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

Hydrolysis efficiency of beta-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 beta-galactosidases, using computational techniques. The enzymes of Aspergillus niger (AnbetaGal), Aspergillus oryzae (AobetaGal), Bacillus circulans (BcbetaGal), Bifidobacterium bifidum (BbbetaGal), and Kluyveromyces lactis (KlbetaGal) were used. The mutations were carried out in all residues that were up to 4.5 A from the galactose/lactose molecules and binding energy was computed. The mutants Tyr96Ala (AnbetaGal), Asn140Ala and Asn199Ala (AobetaGal), Arg111Ala and Glu355Ala (BcbetaGal), Arg122Ala and Phe358Ala (BbbetaGal), Tyr523Ala, Phe620Ala, and Trp582Ala (KlbetaGal) 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 BcbetaGal, BbbetaGal, and KlbetaGal. The double mutations in BcbetaGal and BbbetaGal caused the highest reduction in galactose affinity, while no satisfactory results were observed to KlbetaGal. 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 beta-galactosidases and rendering them promising for industrial applications.