 cross-diffusion coefficients.…”

“…34,[37][38][39][40] These interactions are responsible for the phase separation of protein solutions and stabilize the protein native form with respect to unfolded states. Since the presence of NaCl has no detectable effects on (D 21 ) V , we conclude that this salt does not significantly affect lysozyme-PEG2000 thermodynamic interactions within our experimental concentration domain.…”

“…Finally, we examine the effect of PEG molecular weight on (D 21 ) V by comparing our results at C 2 ) 0.050 M and C 3 ) 0.50 M with those obtained for the lysozyme-TEG-NaCl-water system. In limit of dilute solutions, (D 21 ) V becomes directly proportional to both C 2 and (D 22 ) V .…”

“…27,24 In relation to crystal growth, diffusion is driven mainly by the depletion of protein at the crystal surface with respect to the bulk solution. Here the cross terms (D 31 ) V and (D 21 ) V , which have been found to be positive, characterize salt and polymer transport toward the crystal induced by the protein concentration gradient. These calculations have shown that the concentration of precipitant (salt or polymer) at the crystal surface is enhanced if the precipitant cross-term diffusion coefficient is large compared to precipitant and protein main-term diffusion coefficients.…”

Characteristics of nearly dry enzymes in organic solvents: implications for biocatalysis in the absence of water

“…Indeed, the salt-activation strategy was highly successful (Khmelnitsky et al 1994;Ru et al 1999Ru et al , 2000Ru et al , 2001Lindsay et al 2004). For example, when subtilisin Carlsberg was freeze-dried together with KCl, a dramatic activation effect was observed in several solvents, with a rate enhancement of nearly 4000-fold observed in hexane for a preparation containing 98% w/w KCl salt (Khmelnitsky et al 1994).…”