Extraction, purification et caractérisation des lectines produites par des souches pures d’actinomycètes isolés à partir de la rhizosphère de Lactuca sativa, Vicia fabae, Prunus domestica et P inus halepensis.

Abstract

Lectins are present in all living organisms and are used as tools in research and in the biomedical sector. Currently, the microbial lectins are attracting more and more of interest because of their diverse biological activities. Some strains of actinomycetes Rhizospheric promoters of plant growth and antagonistic fungi and pathogens of these plants were chosen to highlight the production of lectins to purify characterize and study their biological activities. The six strains studied were identified to genera Streptomyces and Nocardiopsis. The strains were cultured on two liquids (Bennet and ISP2). The adhesive strength of the extracellular and intracellular extracts of the culture media is evidence placed in the presence of seven types of erythrocytes. Both extracts of six strains, in any case of the medium, have no agglutinate human erythrocytes. Strongest agglutination was obtained with the presence of intracellular extracts and rat erythrocytes of rabbits. The ISP2 environment better promotes the production of lectins. Intracellular fractions strongly caking rat erythrocytes were retained. That of Pru16 strain for purification and characterization and that of the Vic8 strain to study the immunomodulatory effect. Lectins, the intracellular fraction of strain Pru16 underwent a first primary purification with ammonium sulphate and gel filtration G75. Differential precipitation showed that the first three fractions exhibit HA activity. After filtration, two peaks were observed (peaks A and B). Peak B shows no HA activity. However, the specific activity of the crude extract was 8.37 HU / mg, and it increased to 14.95 UH / mg after ammonium sulfate precipitation, and 20 UH / mg (picA) .after G75 gel filtration. The HA activity of this fraction remains at alkaline pH and high concentrations of EDTA. After column chromatography mucin sepharose 4B (second purification), two peaks are observed. The proteic fraction 33 peak 2 has a high specific HA activity about twelve times greater than that observed for the crude extract (8.37 111.49 against UH / mg), however the first peak shows no HA activity. The affinity test conducted with the proteic fraction 33 of Peak 2 showed that the Pru16 strain binds specifically to protein. The SDS-PAGE analysis showed that the hemagglutinin contained in the portion 33 of the P2 peak is located in the band 16kDa The identification of proteins 16kDa band, after digestion and elution of peptides by LC, was top by tandem mass spectrometry proteomics comparing with the database related strains. The identified analysis of 6 different proteins similar, one of them has a high score, with a molecular weight similar to that for protein band. This is the lysozyme C who did him no HA activity. Our protein of 16kDa presents antimicrobial activity. The enhancement of the phagocytic index of the corrected phagocytic index, and decreased half-life time in circulation of carbon particles in the blood indicate that the Vic8 strain of the crude extract possess one or more factors able to significantly boost the phagocytic activity of macrophages and reticuloendothelial system in mice. This effect is comparable to that exerted by the pure lectins.