Background The bacterium Propionibacterium freudenreichiiis is used for the manufacture of Swisstype cheeses and is responsible for the typical nutty flavor and eye formation. Aspartate ammonialyase catalyzes the degradation of aspartate to fumarate and ammonia. Previous reports have demonstrated that specific aspartate-ammonia lyase activity varies between P. freudenreichii strains. Strains exhibiting high aspartate ammonia-lyase activity are regarded as ambivalent for cheese production since on the one hand they can enhance flavor formation but on the other hand they can provoke gas production that leads to splits and cracks in the cheese loaf. Interestingly, genome data obtained from the GenBank database showed that the aspA gene, which encodes aspartate ammonia-lyase, is ubiquitous present in P. freudenreichii. The gene itself shows considerable between-strain heterogeneity. Objectives To better understand the link between genotype and phenotype, the specific aspartateammonia lyase activity of three isoenzymes originating from P. freudenreichii were studied. Methods Three aspA genes, which encoded isoenzymes of aspartate ammonia-lyases, were cloned into a vector encoding a his-tag and expressed in Escherichia coli. The recombinant proteins were purified and their enzymatic properties were studied using photometric assays and HPLC analysis. Results: The amino acid sequences of the three his-tagged isoenzymes shared between 85 and 95 % identity. All three isoenzymes, which were purified to apparent homogeneity, catalyzed the degradation of aspartate to fumarate and ammonia. Considerable diffenerences were observed regarding the Michaelis-Menten kinetics.