Microbial β-Galactosidases of industrial importance: Computational studies on the effects of point mutations on the lactose hydrolysis reaction.

Hydrolysis efficiency of β-galactosidases is affected due to a strong inhibition by galactose, hampering the complete lactose hydrolysis. One alternative to reduce this inhibition is to perform mutations in the enzyme's active site. The aim of this study was to evaluate the effect of point mutations on the active site of different microbial β-galactosidases, using computational techniques. The enzymes of Aspergillus niger (AnβGal), Aspergillus oryzae (AoβGal), Bacillus circulans (BcβGal), Bifidobacterium bifidum (BbβGal), and Kluyveromyces lactis (KlβGal) were used. The mutations were carried out in all residues that were up to 4.5 Å from the galactose/lactose molecules and binding energy was computed. The mutants Tyr96Ala (AnβGal), Asn140Ala and Asn199Ala (AoβGal), Arg111Ala and Glu355Ala (BcβGal), Arg122Ala and Phe358Ala (BbβGal), Tyr523Ala, Phe620Ala, and Trp582Ala (KlβGal) had the best results, with higher effect on galactose binding energy and lower effect on lactose affinity. To maximize enzyme reactions by reducing galactose affinity, double mutations were proposed for BcβGal, BbβGal, and KlβGal. The double mutations in BcβGal and BbβGal caused the highest reduction in galactose affinity, while no satisfactory results were observed to KlβGal. Using computational tools, mutants that reduced galactose affinity without significantly affecting lactose binding were proposed. The mutations proposed can be used to reduce the negative feedback process, improving the catalytic characteristics of β-galactosidases and rendering them promising for industrial applications.