ABSTRACT
Transgenic hairy root (HR) systems constitute an interesting alternative to improve the efficiency of phytoremediation process. Since peroxidases (Px) have been associated with phenolic compounds removal, in the present work, transgenic tobacco HR, which expressed basic Px genes from tomato (tpx1 and tpx2), were established and assayed for phenol removal. Tobacco HR clones were obtained, including those transgenic for TPX1 or TPX2, those double transgenic (DT) for both Px and the corresponding controls. Based on growth index, the presence of rol C sequence, tpx1 and/or tpx2 genes and the coded proteins, as well as Px activity determinations, we selected 10 tobacco HR clones for phenol removal assays. The removal efficiencies were high for all the HR, although, some transgenic HR showed significantly higher removal efficiencies compared with controls. The results demonstrate that TPX1 is involved in phenol removal not only when it was overexpressed in tomato, but also when it was expressed in other plant, such as tobacco. The higher efficiency of TPX2 transgenic HR showed that this Px also participates in the process. The contribution of other mechanisms (adsorption, H2O2 independent enzymatic processes) could be considered depreciable, which establishes the great implication of Px in phenol removal.
Subject(s)
Biodegradation, Environmental , Nicotiana/enzymology , Peroxidases/metabolism , Phenols/metabolism , Plant Roots/enzymology , Plants, Genetically Modified/enzymology , Data Interpretation, Statistical , Isoenzymes/genetics , Isoenzymes/metabolism , Solanum lycopersicum/enzymology , Solanum lycopersicum/genetics , Peroxidases/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Roots/genetics , Plant Roots/growth & development , Plant Roots/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/growth & development , Plants, Genetically Modified/metabolism , Soil Pollutants/metabolism , Nicotiana/genetics , Nicotiana/growth & development , Nicotiana/metabolismABSTRACT
We have obtained hairy root cultures of Brassica napus with high biomass and genetic stability which produce peroxidases, enzymes involved in biodegradation processes. In this work, these hairy root cultures were used to study the removal of 2,4-dichlorophenol (2,4-DCP), a common contaminant in industrial effluents that is highly toxic for human and aquatic life. The optimum conditions to obtain high efficiency in the removal process were established. Roots were able to remove 2,4-DCP from aqueous solutions containing 100-1000 mg/l, in the presence of H(2)O(2) concentrations ranging from 5 to 10 mM. After a short period of incubation (15 min), high removal efficiencies were achieved (91-94%) and maximal removal, of approx. 97-98%, was obtained with 1 h of reaction. High removal efficiencies (93-95%) were observed in a broad pH range (pH 3-9), reaching 98-99% in the range pH 4-8. Moreover, roots could be re-used, almost for six consecutive cycles, to remove 2,4-DCP. The oxidation catalysed by peroxidases would be the main mechanism involved in this process. The results suggest that these cultures could be useful tools for phytoremediation.
Subject(s)
Brassica napus/metabolism , Chlorophenols/pharmacokinetics , Environmental Pollution/prevention & control , Peroxidase/metabolism , Plant Roots/metabolism , Biodegradation, Environmental , Brassica napus/drug effects , Brassica napus/enzymology , Culture Techniques , Hydrogen Peroxide/pharmacology , Plant Roots/drug effects , Plant Roots/enzymology , Soil Pollutants/metabolism , Water Pollutants, Chemical/metabolismABSTRACT
We have purified various peroxidase isoenzymes from roots and hairy-root cultures of turnip (Brassica napus) which could potentially be used for commercial applications such as an enzyme immunoassays, diagnostic test kits, wastewater treatment and soil remediation. One of them, a basic peroxidase called HR2, was secreted into the medium of turnip hairy-root cultures. HR2 had a pI of 9.6, a molecular mass of 39.3 kDa and showed great thermostability. The inactivation of HR2 by H2O2 in the absence of reductant substrates was studied. Under these conditions H2O2 acted as a suicide substrate. The kinetic constants calculated have been compared with those of a basic isoperoxidase from horseradish (Armoracia sp.) roots (HRP-C), which is commonly used in commercial kits. The results for HR2 indicated that it was more resistant to inactivation because it presented a lower inactivation efficiency and a higher value for the partition ratio (r=1250) than those described for HRP-C. These results make turnip peroxidase HR2 suitable for use in systems in which high H2O2 concentrations are found. Such an application is demonstrated, namely an enzymic diagnostic kit for determination of uric acid in which HR2 was found to be as efficient as the enzyme originally included in standard kits.
