CIDCA   05380
CENTRO DE INVESTIGACION Y DESARROLLO EN CRIOTECNOLOGIA DE ALIMENTOS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Role of surface biomolecules in the heavy metals sequestrant capacity of lactic acid bacteria. A Vibrational Spectroscopy study
Autor/es:
GERBINO, E.; MOBILI, P; TYMCZYSZYN E,; ARAUJO-ANDRADE C.; FRAUSTO-REYES C.; FAUSTO RUI; GOMEZ ZAVAGLIA, A.
Lugar:
Lisboa
Reunión:
Simposio; ISMEC 2012; 2012
Institución organizadora:
International Symposia on Metal Complexes
Resumen:
Introduction: Toxic metals are not degradable and tend to accumulate in the exposed organisms causing serious health effects. The use of inactivated microbial biomass as adsorbents (biosorption) has become an efficient tool to remove such metals from different substrates (i.e. water, food, etc.) [1,2]. For this purpose, several species of microorganisms, including lactic acid bacteria, have been successfully employed. In spite of the relevance of biosorption in a sustainable development, the molecular bases involved have not been properly explored. For this reason, the aim of this work was to evaluate the interaction bacteria/heavy metal from a chemical point of view. Materials and methods: Lactobacillus kefir CIDCA 8348 and L. kefir JCM 5818 were cultured in de MRS broth [3] 48 h at 30 oC (stationary phase). One millilitre of cultures in the stationary phase was harvested and washed twice with ultra pure water (Milli-Q plus; Millipore Cop., USA). The pellets were resuspended into 1 ml milli Q water containing Pb+2 [from Pb(NO3)2], Cd+2 [from Cd(NO3)2] or Ni+2 [from Ni(NO3)2.6H2O] ranging from 0 to 0.9 mM. The suspensions were further incubated 1 h at 30oC (pH 5.5) and then centrifuged 4 min at 6600 g. The pellets were kept to register the Raman spectra. Raman Spectroscopy: The Raman spectra of both bacteria containing the adsorbed heavy metals and nontreated bacteria (controls) were measured by placing them onto an aluminium substrate and then under a Leica microscope integrated to the Raman system (Renishaw 1000B). The wavelength of excitation was 830 nm and the power irradiation over the samples,45 mW. Each spectrum was registered with an exposure of 30 s, two accumulations, and collected in the 1800-200 cm-1 region (spectral resolution: 2 cm-1). S-layer proteins preparation: Bacterial cells harvested in the stationary phase were mixed with 5 M LiCl and the S-layers were extracted and purified as described previously [4]. The pure S-layers were suspended in 1 ml milli Q water containing 0.3 mM Pb(NO3)2, Cd(NO3)2 or Ni(NO3)2.6H2O. The suspensions were incubated 24 h at 30 ºC and then, centrifuged at 4 min at 6600 g. The pellets were used for the FTIR determinations. FTIR spectroscopy: 30 ml of the S-layer pellets were put on a CaF2 window and further dried 15 min at 45 ºC to get a transparent film, used for FTIR experiments. FTIR spectra were recorded in the 4000-500 cm-1 range in transmission mode. The IR spectra were obtained co-adding 128 scans at room temperature (spectral resolution: 4 cm-1). Deconvolution of amide I band and assignment of protein secondary structures was as previously reported [4]. Results: The Raman spectra of both nontreated L. kefir strains (controls) were compared with those obtained after the treatment with each heavy metal. The main differences observed between controls and bacteria/metal samples clearly denote the sites where metals are attached. Modifications of both nCOO- s (1445 cm-1) and nCOO- as (1655 cm-1) bands indicate that bacteria⁄metal interaction occurs through carboxylate groups. The band at 1260 cm-1, related with phosphate groups disappears after the treatment with metals and in the region  1000-850 cm-1, ascribed to polysaccharides, further changes are observed. The most outmost structures in L. kefir strains are the S-layer proteins. They are macromolecular paracrystalline arrays that completely cover the bacterial cell surface [5,6]. They are attached to the underlying cell wall by non-covalent bonds and usually may be dissociated and solubilized into protein monomers by chaotropic agents (i.e.: LiCl). Considering this background, the S-layers of the two strains under study were purified and the interaction S-layer/heavy metals, analyzed using FTIR spectroscopy. The metal/protein interaction occurs mainly through the COO- groups of the side chains of Asp and Glut residues, with some contribution of the NH groups of the peptide backbone. The frequency separation between the  nCOO- as and nCOO- s vibrations in the spectra of the S-layers in presence of the metal ions was found to be ca. 190 cm-1 for S-layer CIDCA 8348 and ca. 170 cm-1 for JCM 5818, denoting an unidentate coordination in both cases [7]. The secondary structure of the proteins was also altered after the interaction with heavy metals: a general trend to increase the amount of b-sheet structures and to reduce the amount of a-helices was observed. These changes allow the proteins to adjust their structure to the presence of the metal ions at minimum energy expense. Conclusion: This analysis allowed obtaining a deeper insight on the molecular interactions involved when heavy metals are attached to the bacterial surface.