Estudios dirigidos a la mejora funcional de enzimas de levaduras no convencionales capaces de sintetizar oligosacáridos prebióticos

Resumen

Prebiotics have been recognized as functional food ingredients since they selectively stimulate the growth/or activity of potentially health-promoting colonic bacteria (Lactobacilli and Bifidobacteria). The byproducts generated by these microorganisms during the metabolism of these prebiotic oligosaccharides are associated to promote beneficial effects like the improvement of the immune response and potentially reducing the risk of acquiring several diseases (cancer, osteoporosis, cardiovascular disease or obesity, among others). In this project, two glycosylhydrolases from the Schwanniomyces occidentalis and the Kluyveromyces lactis yeasts, which are capable of synthesizing oligosaccharides with potential prebiotics properties, have been studied in order to improve the oligosaccharides production yield by various methods. The ¿-fructofuranosidase (Ftase) from Sw. occidentalis is involved in the hydrolysis of sucrose, but also participate in the release of fructose from various ¿-D-fructofuranoside substrates. In particular, this enzyme is capable to synthesize 6-kestose as the mayor product of the reaction, which is a 6F series short-chain fructooligosaccharides (FOS). The other enzyme studied was the ¿-galactosidase from K. lactis that catalyzes the hydrolysis of the galactosyl moiety from non-reducing termini of oligosaccharides and the enzyme is capable to carry out the synthesis of ß(1¿6), ß(1¿3), and ß(1¿4) linked galactooligosaccharides (GOS). In an attempt to enhance the synthesis of FOS, the heterologous expression of functional Ftase was carried out in Saccharomyces cerevisiae and Pichia pastoris. The expression system of S. cerevisiae was used to develop a directed evolution approach in order to improve the Ftase transferase activity. A high-throughput screening (HTS) assay that combined two colorimetric methods was designed and validated to increase the transfructosylation: hydrolysis ratio of the enzyme. The mutant libraries were created by error-prone PCR and recombination techniques. The selected variants were further rescreened by HPAEC-PAD to accurately estimate the FOS production by the Ftase mutants. After three consecutive error-prone PCR libraries and one last in vivo recombination library, the 6-kestose synthesis was enhanced by 5 times and 2 times in short and long times of reactions, respectively. Therefore, the HTS method used selected clones with faster hydrolase/transferase activity. Lastly, in order to improve the ¿-galactosidase stability for the synthesis of GOS, cell permeabilization techniques and directed evolution strategies have been considered. The stability of the enzyme increased considerably using cell ethanol permeabilization and lyophylization techniques. A high-throughput screening assay was developed and validated to increase the thermostability of the enzyme.