title: Testing for diffusion limitations in salt-activated enzyme catalysts operating in organic solvents.
abstract: The dramatic activation of serine proteases in nonaqueous media resulting from lyophilization in the presence of KCl is shown to be unrelated to relaxation of potential substrate diffusional limitations. Specifically, lyophilizing subtilisin Carlsberg in the presence of KCl and phosphate buffer in different proportions, ranging from 99% (w/w) enzyme to 1% (w/w) enzyme in the final lyophilized solids, resulted in biocatalyst preparations that were not influenced by substrate diffusion. This result was made evident through use of a classical analysis whereby initial catalytic rates, normalized per weight of total enzyme in the catalyst material, were measured as a function of active enzyme for biocatalyst preparations containing different ratios of active to inactive enzyme. The active enzyme content of a given biocatalyst preparation was controlled by mixing native subtilisin with subtilisin preinactivated with PMSF, a serine protease inhibitor, and lyophilizing the enzyme mixture in the presence of different fractions of KCl and phosphate buffer. Plots of initial reaction rates as a function of percent active subtilisin in the biocatalyst were linear for all biocatalyst preparations. 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 preparations. Similar activation is reported herein for thermolysin, a nonserine protease, thereby demonstrating that enzyme activation due to lyophilization in the presence of KCl may be a general phenomenon for proteolytic enzymes.
authors: Department of Chemical and Biochemical Engineering, University of Iowa, Iowa City, Iowa 52242, USA.
authors: B A
authors: BA
authors: Bedell
authors: V V
authors: VV
authors: Mozhaev
authors: D S
authors: DS
authors: Clark
authors: J S
authors: JS
authors: Dordick
mesh_terms: D001407:Bacillus / Q000201:enzymology; D002384:Catalysis; D004058:Diffusion; D004789:Enzyme Activation; D007700:Kinetics; D012492:Salts; D012997:Solvents; D013379:Substrate Specificity; D013381:Subtilisins / Q000378:metabolism*; D013820:Thermolysin / Q000378:metabolism*
chemical_list: D012492:Salts; D012997:Solvents; D013381:Subtilisins; D013820:Thermolysin