Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents

Through sequential generations of random mutagenesis and screening, we have directed the evolution of an esterase for deprotection of an antibiotic p-nitrobenzyl ester in aqueous-organic

solvents. Because rapid screening directly on the desired antibiotic (loracarbef) nucleus p-nitrobenzyl ester was not feasible, the p-nitrophenyl ester was employed. Catalytic performance on

the screening substrate was shown to reasonably mimic enzyme activity toward the desired ester. One p-nitrobenzyl esterase variant performs as well in 30% dimethylformamide as the wildtype

enzyme in water, reflecting a 16-fold increase in esterase activity. Random pairwise gene recombination of two positive variants led to a further two-fold improvement in activity.

Considering also the increased expression level achieved during these experiments, the net result of four sequential generations of random mutagenesis and the one recombination step is a

50–60-fold increase in total activity. Although the contributions of individual effective amino acid substitutions to enhanced activity are small (