Background: β-Galactosidases catalyze both hydrolytic and transgalactosylation reactions and therefore deliver many applications in food, medical and biotechnological fields. Aspergillus niger has been a main source of β-galactosidase but the properties are incompletely studied.

Results: Three new β-galactosidases belonging to glycosyl hydrolase family 35 in A. niger strain F0215 were cloned, expressed and biochemically characterized. In addition to the known activity of LacA encoded by lacA, three putative β-galactosidases, designated as LacB, LacC and LacE encoded by the genes lacB, lacC and lacE, respectively, were successfully cloned, sequenced and expressed and secreted by Pichia pastoris. These three proteins as well as LacA have N-terminal signal sequences and are therefore predicted to be extracellular enzymes. They have a typical structure of the fungal β-galactosidases with defined hydrolytic and transgalactosylation activities on lactose. However, their activity properties differed. In particular, LacB and lacE displayed maximum hydrolytic activity at pH 4~5 and 50ºC, while LacC exhibited maximum activity at pH 3.5 and 60ºC. All β-galactosidases carried out transgalactosylation activity optimally in an acidic environment.

Conclusions: Three new β-galactosidases belonging to glycosyl hydrolase family 35 in A. niger strain F0215 were cloned, and biochemically characterized. In addition to the known LacA, A. niger has at least 3 family members of β-galactosidases with remarkably different biochemical properties.