Among the members of the
Among the members of the GH106 family, which groups 319 different sequences, a single 3D-structure has been reported, the BT0986 from Bacteroides thetaiotaomicron that shows a (β/α)8 barrel and catalyzes the hydrolysis of an α-l-rhamnopyranoside bound to the C2 position of an arabinofuranoside (L-Rhap-α-1,2-L-Araf). In this same family of glycosidases only two Amikacin mg have been characterized so far, and the reaction mechanism and the catalytic residues have been inferred from the 3D-structure of BT0986 .
In our previous work, a novel α-l-rhamnosidase was isolated from the marine microorganism Novosphingobium sp. PP1Y, a Gram-negative bacterium isolated from a polluted marine environment in the Pozzuoli harbor (Naples, Italy) [, , ]. The α-RHA from N. sp. PP1Y was isolated from the native bacterium, expressed in E. coli and partially characterized . This enzyme, named RHA-P, was characterized as an inverting monomeric glycosidase of ca. 120 kDa belonging to the GH106 family. A preliminary biochemical characterization using the synthetic substrate pNPR (para-nitrophenyl-α-l-rhamnopyranoside) showed that RHA-P has moderate tolerance to organic solvents and optimal activity between pH 6.0–7.5 at 35–45 °C. Moreover, TLC analysis showed that RHA-P is able to hydrolyze rhamnose from natural flavonoids such as naringin, rutin and neohesperidin dihydrochalcone .
Materials and methods
Discussion RHA-P is a bacterial α-l-rhamnosidase recently isolated from the microorganism Novosphingobium sp. PP1Y . This enzyme is an inverting glycosidase, belonging to the GH106 family, for which an initial biochemical characterization has highlighted an interesting biotechnological potential that needs to be better defined through a deeper functional and structural characterization of the protein. Due to previous low purification yields related to the excessive number of purification steps used, a His-tag was added to the C-terminus of the recombinant protein expressed in the strain BL21(DE3) of E.coli. In our previous work we showed that “standard” conditions of recombinant expression led to the foremost presence of the untagged rRHA-P in the insoluble fraction of the induced cultures. The use of a lower induction temperature (23 °C instead of 37 °C), and a high-salt (0.5 M NaCl) LB medium, containing both betaine and sorbitol, allowed to markedly increase the yield of active rRHA-P in the soluble fraction of induced cultures . RHA-Phis in standard expression conditions is also mostly expressed as inclusion bodies. Once having optimized the expression conditions, which involved again the use of a lower induction temperature and the addition of both betaine and sorbitol to the growth medium, we verified the presence of the recombinant protein in the periplasmic space of induced cells, as suggested by the identification of a signal peptide at the N-terminus of the protein. The presence of a similar peptide has also been described for the α-RHA isolated from Sphingomonas paucimobilis FP2001, which shares a 48% sequence identity with RHA-P . Signal peptides have been described in α-RHAs isolated from fungal sources, such as the enzymes from Aspergillus kawachii and Aspergillus aculeatus [24,47]. In these latter, a slight difference was found in the sequence features compared to their bacterial counterparts; nonetheless, the efficient cleavage of a 17–20 aminoacids long N-terminal peptide was observed in the recombinant proteins. Although the functional reason for this specific sorting of an α-RHA is still not clear, this might be related to the engagement of this enzymatic activity in the complex molecular machinery involved in the biosynthesis and maintenance of the bacterial cell wall, where l-rhamnose appears to be a component of membrane rhamnolipids and polysaccharides [48,49]. The presence of a putative N-terminal signal peptide in RHA-P had been previously suggested by the lack of an expected 22 aminoacid-long peptide, presumably cleaved through a post-translational proteolytic processing, in both the native and the recombinant protein .