Effect of trehalose on the secondary structure of L. kefir S-layer upon liophilization

P. MOBILI, A. GÓMEZ-ZAVAGLIA, A. ABRAHAM, G. DE ANTONI, R. FAUSTO

Luso, Portugal

Congreso; 8º Encontro Nacional da Sociedade Portuguesa de Química.; 2007

Sociedade Portuguesa de Química

The S-layer structures are macromolecular paracrystalline arrays that completely cover the bacterial cell surface. 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 such as guanidine hydrochloride and dissociating agents such as 5 M-LiCl. The S-layer has been described for some lactobacilli, among them L. kefir. It could play an important role in the surface properties of these bacteria and structural differences in S-layer proteins could be responsible for the observed variability in surface properties of different strains of lactobacilli. It has been reported that trehalose is able to protect both cell membranes and proteins from dehydration [1]. Taking into account that the S-layer represents the most external bacterial layer and has a crucial role in the maintenance of the surface properties, the maintenance of its structural properties upon liophylization (a very common procedure to preserve microorganisms) is very important to assure the preservation of the surface properties after this process. In this work, the interaction between trehalose and S-layer proteins extracted from a reference strain of L. brevis ATCC 8287 and from 2 strains of L. kefir (CIDCA 83113 and CIDCA 8348) has been evaluated. These bacterial strains have been chosen because they showed differences in surface properties and in S-layer protein molecular weight, glycosilation and pattern of recognition by antibodies. S-layer proteins were extracted from bacteria using LiCl 5 M; extracts were concentrated by ultrafiltration, dialyzed against bidistilled water and lyophilized in the presence or absence of different concentrations of trehalose. The effect of trehalose has been evaluated by FTIR analysis of the liophylized proteins in KBr pellets (ratio 1 mg protein / 100mg KBr). A total of 512 scans were coadded at a 4 cm-1 resolution. The effect of the protein on the specific bands of trehalose has been analyzed in two regions: a) stretching CH and b) the fingerprint region of carbohydrates: 900-1200 cm-1. In the stretching CH region, the presence of trehalose induces a fusion of the 6 bands corresponding to trehalose itself. On the other hand, the band of trehalose at 995 cm-1 experienced a blueshift from 994.1 cm-1 at the lowest trehalose concentration and to 998.1 cm-1 when the trehalose concentration attains 96 %. In addition, in this region, a fusion of the bands corresponding to trehalose alone was also observed in the presence of the S-layers. These observations indicate that the S-layers analyzed interact with trehalose at all the concentrations. It has been reported that the protein-sugar interaction may maintain proteins in a conformation similar to that of the hydrated protein, perhaps by binding to the hydrophilic domains of the proteins in replace of the water molecules and preventing inter- and intraprotein hydrogen bonding during drying and rehydration [2,3]. References 1. S. Leslie, E. Israeli, B. Lighthart, J. H. Crowe and L. M. Crowe. Appl. Environ. Microbiol. 61 (1995) 3592. 2. J. F. Carpenter, J.H. Crowe and T. Arakawa. J. Dairy Sci. 73 (1990) 3627. 3. J. F. Carpenter, T.  Arakawa and J. H. Crowe. 74 (1992) 225. Acknowledgements: The research was supported by the Portuguese Fundação para a Ciência e a Tecnologia (Projects POCI/QUI/59019/2004 and POCI/QUI/58937/2004), the Program Alban, the European Union Program of High Level Scholarships for Latin America, scholarship Nº. E06D102040AR. AGZ is member of the research career Conicet (National Research Council, Argentina).