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Cloning, expression, and characterization of a peculiar choline-binding beta-galactosidase from Streptococcus mitis

AutorCampuzano, Susana ; Serra, Beatriz; Llull, Daniel ; García, José Luis ; García, Pedro
Fecha de publicaciónsep-2009
EditorAmerican Society for Microbiology
CitaciónApplied and Environmental Microbiology, 75 (18) : 5972-80 (2009)
ResumenA Streptococcus mitis genomic DNA fragment carrying the SMT1224 gene encoding a putative β-galactosidase was identified, cloned, and expressed in Escherichia coli. This gene encodes a protein 2,411 amino acids long with a predicted molecular mass of 268 kDa. The deduced protein contains an N-terminal signal peptide and a C-terminal choline-binding domain consisting of five consensus repeats, which facilitates the anchoring of the secreted enzyme to the cell wall. The choline-binding capacity of the protein facilitates its purification using DEAE-cellulose affinity chromatography, although its complete purification was achieved by constructing a His-tagged fusion protein. The recombinant protein was characterized as a monomeric β-galactosidase showing a specific activity of around 2,500 U/mg of protein, with optimum temperature and pH ranges of 30 to 40°C and 6.0 to 6.5, respectively. Enzyme activity is not inhibited by glucose, even at 200 mM, and remains highly stable in solution or immobilized at room temperature in the absence of protein stabilizers. In S. mitis, the enzyme was located attached to the cell surface, but a significant activity was also detected in the culture medium. This novel enzyme represents the first β-galactosidase having a modular structure with a choline-binding domain, a peculiar property that can also be useful for some biotechnological applications.
Streptococcus mitis belongs to the viridans group of streptococci and is a relevant microorganism because it is both an opportunistic pathogen and phylogenetically close to Streptococcus pneumoniae, a major respiratory human pathogen. Although S. mitis isolates usually produce only mild infections, some S. mitis strains have acquired increased virulence and are one of the main causes of infectious endocarditis (15, 36). Remarkably, S. mitis, like only a few other streptococci, displays phosphorylcholine residues in its cellular envelope (3). This aminoalcohol is used for the anchorage of proteins belonging to the so-called “choline-binding proteins” (CBPs), which fulfill important physiological functions in these bacteria. CBPs bind to phosphorylcholine residues present in the teichoic and lipoteichoic acids located at the surface of S. pneumoniae and some streptococci of the mitis group. CBPs share a modular organization consisting of a biologically active domain and a conserved choline-binding domain (CBD), which contains 6 to 18 imperfect 20-amino-acid tandem repeats each located either at the carboxy- or amino-terminal ends of the proteins (26). This CBD is able to specifically bind to choline or its structural analogues like DEAE (diethylaminoethanol), which permits purification by affinity chromatography in a single step using DEAE-cellulose supports (38). Crystallographic studies of CBPs have shown that a typical CBD consists of several β-hairpins organized as a left-handed superhelix and that the linkage of CBPs to the choline-containing cell wall substrate is carried out through the binding of choline residues to the interface of two consecutive choline-binding repeats, named choline-binding sites (9, 13, 14).
β-d-Galactosidases (β-d-galactoside galactohydrolase; EC 3.2.1.23) constitute a large family of proteins that cleave the glycosidic bond between two or more carbohydrates or between a carbohydrate and a noncarbohydrate moiety, e.g., lactose and related chromogens, like o-nitrophenyl-β-d-galactopyranoside (ONPG), p-nitrophenyl-β-d-galactopyranoside (PNPG), or 6-bromo-2-naphthyl-galactopyranoside. β-d-galactosidases belong to the glycosyl hydrolase (GH) superfamily, which contains 114 families (see http://www.CAZY.org) classified on the basis of amino acid sequence similarity (12). The enzymes exhibiting β-galactosidase activity are currently classified within four different families: GH-1, GH-2, GH-35, and GH-42. β-Galactosidases are widely distributed in nature and are present in numerous microorganisms (yeasts, fungi, bacteria, and archaea), plants, and animals (34, 44). These enzymes are of great interest for several industrial or biotechnological processes; the hydrolytic activity has been applied in the food industry for decades to reduce the lactose content of milk products in order to circumvent lactose intolerance, which is prevalent in more than half of the world's population (27). More recently, interest in β-galactosidases has increased due to their ability to synthesize β-galactosyl derivatives that have received a great deal of attention owing to their important roles in many biological processes (27). In this study, we report the purification and biochemical characterization of a peculiar β-galactosidase encoded by the SMT1224 gene of S. mitis that represents a new type of β-galactosidase within this paradigmatic enzyme family
Descripción9 páginas, 4 figuras, 4 tablas -- PAGS nros. 5972-5980
Versión del editorhttp://dx.doi.org/10.1128/​AEM.00618-09
URIhttp://hdl.handle.net/10261/53479
DOI10.1128/​AEM.00618-09
ISSN0099-2240
E-ISSN1098-5336
ISMN10.1128/​AEM.00618-09
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