Structural features of Aspergillus niger β-galactosidase define its activity against glycoside linkages

beta-Galactosidases are biotechnologically interesting enzymes that catalyze the hydrolysis or transgalactosylation of beta-galactosides. Among them, the Aspergillus niger beta-galactosidase (AnbGal) belongs to the glycoside hydrolase family 35 (GH35) and is widely used in the industry due to its high hydrolytic activity which is used to degrade lactose. We present here its threedimensional structure in complex with different oligosaccharides, to illustrate the structural determinants of the broad specificity of the enzyme against different glycoside linkages. Remarkably, the residues Phe264, Tyr304, and Trp806 make a dynamic hydrophobic platform that accommodates the sugar at subsite + 1 suggesting a main role on the recognition of structurally different substrates. Moreover, complexes with the trisaccharides show two potential subsites + 2 depending on the substrate type. This feature and the peculiar shape of its wide cavity suggest that AnbGal might accommodate branched substrates from the complex net of polysaccharides composing the plant material in its natural environment. Relevant residues were selected and mutagenesis analyses were performed to evaluate their role in the catalytic performance and the hydrolase/transferase ratio of AnbGal. Thus, we generated mutants with improved transgalactosylation activity. In particular, the variant Y304F/Y355H/N357G/W806F displays a higher level of galactooligosaccharides production than the Aspergillus oryzae beta-galactosidase, which is the preferred enzyme in the industry owing to its high transferase activity. Our results provide new knowledge on the determinants modulating specificity and the catalytic performance of fungal GH35 beta-galactosidases. In turn, this fundamental background gives novel tools for the future improvement of these enzymes, which represent an interesting target for rational design.