Chemical modification of enzymes for enhanced functionality

The explosion in commercial and synthetic applications of enzymes has stimu lated much of the interest in enhancing enzyme functionality and stability. Covalent chemical modification, the original method available for altering protein properties, has now re-emerged as a powerful complementary approac h to site-directed mutagenesis and directed evolution for tailoring protein s and enzymes. Glutaraldehyde crosslinking of enzyme crystals and polyethyl ene glycol (PEG) modification of enzyme surface amino groups are practical methods to enhance biocatalyst stability. Whereas crosslinking of enzyme cr ystals generates easily recoverable insoluble biocatalysts, PEGylation incr eases solubility in organic solvents. Chemical modification has been exploi ted for the incorporation of cofactors onto protein templates and for atom replacement in order to generate new functionality, such as the conversion of a hydrolase into a peroxidase. Despite the breadth of applicability of c hemically modified enzymes, a difficulty that has previously impeded their implementation is the lack of chemo- or regio-specificity of chemical modif ications, which can yield heterogeneous and irreproducible product mixtures . This challenge has recently been addressed by the introduction of a uniqu e position for modification by a site-directed mutation that can subsequent ly be chemically modified to introduce an unnatural amino acid sidechain in a highly chemo- and regio-specific manner.