ARCHAEAL CAROTENOIDS: OPTIMIZATION OF A PROTOCOL FOR THE PURIFICATION OF BACTERIORUBERINS FROM THE HALOARCHAEON Haloferax volcanii

PABLO PAGOLA; ROSANA DE CASTRO; CHURIO, MARIA SANDRA

San Miguel de TUCUMAN

Congreso; XII CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL; 2017

SAMIGE Asociación Argentina de Microbiología General

Carotenoids are isoprenoid pigments produced by plants and microorganisms. Due to their antioxidant capacity they are benefcial to human health and have numerous applications. Haloarchaea are prokaryotic organisms that thrive in high salt environments (> 2 M NaCl) and produce orange/red carotenoids with C50 (bacterioruberins, Bctr). Haloarchaea represent a novel source of bioactive compounds, including carotenoids, which have been characterized to a limited extent, and thus, still remain unexploited. The aim of this work was to optimize a protocol for the purifcation of carotenoids from the haloarchaeon Haloferax volcanii and characterize their antioxidant properties. This procedure mayfacilitate obtaining samples enriched in haloarchaeal carotenoids to explore their bioactive properties and evaluate their potential applications. Two protocols were assayed for the extraction of carotenoids from cells of a genetically modifed overpigmented H. volcanii strain (HVLON3), previously constructed in our laboratory: PI (acetone: methanol, 1:1, v/v) and PII (100% acetone). The carotenoid-enriched extracts were then fractionated by thin-layer chromatography (TLC) obtaining similar yields (84-91 %) of the carotenoid fraction, denoted as BR1, with an estimated Rf = 0.25. Both protocols coupled to TLC removed several contaminants, particularly PII, which allowed elimination of the spot BR2 (Rf = 0.29).Identifcation of carotenoids, (mainly Bctr) in BR1 as well as in the whole extracts was performed by HPLC analysis. Two main peaks with retention times (Rt) of 3.00min and 4.00-5.14 min were evidenced.Based on their spectral analysis, that with Rt = 4.00-5,14 corresponded to Bctr showing 3 peaks with maximal absorbance in the range of 467-526 nm. The former with Rt = 3.00 min (maximal absorbance at 270-290 nm) so far could not be identifed but may be a precursor or degradation product of Bctr.The antioxidant capacity of the H. volcanii Bctr was evaluated based on their radical reactivity using the DPPH method coupled to EPR detection and compared to that of b-carotene (BC) as a reference, using various carotenoid concentrations. The antioxidant capacity was 3 fold higher for Bctr compared to BC based on estimated EC50 of 4.46 x 10-5 M and 1.39 x 10-4 M, respectively. Supported by EXA-731/15- UNMdP, PIP1106-CONICET and PICT1477 (RDC); PIP 0804/14 and 15/E710 UNMDP (MSC)