Pigmentos azul (ficocianina) e vermelho (r-ficoeritrina) da macroalga marinha vermelha Hypnea musciformis (Wulfen) Lamouroux (Gigartinales, Rhodophyta): purificação e caracterização parcial

Abstract

Recent researches have shown that substances produced by seaweeds have an amazing biotechnological potential with several industrial uses. However, the natural pigments stand out, but they have not been intensively explored yet, especially when obtained from macroalgae. Because of the need to use natural substances, this work intends to extract and purify the pigments of the Hypnea musciformis red sea macroalgae, as known as phycobiliproteins, and characterize the red pigment R-phycoerythrin (R-FE). The pigments were extracted from the fresh macroalgae in potassium phosphate buffer 25 mM (pH 6.5), followed by fractionation by ammonium sulfate 0-30% and 30-45% saturated for posterior application in chromatographic columns in DEAE-Sephacel and Sephacryl S-300 matrix. The spectrophotometric analysis of the obtained fractions showed pigments related to the said macroalgae species, R-phycocyanin (λ = 550 and 610 nm), and R-FE, with absorption peaks at 495, 540 and 564 nm and fluorescence emission at 575 nm. It was obtained pure R-FE with 15.75% recovery, 8.56 μg/g productivity (seaweed dry weight) and 4.17 purity index. By electrophoresis (PAGE-SDS) in reducing conditions, the R-FE showed three protein bands with 18, 20, and 37 kDa apparent molecular mass, relative to the α, β and γ subunits. The R-FE showed 95.35% of its secondary structure in α-helix, maintaining this structure stability until 60 °C. Starting from 70 °C, it was observed changes in the absorption specter, occurring reductions in absorption (28.26%) and fluorescence emission (49.01%). The R-FE absorption specter, when stored for 20 days, remained stable when in refrigerated conditions. However, when stored at 25 °C, the maximum absorption peaks at 495, 540 and 564 nm showed an 89.7, 89.7 and 92.8% decrease, respectively. Concerning its fluorescence emission, it was observed the loss of 38.8, 73.7 and 100% under the temperature of -20, 4 and 25 °C respectively within the first five days. It was possible to extract phycobiliproteins and purify specifically the R-FE fluorescent pigment with a high purity rate from Hypnea musciformis, showing thermostable α-helix structure predominance, highly stable spectroscopic properties and especially in proper storage conditions.