Combinatorial formulation of biocatalyst preparations for increased activity in organic solvents: Salt activation of penicillin amidase

Abstract

A combinatorial experimental technique was used to identify salts and salt mixtures capable of activating penicillin amidase in organic solvents for the transesterification of phenoxyacetate methyl ester with 1-propanol. Penicillin amidase was lyophilized in the presence of various chloride and acetate salts within 96-deep-well plates and catalytic rates measured to determine lead candidates for highly salt-activated preparations. The kinetics of the most active formulations were then further evaluated. These studies revealed that a formulation consisting of 98% (w/w) of a 1:1 KAc:CsCl salt mixture, 1% (w/w) enzyme, and 1% (w/w) potassium phosphate buffer was approximately 35,000-fold more active than the salt-free formulation in hexane, as reflected in values of V(max)/K(m). This extraordinary activation could be extended to more polar solvents, including tert-amyl alcohol, and to formulations with lower total salt contents. A correlation was found between the kosmotropic/chaotropic behavior of the salts (as measured by the Jones-Dole B coefficients) and the observed activation. Strongly chaotropic cations combined with strongly kosmotropic anions yielded the greatest activation, and this is likely due to the influence of the ions on protein-water and protein-salt interactions.