Characterization of cobamides produced by lactic acid bacteria of technological interest

VANNINI, VERÓNICA; SESMA, FERNANDO; TARANTO, MARIA PIA

San Miguel de Tucuman

Simposio; III Simposio Internacional de Bacterias Lácticas. Red BAL Argentina; 2009

CERELA-CONICET

Cobamides (Cba) are complete corrinoid, namely, a corrinoid containing thenucleotide loop. The name of the Cba depends on the nature of the lower ligand.Cobamides (e.g., coenzyme B12) are unique for their structural complexity and their denovo synthesis requires a great deal of genetic information, which is only found inprokaryotes.Previous studies in our laboratory showed that Lactobacillus (Lb) reuteri CRL 1098produces cobalamin (Taranto y col. 2003). A screening of several lactic acid bacteria(LAB) showed that the cell extract (CE) of Lb. curvatus CRL 1000, Lb. coryniformisCRL 1001 and Lb. murinus CRL 1104, were able to correct the cobalaminrequirement of Lb. delbrueckii subsp. lactis ATCC 7830, a strain that requires B12;Salmonella enterica serovar Typhimurium AR2680 (metE cbiB) and Escherichia coli113 (metE) used as indicators strains.Therefore the aim of this work was to isolate and characterized the nature of thecobamides produced by the LAB strains selected.Firstly, the each CE was passed through a C18 solid-phase extraction column andpurified by reverse-phase (RP)-HPLC with a diode array detector. The HPLCchromatogram of the standard CN-Cbl showed a peak with a retention time (RT) of40.5 min; all the strains assayed showed peaks with RT similar to the standar CN-Cbl.In addition, the peaks that eluted within 35 min and 45 min were collected andanalyzed by the Salmonella bioassay. Lb curvatus, Lb coryniformis and Lb murinusshowed two peaks (RT 38 min and 40.5 min) with the same B12 complementationproperty as the crude CE.In order to preliminary confirm the nature of these cobalamin like compounds, weperformed an Enzyme inmunoassay using specific antibodies against vitamin B12.All the peaks isolated able to correct the autotrophy of the indicator strain, showed apositive result with this assay. The mayor cobinamide produced eluted with retentiontimes of approximately 40.5 min in all the LAB assayed.Based on these results, we conclude that Lb curvatus CRL 1000, Lb coryniformis CRL1001 and Lb murinus CRL 1104 produces a vitamin B12 analogue.