Characterization of S-layer proteins from lactobacilli isolated from kefir

ANDREA GÓMEZ-ZAVAGLIA

Londres (UK)

Conferencia; Invitación Department of Chemical Engineering. Imperial College London. Reino Unido.; 2008

Bacteria of genus Lactobacillus are commonly found in dairy products, and are usually associated with the beneficial effects of these products to human health. This beneficial activity is at least partially related with bacterial surface properties as adhesion and aggregation. Some lactobacilli bear an S-layer, a crystalline bidimensional array of glycoproteic subunits linked non-covalently to each other and to the underlying cell-wall. As the outermost envelope in lactobacilli, S-layer could play an important role in the surface properties of these bacteria. Structural differences in S-layer proteins could be responsible for the observed variability in surface properties of different strains of lactobacilli.  FTIR has proved to be a useful tool for the study of secondary structure of proteins by means of peak-fitting and/or analysis of second derivative spectra of amide I region (1720–1600 cm-1). Empirical association was made between position of amide I band and/or position and relative areas of component bands in peak-fitting and the type and relative content of secondary structure in the protein. In the present work, FTIR has been used to study the secondary structure of S-layer proteins from different strains of lactobacilli and the changes induced on these structures after heating. S-layer proteins from reference strains L. brevis ATCC 8287 and L. kefir JCM 5818 and from four strains of L. kefir isolated from kefir at CIDCA (L. kefir CIDCA 83113, CIDCA 8321, CIDCA 8344 y CIDCA 8348) have been analyzed. These bacterial strains 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 5M, extracts were concentred by ultrafiltration, dialyzed against bidistilled water and lyophilized. The infrared spectra, in the 4000-500 cm-1 range, were recorded by transmission spectroscopy of solid proteins in KBr pellets, using a variable temperature infrared cell connected to a digital controller. FTIR spectra demonstrated a correlation between the secondary structures of S-layer proteins and the surface properties of L. kefir strains. A high percentage of b-sheet contents (40-50 %) was found for non-aggregating strains, whereas this percentage decreased to 25-30 % for aggregating ones. A quantitative comparison of the S-layers was performed by means of cluster analysis based on the obtained spectroscopic data. This analysis enabled the strains to be grouped in clusters according to the spectral diversity in the Amide I region. The non-aggregating strains of L. kefir cluster at Ssm > 0.943 and the aggregating strains form another cluster, with Ssm > 0.769. L. brevis ATCC 8287 appears clearly separated from these two clusters: the similarity with the aggregating strains is 0.658 and the similarity with the non-aggregating ones, 0.665. The thermal analysis of the lyophilized S-layer proteins was performed by means of differential scanning calorimetry (DSC) and FTIR. DSC analysis within the 30-130 ºC range showed two phase transitions with maxima located at ca. 58 and 98 ºC for L. brevis and in the 67-70 and 110-119 ºC ranges for the different strains of L. kefir (CIDCA 8344 only shows the lowest temperature phase transition). FTIR spectra obtained reveal that for all the L. kefir S-layer proteins the major secondary structure modifications upon heating occur nearly at the first phase transitions observed by DSC, with the thermal stability increasing with the percentage of b-sheets structures. The S-layer protein of L. brevis ATICC 8287, which among all protein studied is that with maximum b-sheet contents (and no a-helix structure) was then found to be the protein showing a greater thermal stability.