br Acknowledgements br Introduction L
Introduction α-L-Rhamnosidases (EC 22.214.171.124) are glycosyl hydrolases (GHs) that cleave terminal α-l-rhamnose from a large number of natural products . The action of α-L-rhamnosidases (α-RHAs) has been reported, among others, on different complex substrates such as heteropolysaccharides or glycosylated proteins containing rhamnose units (gellan gum, rhamnogalacturonan and arabinogalactan-proteins) [, , , ]. Among α-RHAs substrates, natural flavonoids are gaining much interest among the food and nutraceutic industry. Natural flavonoids are polyphenolic compounds generally characterized by a three-ring structure, which consists of two phenyl rings (A and B) and a heterocyclic ring (C). These molecules are naturally produced in plants in glycosylated forms, showing the presence of either a rutinoside (6-α-l-rhamnosyl-β-D-glucoside) or a neohesperidoside (2-α-l-rhamnosyl-β-D-glucoside) disaccharidic unit bound in different positions. These molecules are very interesting due to their potential antioxidant, antitumor and anti-inflammatory properties [6,7]. In humans, endogenous β-glycosidases and α-L-arabinosidases in the small intestine are responsible for removing the glucose (or possibly arabinose or xylose) moiety from flavonoids thus facilitating their absorption. These enzymes, however, are not able to cleave terminal rhamnose units, thereby limiting the bioavailability of rhamnosylated flavonoids that are converted to more bioactive forms by the colon microflora [8,9]. Therefore, enzymatic rhamnose removal from potentially bioactive flavonoids may be the key for improving their intestinal Conessine and thus their beneficial properties for human health [10,11]. Due to their ability to hydrolyze rhamnose from natural flavonoids, α-RHAs are used in several biotechnological applications. Some examples include the hydrolysis of naringin to improve beverage quality by debittering grapefruit and citrus juices [12,13], and the removal of hesperidin crystals from orange-derived preparations . Other applications of α-RHAs are gaining popularity in the oenological industry, where these enzymes are used to hydrolyze terpenyl glycosides to enhance aroma in wine, grape juices and derived beverages . Moreover, an α-RHA has been implemented for the synthesis of rhamnose-containing chemicals by reverse hydrolysis, suggesting a yet unexplored potential of this enzymatic class in the chemical and pharmaceutical industry . Despite their potential as industrial biocatalysts, to date only a limited number of bacterial rhamnosidases has been fully characterized [, , , , ]. The commercial preparations of α–L-rhamnosidases, naringinases and hesperidinases available and currently used in oenology, are all isolated from fungal sources such as Aspergillus niger and Penicillium decumbens [, , ]. The different operational parameters observed among the bacterial and fungal sources, with bacterial RHAs being more efficient at higher pH and temperature, suggest this class of α-RHAs to be an alternative source of biocatalysts to use for carbohydrates biotransformation at high pH values. Few attempts have been made so far to engineer bacterial α-RHAs to unravel the molecular details underlying their catalytic mechanism, to modify their substrate specificity or to optimize their catalytic efficiency towards different substrates. A major obstacle is the limited number of α-RHAs crystal structures that are currently available among the different families of GH enzymes, which include the GH28, GH78, and GH106 families according to the CAZy database [, , ]. To the best of our knowledge only five crystal structures of bacterial α-L-rhamnosidases are currently available, four of which belong to the GH78 family, which show an (α/α)6 3D-structure, and include the putative α-l-rhamnosidase BT1001 from Bacteroides thetaiotaomicron VPI-5482 , the α-l-rhamnosidase B (BsRhaB) from Bacillus sp. GL1 , the α-l-rhamnosidase (SaRha78A) from Streptomyces avermitilis , and the α-l-rhamnosidase (KoRha) from Klebsiella oxytoca .