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  • Finally naringin and hesperidin have different glycosidic mo


    Finally, naringin and hesperidin have different glycosidic moieties (neohesperidose α-1,2 and rutinose α-1,6 respectively), bound in the same C7 position on the A ring of the flavonoid. The higher value of k/KM of RHA-Phis towards the former substrate suggests that the enzyme shows a preferential hydrolysis of the neohesperidose α-1,2 unit. Although a slight difference in the substituents of the flavonoidic rings involved should be taken into account in the discussion of these results, several conclusions can be drawn from these experiments. Specifically, the data obtained suggest that i) a rhamnose-containing disaccharidic unit is better accommodated in the active site compared to rhamnose alone, ii) when bound to the C7 of the A ring of the flavonoid, the neohesperidose moiety is hydrolyzed more efficiently than rutinose, and iii) when rutinose is present as the disaccharidic moiety, the preferential position for its hydrolysis is the C3 of the flavonoid C ring. All the hydrolyzed flavonoids used in this work share the presence of a polyphenolic aromatic portion that can be accommodated in the active site of RHA-Phis. Conversely, the rhamnose oligosaccharides tested (a penta-, hexa- and octasaccharide, Fig. S1 of the Supplementary Material), which lack the polyphenolic portion, were not hydrolyzed by RHA-Phis. It should be noted, however, that all these oligosaccharides are characterized by the same α-1,3 glycosidic Captopril at the non-reducing terminal of the chain. The lack of hydrolysis could be due either to the inability of the enzyme to hydrolyze α-1,3 bonds, or to the impossibility to efficiently accommodate these oligosaccharidic chains in the active site.
    Conclusions In this work we have gained a substantial amount of structural and functional information on RHA-P, a novel recombinant rhamnosidase of the GH106 family recently isolated from the marine bacterium N. sp. PP1Y. The GH106 is a still poorly characterized family of glycosyl hydrolases and, to the best of our knowledge, only the crystal structure of protein BT0986 from Bacteroides thetaiotaomicron, has been solved so far. Therefore, in the absence of RHA-P crystal structure, the insights obtained from the combined approach involving a multiple sequences alignment and the homology modeling described in this work allowed to identify critical residues in the active site of RHA-P, which is of fundamental importance for the future fine-tuning of this enzymatic activity for biotechnological applications. Our results have highlighted that the biotechnological use of RHA-P, which has shown a remarkable catalytic efficiency on several flavonoids, encompass the use of either the purified protein or the whole recombinant cells expressing the His-tagged protein. Lastly, the optimization of the recombinant expression and purification protocol will undoubtedly pave the way in the near future to crystallization experiments that are required to shed light on the catalytic and structural features of this unique family of glycosyl hydrolases.
    Author contributions
    Introduction Aggressive agents of chloride ions penetration into the concrete reinforcement is one of the most usual corrosive attacks in harsh environments that leads to several destructive consequences such as reduction of serviceability and strength. Cracks are the facilitator of the penetration process and certain routes for accession of moisture and oxygen to the reinforcement. Furthermore, the presence of enough chloride ions would cause the deterioration of passive layer adhered to steel rebar [1]. It is highly accepted that the incorporation of a pozzolanic material can reduce the permeability of concrete and enhance its resistance against chloride ions penetration [2]. The usage of supplementary cementitious materials such as rice husk ash has not only reduced the fuel demand for cement production and environmental pollution but also improved the mechanical properties and durability of concrete especially in aggressive environments [3].