Pig liver esterase (PLE) is probably the most important carboxyl esterase in organic synthesis and is commercially obtained by extraction of the animal tissue. However, problems occur in its application due to the presence of several isoenzymes (alpha-, beta- and gamma-PLE). The functional expression of the gamma-isoenzyme was already shown and differences in the enantioselectivity compared to the commercial preparations were confirmed. The amino acid and nucleotide sequences of the alpha- and beta-PLE are still unknown. In this work, putative sequences of the alpha-isoenzyme were identified from a commercial PLE preparation by 2D gel electrophoresis, digestion with proteases and analysis using Matrix-assisted laser desorption/ionization-time of flight (TOF) and electrospray ionisation quadrupole-TOF mass spectrometry. Based on these results, three amino acid exchanges were introduced into the gene encoding gamma-rPLE by site-directed mutagenesis, and the proteins were expressed in E. coli Origami (DE3). The produced PLE mutants were characterised with respect to their substrate specificity and enantioselectivity. No significant differences in the activity towards methyl butyrate were found, but several variants showed substantially enhanced enantioselectivity in the resolution of (R,S)-1-phenyl-2-butyl acetate with E = 100 for the best mutant V236P/A237G.