Structural characterization of the β-galactosidase from 'Kluyveromyces lactis' and expression and directed evolution of β-galactosidases with high biotechnological interest

摘要

[Abstract] ß-galactosidases are hydrolase enzymes that catalyze the hydrolysis of ß-galactosides into their monosaccharides. Due to this ability these proteins are very important in food, clinical and pharmaceutical industries.In this thesis the Kluyveromyces lactis ß-galactosidase was cloned in a Saccharomyces cerevisiae strain, expressed, purified, and crystallized. Its free state structure and its complex with the product galactose were determined to 2.75 and 2.8 Å, respectively. K. lactis ß-galactosidase folds into 5 domains in a pattern conserved with other prokaryote enzymes solved for GH2 family, although two long insertions in domains 2 (264-274) and 3 (420-443) are unique and seem related to oligomerization and specificity. K. lactis ß-galactosidase tetramer is an assembly of dimers, with higher dissociation energy for the dimers than for its assembly, which can explain that equilibrium exists in solution between the dimeric and tetrameric form of the enzyme.On the other hand, a hybrid K. lactis-Aspergillus niger ß-galactosidase was constructed, expressed and characterized. The hybrid protein between K. lactis and A. niger ß-galactosidases increases the yield of the protein released to the growth medium and the modifications introduced in the construction conferred to the protein biochemical characteristics of biotechnological interest. The production of this hybrid K. lactis - A. niger ß-galactosidase was also tested in a continuous immobilized culture using spent grains (a by-product of brewery industry) as an immobilizing material in an airlift fermenter.Finally, different A. niger ß-galactosidase constructions were expressed and studied in a S. cerevisiae strain and directed evolution techniques were applied to modify the optimal pH of the protein to a more neutral one.