Functional-based screening of crude beta-galactosidase activities from 42 yeast strains resulted in the selection of a single enzyme of potential interest as a digestive supplement. beta-Galactosidase produced by Kluyveromyces marxianus DSM5418 was purified to homogeneity by a combination of gel filtration, ion-exchange, and hydroxylapatite chromatographies. The denatured (123 kDa) and native molecular masses (251 kDa) suggest that the enzyme is a homodimer. The optimum pH and temperature of the purified enzyme were 6.8 and 37 degrees C, respectively. The unpurified beta-galactosidase in particular displayed a high level of stability when exposed to simulated intestinal conditions in vitro for 4 h. Matrix-assisted laser desorption ionization mass sectrometry analysis revealed that the enzyme's trypsin-generated peptide mass fingerprint shares several peptide fragment hits with beta-galactosidases from Kluyveromyces lactis. This confirms the enzyme's identity and indicates that significant sequence homology exists between these enzymes.