Bioengineering of β-galactosidases for their use in the food industry

Abstract

β-galactosidases are enzymes required in a large number of applications by food industry. In this thesis we engineered the β-galactosidases from Aspergillus niger and Kluyveromyces lactis in order to obtain more suitable forms for industrial uses. Aspergillus niger β-galactosidase was expressed in yeast, purified by affinity chromatography and crystallized to perform Xray diffraction experiments. The three-dimensional structures of the enzyme in its native form and in complexes with five oligosaccharides were solved. The results gave insights into specificity determinants of GH35 β- galactosidases catalysis and this knowledge allowed engineering of the enzyme to achieve new variants with increased transgalactosylation activity. Moreover, thermostable mutants of β-galactosidase from Kluyveromyces lactis were obtained by performing a rational design strategy. The introduction of new disulfide bonds among subunit interfaces also produced a noticeable rise in the catalytic efficiency of the mutants. This effect was attributable to quaternary structure stabilization, which increases the proportion of the highest active oligomeric forms in the equilibrium. Finally, immobilization studies using two different supports showed that in the mutant the loss of catalytic activity after immobilization, even after several reutilization cycles, was lower than in the native protein. Therefore, this mutant variant performed better than the native form during immobilization, which represents a new advantage for industrial applications.