Extraction, Partial Characterization And Susceptibility To Hg2+ Of Acid Phosphatase From The Microalgae Pseudokirchneriella Subcapitata [extração, Caracterização Parcial E Susceptibilidade Ao Hg2+ Da Fosfatase ácida Da Microalga Pseudokirchneriella Subcapitata]

Pseudokirchneriella subcapitata is a unicellular green algae widely distributed in freshwater and soils. Due to its cosmopolitan characteristic, its use is recommended by national and international protocols in ecotoxicity studies. The alteration of phosphatase activities by agriculture pollutants like heavy metals has been extensively used as a biomarker in risk assessment and biomonitoring. In this study, we compared the extraction of acid phosphatase from P. subcapitata by different procedures and we studied the stability, substrates specificity, kinetics and the effect of Hg2+ in the crude extract. The freezing and thawing technique associated with probe sonication was the most suitable method of extraction. The enzyme was stable when frozen at -20°C for at least six months, showed an optimum pH of 5 and a Km value of 0.27 mM for p-nitrophenylphosphate (pNPP) as substrate. Some natural organic substrates were cleaved by a similar extent as the synthetic substrate pNPP. Short term exposure (24 hours) to Hg2+ had little effect but inhibition of the specific activity was observed after 7 days with EC50 (concentration of Hg2+ that promotes 50% decrease of specific activity) value of 12.63 μM Hg2+.665634642Abd Elaah, G.A., El Nagdy, M.A., Seasonal variations of zoosporic and terrestrial fungi in cultivated soil Sohag Governorate and isozyme differences among Achlya species (1999) African Journal of Mycology and Biotechnology, 7, pp. 7-23Alamillo, J.M., Cardenas, J., Pineda, M., Purification and molecular properties of urate oxidase from Chlamydomonas reinhardtii (1991) Biochimica et Biophysica Acta, 1076, pp. 203-206Amani, M., Moosavi-Movahedi, A.A., Floris, G., Longu, S., Mura, A., Moosavi-Nejad, S.Z., Saboury, A.A., Ahmad, F., Comparative study of the conformational lock, dissociative thermal inactivation and stability of Euphorbia latex and lentil seedling amine oxidases (2005) Protein Journal, 24, pp. 183-191Assche, F., Clijsters, H., Effect of metals on enzyme activity in plants (1990) Plant, Cell and Environment, 13, pp. 195-206Baun, A., Justesen, K.P., Nyholm, N., Algal tests with soil suspensions and elutriates (2002) A comparative evaluation for PAH-contaminated soils. Chemosphere, 46 (2002), pp. 251-258Bennun, A., Blum, J.J., Properties of the induced acid phosphatase and of the constitutive acid phosphatase of Euglena (1966) Biochimica. et Biophysica Acta, 128, pp. 106-123Chen, Q.X., Zheng, W.Z., Lin, J.Y., Shi, Y., Xie, W.Z., Zhou, H.M., Effect of metal ions on the activity of green crab (Scylla serrata) alkaline phosphatase (2000) International Journal of Biochemistry and Cell Biology, 32, pp. 879-885(1992) Procedimentos para a utilização de testes de toxicidade no controle de efluentes líquidos, p. 17. , COMPANHIA DE TECNOLOGIA DE SANEAMENTO AMBIENTAL - CETESB, São Paulo: CETESB, Série Manuais(2005) Resolução n. 357 de 17 de março de 2005. Diário Oficial da União, , CONSELHO NACIONAL DO MEIO AMBIENTE - CONAMA., Brasília, 18 de março deCooper, A., Bowen, I.D., Lloyd, D., The properties and subcellular localization of acid phosphatases in the colourless alga Polytomella caeca (1974) Journal of Cell Science, 15, pp. 605-618Demeke, T., Chang, H.G., Morris, C.F., Effect of germination, seed abrasion and seed size on polyphenol oxidase assay activity in wheat (2001) Plant Breeding, 120, pp. 369-373Dixon, M., Webb, E.C., (1979) Enzymes, p. 476. , 3 ed, London: LongmansDuff, S.M.G., Sarath, G., Plaxton, W.C., The role of acid phosphatases in plant phosphorous metabolism (1994) Physiologia Plantarum, 90, pp. 791-800El Demerdash, F.M., Elagamy, E.I., Biological effects in Tilapia nilotica fish as indicators of pollution by cadmium and mecury (1999) International Journal of Environmental Health Research, 9, pp. 173-186Fathi, A.A., Toxicological response of the green algae Scenedesmus bijuga to mercury and lead (2002) Folia Microbiologica, 47, pp. 667-671Gellatly, K., Moorehead, G.B.G., Duff, S.M.G., Lefebvre, D.D., Plaxton, W.C., Purification and characterization of a potato tuber acid phosphatase having significant phosphotyrosine phosphatase activity (1994) Plant Physiology, 106, pp. 223-232Geoffroy, L., Teisseire, H., Couderchet, M., Vernet, G., Effect of oxyfluorfen and diuron alone and in mixture on antioxidative enzymes of Scenedesmus obliquus (2002) Pesticide Biochemistry and Physiology, 72, pp. 178-185Gill, T.S., Tewari, H., Pande, J., In vivo and in vitro effects of cadmium on selected enzymes in different organs of the fish Barbus conchonius Ham. (Rosy barb) (1991) Comparative Biochemistry and Physiology, Part C, 100, pp. 501-505Granbom, M., Chowb, F., Lopes, P.F., Oliveira, M.C., Colepicolo, P., de Paula, E.J., Pedersén, M., Characterisation of nitrate reductase in the marine macroalga Kappaphycus alvarezii (Rhodophyta) (2004) Aquatic Botany, 78, pp. 295-305Granjeiro, J.M., Ferreira, C.V., Juca, M.B., Taga, E.M., Aoyama, H., Bovine kidney low molecular weight acid phosphatase (1997) FMN-dependent kinetics. Biochemistry and Molecular Biology International, 41, pp. 1201-1208Gratão, P.L., Polle, A., Lea, P.J., Azevedo, R.A., Making the life of heavy metal-stressed plants a little easier (2005) Functional Plant Biology, 32, pp. 481-494Huang, C.C., Chen, M.W., Hsieh, J.L., Lin, W.H., Chen, P.C., Chien, L.F., Expression of mercuric reductase from Bacillus megaterium MB1 in eukaryotic microalga Chlorella sp. DT: An approach for mercury phytoremediation (2006) Applied Microbiology and Biotechnology, 72, pp. 197-205Jonsson, C.M., Aoyama, H., In vitro effect of agriculture pollutants and their joint action on Pseudokirchneriella subcapitata acid phosphatase (2007) Chemosphere, 69, pp. 849-855Keddy, C.J., Greene, J.C., Bonnel, M.A., Review of whole-organism bioassay: Soil, freshwater, sediment, and freshwater assessment in Canada (1995) Ecotoxicology and Environmental Safety, 30, pp. 221-251Kong, F.X., Chen, Y., Effect of aluminium and zinc on enzyme activities in the green alga Selenastrum capricornutum (1995) Bulletin of Environmental Contamination and Toxicology, 55, pp. 759-765Lien, T., Knutsen, G., Synchronous cultures of Chlamydomonas reinhardtii: Properties and regulation of repressible phosphatases (1973) Physiologia Plantarum, 28, pp. 291-298Lowry, O.H., Lopez, J.A., The determination of inorganic phosphate in the presence of labile phosphate esters (1945) Journal of the Biological Chemistry, 162, pp. 421-424Lowry, O.H., Rosebrough, N.J., Farr, A.F., Randall, R.J., Protein measurement with the Folin phenol reagent (1951) Journal of the Biological Chemistry, 193, pp. 265-275Mamais, D., Kouzeli-Katsiri, A., Christoulas, D.G., Andreadakis, A., Aftias, E., Evaluation of agricultural utilisation of the sludge produced at Psyttalia WWTP (2000) Water Science and Technology, 42, pp. 21-27Matagne, R.F., Loppes, R., Deltour, R., Phosphatases of Chlamydomonas reinhardi: Biochemical and cytochemical approach with specific mutants (1976) Journal of Bacteriology, 126, pp. 937-950Nakazato, H., Okamoto, T., Ishikawa, K., Okuyama, H., Purification and characterization of phosphatase inducibly synthesized in Spirodela oligorrhiza grown under phosphate-deficient conditions (1997) Plant Physiology and Biochemistry, 35, pp. 437-446Okamoto, O.K., Pinto, E., Latorre, L.R., Bechara, E.J.H., Colepicolo, P., Antioxidant modulation in response to metal-induced oxidative stress in algal chloroplasts (2001) Archives of Environmental Contamination and Toxicology, 40, pp. 18-24Okamura, H., Mingyu, P., Aoyama, I., Sudo, M., Okubo, T., Nakamura, M., Algal growth inhibition by river water pollutants in the agricultural area around lake Biwa, Japan (2002) Environmental Pollution, 117, pp. 411-419Omar, H.H., Bioremoval of zinc by Scenedesmus obliquus and Scenedesmus quadricauda and its effects on growth and metabolism (2002) International Biodeterioration and Biodegradation, 50, pp. 95-100(1981) Guidelines for testing of chemicals: Alga, growth inhibition test, , ORGANIZATION FOR ECONOMIC COOPERATION AND DEVELOPMENT - OECD, Paris:, OECDOyedele, D.J., Asonugho, C., Awotoye, O.O., Heavy metals in soil and accumulation by edible vegetables after phosphate fertilizer application. Electronic Journal of Environmental (2006) Agricultural and Food Chemistry, 5, pp. 1446-1453Patni, N.J., Aaronson, S., Partial characterization of the intra- and extracellular acid phosphatase of an alga, Ochromonas danica (1974) Journal of General Microbiology, 83, pp. 9-20Pompeu, G.B., Gratão, P.L., Vitorello, V.A., Azevedo, R.A., Antioxidant isoenzyme responses to nickel-induced stress in tobacco cell suspension culture (2008) Scientia Agricola, 65, pp. 548-552Peterson, S.M., Stauber, J.L., New algal enzyme bioassay for the rapid assessement of aquatic toxicity (1996) Bulletin of Environmental Contamination and Toxicology, 56, pp. 750-757Prazeres, J.N., Ferreira, C.V., Aoyama, H., Acid phosphatase activities during the germination of Glycine max seeds (2004) Plant Physiology and Biochemistry, 42, pp. 15-20Rai, L.C., Husaini, Y., Mallick, N., pH altered interaction of aluminium and fluoride on nutrient uptake, photosynthesis and other variables of Chlorella vulgaris (1998) Aquatic Toxicology, 42, pp. 67-84Revoredo, M.D., Melo, W.J., Enzyme activity and microbial biomass in an Oxisol amended with sewage sludge contaminated with nickel (2007) Scientia Agricola, 64, pp. 61-67Rivoal, J., Turpin, D.H., Plaxton, W.C., In vitro phosphorylation of phosphoenolpyruvate carboxylase from the green algae Selenastrum minutum (2002) Plant and Cell Physiology, 43, pp. 785-792Schnackenberg, J., Schulz, R., Senger, H., Characterization and purification of a hidrogenase from eukaryotic green algae Scenedesmus obliquus (1993) FEBS Letters, 327, pp. 21-24Simon, D., Helliwell, S., Extraction and quantification of chlorophyll A from freshwater green algae (1998) Water Research, 32, pp. 2220-2223Strmac, M., Braunbeck, T., Cytological and biochemical effects of a mixture of 20 pollutants on isolated rainbow trout (Oncorhynchus mykiss) hepatocytes (2002) Ecotoxicology and Environmental Safety, 53, pp. 293-304Theodorou, M.E., Elrifi, I.R., Turpin, D.H., Plaxton, W.C., Effect of phosphorus limitation on respiratory metabolism in the green algae Selenastrum minutum (1991) Plant Physiology, 95, pp. 1089-1095Tsekova, K., Galabova, D., Phosphatase production and activity in copper (II) accumulating Rizopus delemar (2003) Enzyme Microbiology and Technology, 33, pp. 926-931Verlecar, X.N., Jena, K.B., Chainy, G.B.N., Modulation of antioxidant defences in digestive gland of Perna viridis (L.), on mercury exposures (2008) Chemosphere, 71, pp. 1977-1985Verma, S.R., Tonk, I.P., Chand, R., In vivo accumulation and effects of mercuric chloride on tissue phosphatases of Notopterus notopterus (1985) Clinical Physiology and Biochemistry, 3, pp. 199-203(1989) Mercury: Environmental aspects, , http://www.inchem.org/documents/ehc/ehc/ehc086.htm, WORLD HEALTH ORGANIZATION - WHO, Geneva: WHO/International Programme on Chemical Safety, (Environmental Health Criteria, 86). Avaliable at, Accessed 12 Nov. 2007Yamamoto, M., Acid phosphatase in Enteromorpha (1972) Phytochemistry, 11, pp. 2451-245