It was shown recently that proline-beta-naphthylamidase from pig liver resembles the gamma-subunit of pig liver esterase (PLE), which could be functionally expressed in the yeast Pichia pastoris in recombinant form (rPLE). The gene encoding rPLE shares 97% identity with the published nucleotide sequence of porcine intestinal carboxylesterase (PICE). By site-directed mutagenesis, 22 nucleotides encoding 17 amino acids were exchanged stepwise from the PLE gene yielding the recombinant PICE sequence and eight intermediate mutants. All esterases were successfully produced in P.pastoris as extracellular proteins with specific activities ranging from 4 to 377 U/mg and V(max)/K(m) values from 12 to 1000 l min(-1) x 10(-3) using p-nitrophenyl acetate as substrate. Activity-staining of native polyacrylamide gels followed by molecular mass determination suggests that the most active forms of all variants are present as trimers with a molecular mass of 190-210 kDa. All enzymes exhibit the highest activity in the pH range 8-9 and between 60 and 70 degrees C. Almost all esterases show a higher ratio of methyl butyrate hydrolase activity to proline-beta-naphthylamidase activity than rPLE.