Common limitations to the use of proteins outside their normal biological roles include: solubility, expression, stability and activity. The use of protein engineering to enhance enzymes biophysical properties can yield proteins with greater suitability for industrial and commercial applications.

The α/β hydrolase fold is common to a range of enzymes from highly differing phylogenetic origin and catalytic function. Dienelactone hydrolase (DLH) is monomeric and the simplest of known α/β hydrolase fold enzymes. Here we have used directed evolution to select for DLH variants with improved activity toward the non-physiological substrate p-nitrophenyl acetate. A round of evolution comprised screening a library of 5,000 variants, where the best were selected, isolated and used as the DNA template for the following round. After six rounds of evolution a 45-fold increase in k_cat/K_m was observed for the hydrolysis of p-nitrophenyl acetate. The best variants also showed improved activity toward larger ester substrates with a 84-fold increase in k_cat/K_m for the hydrolysis of p-nitrophenyl butyrate.

The amino acid substitutions responsible for the rate enhancement are located in and around the active site. Crystal structures of the wild-type and more active variants revealed small changes in orientation of active site loops. Substitutions in the loop containing the histidine of the catalytic triad have increased loop flexibility and made the histidine more accessible during catalysis.