TESTING FOR DIFFUSION LIMITATIONS IN SALT-ACTIVATED ENZYME CATALYSTS OPERATING IN ORGANIC-SOLVENTS

The dramatic activation of serine proteases in nonaqueous media result ing from lyophilization in the presence of KCl is shown to be unrelate d to relaxation of potential substrate diffusional limitations. Specif ically, lyophilizing subtilisin Carlsberg in the presence of KCl and p hosphate buffer in different proportions, ranging from 99% (w/w) enzym e to 1% (w/w) enzyme in the final lyophilized solids, resulted in bioc atalyst preparations that were not influenced by substrate diffusion. This result was made evident through use of a classical analysis where by initial catalytic rates, normalized per weight of total enzyme in t he catalyst material, were measured as a function of active enzyme for biocatalyst preparations containing different ratios of active to ina ctive enzyme. The active enzyme content of a given biocatalyst prepara tion was controlled by mixing native subtilisin with subtilisin preina ctivated with PMSF, a serine protease inhibitor, and lyophilizing the enzyme mixture in the presence of different fractions of KCl and phosp hate buffer. Plots of initial reaction rates as a function of percent active subtilisin in the biocatalyst were linear for all biocatalyst p reparations. Thus, enzyme activation (reported elsewhere to be as high as 3750-fold in hexane for the transesterification of N-Ac-L-Phe-OEt with n-PrOH) is a manifestation of intrinsic enzyme activation and not relaxation of diffusional limitations resulting from diluted enzyme p reparations. Similar activation is reported herein for thermolysin, a nonserine protease, thereby demonstrating that enzyme activation due t o lyophilization in the presence of KCl may be a general phenomenon fo r proteolytic enzymes. (C) 1998 John Wiley & Sons, Inc.