Polyethylene glycol-modified subtilisin forms microparticulate suspensions in organic solvents

Mono-methoxy polyethylene glycol chains may be attached to subtilisin Carlsberg by reaction with their tresyl esters. The resulting PEG-subtilisin can be dispersed in toluene and 1,1,1-trichloroethane to give optically clear systems in which it is catalytically active. However, these contain large aggregates of the PEG-subtilisin monomers: dynamic light scattering indicates mean diameters of at least about 300 nm, while small-angle x-ray scattering and ultracentrifugation confirm sizes greater than 20 nm or so (in the latter method there is a tendency to neutral buoyancy). Hence it seems that this mainly organic reaction system is closer to the well-known suspensions of dried enzyme particles than to a molecular solution. Probably because of this aggregation, the precise dispersibility of the PEG-subtilisin is poorly reproducible, though a reversible temperature-dependent apparent solubility limit is sometimes observed. The UV spectrum of the enzyme in trichloroethane can be close to that in water, though circular dichroism suggest some structural change when dispersed in the solvent.