Extraction, partial characterization and susceptibility to Hg2+ of acid phosphatase from the microalgae Pseudokirchneriella subcapitata

Claudio Martín Jonsson,Hiroshi Aoyama

doi:10.1590/s0103-90162009000500008

Claudio Martín Jonsson, Hiroshi Aoyama

Open Access

https://doi.org/10.1590/s0103-90162009000500008

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Abstract

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 -20ºC 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+.

Highlights

Pseudokirchneriella subcapitata is a chlorophyceae algae present in freshwater and soil, which is widely used in studies of contamination by agriculture pollutants (Keddy et al, 1995; Baun et al, 2002; Okamura et al, 2002)
We compared some methods of extraction of acid phosphatase from P. subcapitata and we investigated some aspects of the enzyme activity focusing on storage stability, pH range, substrate specificity and kinetic parameters
The results demonstrated an increase of about 4-fold in the enzymatic activity in the samples submitted to freezing (N2)/maceration or probe sonication in relation to those submitted to ultra-sound water bath

Summary

Introduction

Pseudokirchneriella subcapitata (formely Selenastrum capricornutum) is a chlorophyceae algae present in freshwater and soil, which is widely used in studies of contamination by agriculture pollutants (Keddy et al, 1995; Baun et al, 2002; Okamura et al, 2002). The alteration of phosphatase activities by chemical pollutants such as heavy metals has been used as a biomarker in risk assessment and biomonitoring (El Demerdash & Elagamy, 1999; Strmac & Braunbeck, 2002). In this context, in vitro and in vivo changes on aquatic and soil acid and alkaline phosphatases activities by contaminants have been reported (Verma et al, 1985; El Demerdash & Elagamy, 1999; Jonsson & Aoyama, 2007; Revoredo & Melo, 2007). Up to now, there are few references reporting the extraction, characterization and sensibility to pollutants of acid phosphatase in relevant primary producers with cosmopolitan distribution and their use as test organisms in ecotoxicity risk assessment (OECD, 1981; CETESB, 1992)

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