Polyproline, which is an amphiphilic polypeptide, was incorporated into the carboxyl terminal of an esterase by the recombinant DNA technique. The hydrophobicity of the esterase increased with increasing chain length of polyproline without inducing significant conformational changes. The mutant esterase catalyzed the hydrolysis of long-chain carboxylic acid ester more efficiently than the native esterase. It is considered that the alteration of substrate specificity is due to enhanced access of the mutant esterases to hydrophobic substrates.