ENZYME BIOPROSPECTING OF MICROBIAL GLYCOSIDASES AND DIOXYGENASES FOR BIOCATALYSIS.

Main aim of this PhD project is the bioprospecting of different microbial enzymatic activities, to evaluate their biotechnological potential in different bioconversion processes. The characterization of four extradiol ring cleavage dioxygenases (ERCDs) and of a α-L-rhamnosidase isolated from Novosphingobium sp. PP1Y (N. sp. PP1Y) is described. In the last part of the project, gut microorganisms have been used for the identification and characterization of novel glycosyl hydrolases able to degrade the arabinogalactan polysaccharide (AG) of M. tuberculosis cell wall. In this work, the marine microorganism N. sp. PP1Y and the gut microorganism Bacteroides finegoldii were used as source for oxygenases and glycosyl hydrolases. More in detail, the optimization of recombinant expression and purification of four novel ERCDs from strain PP1Y allowed to obtain in good yields the corresponding proteins and to carry out their characterization. The activity screening using different catecholic substrates confirmed that these enzymes are able to catalyze the ring cleavage of a variety of mono- and polycyclic aromatic hydrocarbons with different sizes and conformation. In addition, the bioconversion of catechol estrogens, which could be used as precursors in the production of different steroid families and hormones, was evaluated. The results described in this thesis confirmed that these enzymes can be foreseen as a valuable tool for the modification of complex hydroxylated heterocyclic aromatic compounds, which are a starting point in the production pipeline of many pharmacologically active molecules, such as steroid-like molecules. Noteworthy, site-specific cleavage and subsequent modification of aromatic substrates, obtained by enzymatic biocatalysts, is of great advantage for industrial applications when compared to the complex mixture of products that are released instead during chemical modification procedures. In the second part of the PhD project, the biochemical characterization of RHA-P, a bacterial α-L-rhamnosidase isolated from the microorganism Novosphingobium sp. PP1Y, was performed. The active site topology and substrate specificity of RHA-P were investigated by homology modeling. The enzyme, whose recombinant expression and purification was optimized, resulted to be appealing from a biotechnological point of view for the bioconversion and de-rhamnosylation of natural flavonoids. The biotechnological use of either wild-type or mutant rhamnosidases is currently a need for food and beverages industry to improve the organoleptic properties of processed vegetal products. Moreover, the possibility of using efficient whole cells biocatalysts for expressing RHA-P has been described. This is particularly interesting because whole cells biocatalysts have numerous advantages in industrial bioconversion processes, allowing costs and process steps reduction. Finally, the bioprospecting of novel GHs from microorganisms belonging to the human gut microbiome (HGM), able to degrade the AG of M. tuberculosis cell wall, have been carried out. The isolation and characterization of different galactofuranosidases is described, which are able to completely degrade the mycobacteria galactan moiety of AG. Moreover, evidences for the presence of arabinofuranosidases were found for another HGM bacterium, which may be used as biocatalysts for the complete degradation of mycobacteria AG polysaccharide.