Acid phosphatase from the microalgae Selenastrum capricornutum : extraction, characterization and effect of agriculture pollutants / Fosfatase acida da microalga Selenastrum capricornutum : extração, caracterização e efeito de poluentes de origem agricola

AUTOR(ES)
DATA DE PUBLICAÇÃO

2005

RESUMO

Selenastrum capricornutum is an unicellular green algae widely distributed in freshwater and soils among the world. Because this cosmopolitan characteristic its use is recommended by national and international protocols in ecotoxicity studies. Algae acid phosphatase plays important roles in the metabolism such as decomposing organic phosphates into free phosphates and organic compounds; autophagic digestive process recycling cellular materials, and zygote formation during reproduction. Chemicals released into the environment from agriculture activities like pesticides and sewage sludge contaminants may impair algae phosphatase activity. In this work it was studied the extraction, stability, substrates specificity, effect of inhibitors and kinetics of acid phosphatase in the Selenastrum capricornutum crude extract. It was also evaluated the in vitro effect of thirty agriculture pollutants (24 organic and 6 heavy metals) and performed more detailed in vitro studies and in vivo experiments with the more potent inhibitors or activators. The results showed that the extraction increased by freezing/thawing between cycles of probe sonication. The enzyme showed an optimum pH of 5, a Km value of 0.27 mM for p-NPP and stability by freezing storage during approximately 6 months. The enzyme was inhibited by tartrate (35%), fluoride (75%), Pi (45%) and p-CMB (48%). Natural organic substrates like FMN, fructose 1,6 diphosphate and a-glycero-phosphate were cleaved in a similar extent as the synthetic p-NPP, suggesting the possibility of the enzyme involvement in the cellular metabolism of such intermediates. The results demonstrated that only the surfactant linear alkyl benzenesulphonate (LAS) and the heavy metals Hg2+, Al3+ and Cu2+ altered markedly (>50%) the enzyme activity. The order of inhibition at the highest concentration tested was LAS>Hg2+= Al3+>Se3+=Pb2+>Cd2+. Calculated median inhibition concentration (IC50) values for Hg2+ and LAS were 0.085 and 0.289 mM, respectively. A non-competitive inhibition mechanism was attributed for the former (Ki = 0.0365 mM) while LAS behaved as a competitive inhibitor (Ki = 0.010 mM). In vivo studies with treated algae cultures for 24 hours showed that enzyme activity and protein content diminished in Hg2+ treatments whereas increased in LAS exposures. In contrast with short term treatments, the inhibition of specific activity was observed in exposed algae during 7 days with estimated median effect concentration (EC50) value of 12.63 ?M for Hg2+and 39% reduction for LAS 2 mM. The results of in vitro experiments with Cu2+ as an activator demonstrated a EC50 and a KdCu2+ (dissociation constant for Cu2+) equivalent to 1.80 and 1.64 ?M, respectively. When the enzyme was preincubated in the presence of Cu2+ 0.2mM, Km and Ea (activaction energy) decreased whereas Vmax practically did not changed. According to the data of stability as a function of time and the preincubation conditions during some experiments, it was suggested that the activator effect of copper might be explained by the increase on the thermal stability of the enzyme. In vivo experiments showed significantly diminution of the enzyme activity together with protein content at 1 and 10 mM Cu2+ but not at 0.1 mM. This metal did not specifically affect the activity of the enzyme in exposed algae at the experimental conditions. Join action inhibition studies indicated that Hg2+ has a lower IC50 value than Al3+ or LAS , and that the Hg2++Al3+ and Hg3++LAS mixtures have respectively, additive and slight antagonism effects. Copper, which demonstrated an activator effect when was preincubated with the enzyme, behaved as a slight antagonist for the inhibitor effect of Hg2+. It can be expected that the results of this work will help in the experimental design and discussion of results in algae enzyme studies regarding the extraction, characterization and effect of chemicals. We hope that the results presented in this work can improve the understanding of the basic events of the impact of agriculture pollutants at biochemical levels in primary producers organisms

ASSUNTO(S)

fosfatase acida acid phosphatase toxicity - tests poluentes selenastrum capricornutum pollutants toxicidade - testes algae alga enzymes selenastrum capricornutum

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