Enzymes as useful tools for environmental purposes.

In the environment enzymes may play important and different roles at least in three cases: as main agents (as isolated, cell-bound or immobilized enzymes) in charge of either the transformation and/or degradation of compounds polluting the environment and the restoration of the polluted environment; as reliable and sensitive tools to detect and measure the amount and concentration of pollutants before, during and after the restoration process; as reliable, easy and sensitive indicators of quality and health status of the environment subjected to the restoration process. To our knowledge papers or reviews integrating findings on these three functions of enzymes are missing in literature. Therefore the main scope of the present paper is to briefly encompass general and specific concepts about roles of enzymes as decontaminating agents, pollutant assaying agents and indicators of environment safety. Examples chosen among those published very recently, supporting and confirming peculiarities, features, and performance of enzymatic agents will be illustrated.

Bioremediation of a Chilean Andisol contaminated with pentachlorophenol (PCP) by solid substrate cultures of white-rot fungi.

This study provides a first attempt investigation of a serie of studies on the ability of Anthracophyllum discolor, a recently isolated white-rot fungus from forest of southern Chile, for the treatment of soil contaminated with pentachlorophenol (PCP) to future research on potential applications in bioremediation process. Bioremediation of soil contaminated with PCP (250 and 350 mg kg⁻¹ soil) was investigated with A. discolor and compared with the reference strain Phanerochaete chrysosporium. Both strains were incorporated as free and immobilized in wheat grains, a lignocellulosic material previously selected among wheat straw, wheat grains and wood chips through the growth and colonization of A. discolor. Wheat grains showed a higher growth and colonization of A. discolor, increasing the production of manganese peroxidase (MnP) activity. Moreover, the application of white-rot fungi immobilized in wheat grains to the contaminated soil favored the fungus spread. In turn, with both fungal strains and at the two PCP concentrations a high PCP removal (70-85%) occurred as respect to that measured with the fungus as free mycelium (30-45%). Additionally, the use of wheat grains in soil allowed the proliferation of microorganisms PCP decomposers, showing a synergistic effect with A. discolor and P. chrysosporium and increasing the PCP removal in the soil.

Degradation of polycyclic aromatic hydrocarbons by free and nanoclay-immobilized manganese peroxidase from Anthracophyllum discolor.

Manganese peroxidase (MnP) produced by Anthracophyllum discolor, a Chilean white rot fungus, was immobilized on nanoclay obtained from volcanic soil and its ability to degrade polycyclic aromatic hydrocarbons (PAHs) compared with the free enzyme was evaluated. At the same time, nanoclay characterization was performed. Nanoclay characterization by transmission electronic microscopy showed a particle average size smaller than 100 nm. The isoelectric points (IEP) of nanoclay and MnP from A. discolor were 7.0 and 3.7, respectively, as determined by micro electrophoresis migration and preparative isoelectric focusing. Results indicated that 75% of the enzyme was immobilized on the nanoclay through physical adsorption. As compared to the free enzyme, immobilized MnP from A. discolor achieved an improved stability to temperature and pH. The activation energy (Ea) value for immobilized MnP (51.9 kJ mol(-1)) was higher than that of the free MnP (34.4 kJ mol(-1)). The immobilized enzyme was able to degrade pyrene (>86%), anthracene (>65%), alone or in mixture, and to a less extent fluoranthene (<15.2%) and phenanthrene (<8.6%). Compared to free MnP from A. discolor, the enzyme immobilized on nanoclay enhanced the enzymatic transformation of anthracene in soil. Overall results indicate that nanoclay, a carrier of natural origin, is a suitable support material for MnP immobilization. In addition, immobilized MnP shows an increased stability to high temperature, pH and time storage, as well as an enhanced PAHs degradation efficiency in soil. All these characteristics may suggest the possible use of nanoclay-immobilized MnP from A. discolor as a valuable option for in situ bioremediation purposes.

Dephenolization and detoxification of olive-mill wastewater (OMW) by purified biotic and abiotic oxidative catalysts.

The capability of two oxidative catalysts, a laccase from Rhus vernicifera and birnessite, a manganese oxide, in the dephenolization and detoxification of two olive-mill wastewater (OMW) samples, C1 and C2, differing for complexity and composition, was evaluated. OMW phenolic extracts (EC1 and EC2) and mono-substrate solutions of phenols mostly present in OMW samples were also tested. Birnessite was more effective than laccase in removing the phenolic content from mono-substrate solutions (more than 70% of each initial phenolic concentration) and of either OMW samples or EC1 and EC2 extracts. For instance, 60% of the total phenolic content of EC1 was removed after 48-h treatment with 5 mg mL(-1) birnessite and the efficiency was lower as greater was the complexity of the OMW sample (only 17% removal from EC2 over the same time span). Phytotoxicity tests with Lepidium sativum and Lycopersicon esculentum seeds and antibacterial toxicity tests with Bacillus megaterium were performed on crude OMW samples and their extract and exhausted fractions before and after the catalytic treatment. Results demonstrated that (a) monomeric phenols were certainly but not exclusively responsible of OMW phytotoxicity, whereas their removal led to a quite complete elimination of the toxicity toward bacterial growth; (b) other components not removable by the oxidative catalysts very likely contribute to OMW phytotoxicity; and (c) the choice of the vegetal species to use in toxicity tests might be crucial for correct and easily interpretable results. Overall the results provided useful information on the possible use of oxidative catalysts for the efficient treatment of complex aqueous wastes such as those deriving from olive industry.

Impact of river overflowing on trace element contamination of volcanic soils in south Italy: part I. Trace element speciation in relation to soil properties.

Volcanic soils affected by different numbers of polluted river flooding events were investigated. Chromium and Cu were the major soil contaminants. Nickel, Fe, Zn and Mn total content never exceeded the Italian mandatory limits. The distribution of Cr and Cu total contents among studied soils indicated that only Cr contamination was related to overflowing events. In polluted soils, sequential chemical extractions revealed a preferential association of Cr and Cu with organic forms. A progressive Cr insolubilization with ageing was observed. Significant amounts of Cr and Cu were extracted by NH(4)-oxalate, suggesting metals association with short-range-order aluminosilicates and organo-mineral complexes. Possible methodological drawbacks in the use of the EU-BCR chemical speciation protocol on volcanic soils are discussed. Micromorphology and SEM/WDS analyses revealed Cr and Cu enriched silt and clay coatings in surface and subsurface soil horizons, suggesting a transfer of metal-rich sediments along the soil pore network with water movement.

Impact of river overflowing on trace element contamination of volcanic soils in south Italy: part II. Soil biological and biochemical properties in relation to trace element speciation.

The effect of heavy metal contamination on biological and biochemical properties of Italian volcanic soils was evaluated in a multidisciplinary study, involving pedoenvironmental, micromorphological, physical, chemical, biological and biochemical analyses. Soils affected by recurring river overflowing, with Cr(III)-contaminated water and sediments, and a non-flooded control soil were analysed for microbial biomass, total and active fungal mycelium, enzyme activities (i.e., FDA hydrolase, dehydrogenase, beta-glucosidase, urease, arylsulphatase, acid phosphatase) and bacterial diversity (DGGE characterisation). Biological and biochemical data were related with both total and selected fractions of Cr and Cu (the latter deriving from agricultural chemical products) as well as with total and extractable organic C. The growth and activity of soil microbial community were influenced by soil organic C content rather than Cu or Cr contents. In fact, positive correlations between all studied parameters and organic C content were found. On the contrary, negative correlations were observed only between total fungal mycelium, dehydrogenase, arylsulphatase and acid phosphatase activities and only one Cr fraction (the soluble, exchangeable and carbonate bound). However, total Cr content negatively affected the eubacterial diversity but it did not determine changes in soil activity, probably because of the redundancy of functions within species of soil microbial community. On the other hand, expressing biological and biochemical parameters per unit of total organic C, Cu pollution negatively influenced microbial biomass, fungal mycelium and several enzyme activities, confirming soil organic matter is able to mask the negative effects of Cu on microbial community.

Bacterial communities and enzyme activities of PAHs polluted soils.

Three soils (i.e. a Belgian soil, B-BT, a German soil, G, and an Italian agricultural soil, I-BT) with different properties and hydrocarbon-pollution history with regard to their potential to degrade phenanthrene were investigated. A chemical and microbiological evaluation of soils was done using measurements of routine chemical properties, bacterial counts and several enzyme activities. The three soils showed different levels of polycyclic aromatic hydrocarbons (PAHs), being their contamination strictly associated to their pollution history. High values of enzyme activities and culturable heterotrophic bacteria were detected in the soil with no or negligible presence of organic pollutants. Genetic diversity of soil samples and enrichment cultures was measured as bands on denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA sequences from the soil and enrichment community DNAs. When analysed by Shannon index (H'), the highest genetic biodiversity (H'=2.87) was found in the Belgian soil B-BT with a medium-term exposition to PAHs and the poorest biodiversity (H'=0.85) in the German soil with a long-term exposition to alkanes and PAHs and where absence, or lower levels of enzyme activities were measured. For the Italian agricultural soil I-BT, containing negligible amounts of organic pollutants but the highest Cu content, a Shannon index=2.13 was found. The enrichment of four mixed cultures capable of degrading solid phenanthrene in batch liquid systems was also studied. Phenanthrene degradation rates in batch systems were culture-dependent, and simple (one-slope) and complex (two-slope) kinetic behaviours were observed. The presence of common bands of microbial species in the cultures and in the native soil DNA indicated that those strains could be potential in situ phenanthrene degraders. Consistent with this assumption are the decrease of PAH and phenanthrene contents of Belgian soil B-BT and the isolation of phenanthrene-degrading bacteria. From the fastest phenanthrene-degrading culture C(B-BT), representative strains were identified as Achromobacter xylosoxidans (100%), Methylobacterium sp. (99%), Rhizobium galegae (99%), Rhodococcus aetherovorans (100%), Stenotrophomonas acidaminiphila (100%), Alcaligenes sp. (99%) and Aquamicrobium defluvium (100%). DGGE-profiles of culture C(B-BT) showed bands attributable to Rhodococcus, Achromobacter, Methylobacterium rhizobium, Alcaligenes and Aquamicrobium. The isolation of Rhodococcus aetherovorans and Methylobacterium sp. can be consistent with the hypothesis that different phenanthrene-degrading strategies, cell surface properties, or the presence of xenobiotic-specific membrane carriers could play a role in the uptake/degradation of solid phenanthrene.

Oxidative transformation of phenols in aqueous mixtures.

The transformation by an oxidoreductase (a laccase from Rhus vernificera) of a mixture of four phenols (catechol, methylcatechol, m-tyrosol and hydroxytyrosol) that simulates a typical wastewater derived from an olive oil factory was investigated. Results achieved in this study confirm that laccase-mediated transformation of phenols depends on the nature and the initial concentration of the involved phenol, the time course of the reaction, and mainly, on the complexity of the phenolic incubation mixture. Actually, the four phenols each have a completely different response to enzyme action both in terms of quantitative and kinetic transformation. For example, after 24-h incubation, methylcatechol was completely removed, whereas 30% of untransformed hydroxytyrosol and catechol and more than 65% of m-tyrosol were still present in the reaction mixture. A reduction of enzyme activity occurred for all phenols after enzymatic oxidation. No correspondence between phenol transformation and disappearance of enzymatic activity was observed, thus suggesting that different mechanisms are probably involved in the laccase-mediated transformation of the four phenols. The behavior of phenols became more complex when an increasing number of phenols was present in the reaction mixture, and even more so when different concentrations of phenols were used. Competitive effects may arise when more than one phenol is present in the reaction solution and interacts with the enzyme.

Enzymatic oxidative transformation of chlorophenol mixtures.

Chlorinated phenols are major industrial and agricultural xenobiotics that pollute soil and ground water. It has been shown that laccases catalyze the oxidative coupling of phenolic compounds. Therefore, the transformation of one or a mixture of several chlorinated phenols by a laccase from the fungus Trametes villosa was studied. Generally, if more than one phenol was added, the transformation of chlorinated phenols decreased, and if the concentration of the laccase was increased, the transformation of the phenols was enhanced. There were exceptions to these observations: for instance, the transformation of 0.1 mM 4-chlorophenol incubated with 1 mM 2,4-dichlorophenol in buffered salt solutions was not enhanced if the concentration of the laccase was increased from 2 to 20 DMP units/mL. The reason for the reduced transformation of chlorinated phenols in the presence of additional phenols is still unknown. However, in spite of some limitations, the application of laccase to decontaminate wastewater polluted with chlorinated phenols appears feasible.

Pesticide influence on soil enzymatic activities.

The influence of four pesticides, e.g. glyphosate, paraquat, atrazine, and carbaryl, on the activities of invertase, urease and phosphatase of twenty-two soils, numbered as 1-22, was investigated. Soils displayed a general variability of enzyme activities with invertase being more abundant than urease and phosphatase in the order listed. The addition of glyphosate and paraquat activated invertase and urease activities in several soils. Increments of invertase activity ranged from a very low increase (+4%) up to +204% in soils 11 and 14, respectively. Smaller increases were measured for urease. A general inhibitory effect (from 5% to 98%) was observed for phosphatase in the presence of glyphosate. The effects of atrazine and carbaryl on the three soil enzymes were evaluated against that exhibited by methanol, the solvent used for their solubilization. In almost all soils, atrazine further inhibited invertase activity with respect to the inhibitory effect shown by methanol. By contrast, consistent activation effects (from 61% to 10217%) were measured for urease with methanol alone and/or methanol-pesticide mixtures. Contradictory results were observed with phosphatase. Similarities found between the results obtained with enzymes in soils and those measured with synthetic enzyme complexes (e.g. free enzymes and/or clay-, organo-, and organo-clay-enzyme complexes) exposed to the same pesticides allowed some relationships between responses of soil enzymes to pesticides and soil properties to be hypothesized.

Bioavailability of 2,4-D sorbed to a chlorite-like complex.

An Al(OH)x-montmorillonite (chlorite) complex (AM18) was prepared and 2,4-dichlorophenoxyacetic acid (2,4-D) sorbed to saturation. After several washing cycles the 'strongly sorbed' 2,4-D was 507 micrograms g-1 AM18. The bioavailability of sorbed 2,4-D was assessed in a minimal salts medium with the AM18-2,4-D as the sole C and energy source. Over a 28-day period a Pseudomonas sp. degraded 23% more of the sorbed 2,4-D than could be accounted for by desorption from AM18 in the non-inoculated controls. Possible explanations for the increase in bioavailability are presented.

The effect of sorbitol on acid phosphatase deactivation.

Acid phosphatase thermal deactivation follows a complex path: an initial decay toward an equilibrium distribution of at least two intermediate structures, mutually at the equilibrium, followed by a final breakdown toward a completely inactive enzyme configuration. The results obtained in the presence of sorbitol have been compared to those produced in the course of purely thermal deactivation of the native enzyme. For any sobitol concentration, an equivalent temperature is calculated that results in exactly the same activity-versus-time profile. This suggests enzyme deactivation to be controlled by a single, unchanging step. Immobilized enzyme runs have been performed, as well, by entrapping acid phosphates within a polymeric network formed onto the upstream surface of an ultrafiltration membrane. The stabilizing effect of entrapment cumulates with that produced by sorbitol. In this case, however, an equivalent temperature cannot be determined, thus indicating that a different deactivation mechanism is followed.

Enzyme stabilization: state of the art.

'Enzyme stabilization' is one of the most important fields in basic and applied enzymology. In basic enzymology, it is of particular relevance to understand enzyme stabilization principles first elucidating how and why the enzymes lose their biological activity and then deriving structure-stability relationships existing in enzymatic molecules. In applied enzymology, the most significant goal is to achieve useful compounds by biocatalysis. Enzymes are good catalysts in terms of high catalytic and specific activity with ability to function under mild conditions. However, they are not always ideal catalysts for practical applications because they are generally unstable and they inactivate rapidly through several mechanisms. In order to enhance enzyme stability, many strategies have been pursued in recent years. The present article is an attempt to provide detailed information about these strategies.

New experimental technique for detecting the effect of low-frequency electric fields on enzyme structure.

A new experimental approach has been developed to determine kinetic and thermodynamic parameters of the inactivation of an enzyme under labile conditions both with and without exposure to electrical currents as sources of perturbation. Studies were undertaken to investigate if low-frequency electric currents can accelerate the thermal inactivation of an enzyme through interactions with dipole moments in enzymatic molecules and through related mechanical stresses. The experiments were conducted with the enzyme acid phosphatase. The enzyme was exposed to a 50-Hz current at different densities (10 to 60 mA/cm2 rms) or to a sinusoidal or square-wave current at an average density of 3 mA/cm2 and frequencies from, respectively, 50 Hz to 20 kHz and 500 pulses per second (pps) to 50,000 pps. Positive-control experiments were performed in the presence of a stabilizer or a deactivator. The results indicate that the technique is sensitive to conformational changes that otherwise may be impossible to detect. However, exposure to electric currents under the experimental conditions described herein showed no effects of the currents.

Deactivation of free and stabilized acid phosphatase by urea.

Tests on acid phosphatase (E.G. 3.1.3.2) deactivation by urea have been performed at two pH values. Two conditions have been used: native enzyme operating batch-wise in dilute solution and stabilized enzyme in continuous flow ultrafiltration membrane reactor. Stabilization is achieved by confining the enzyme within a concentrated solution of a linear chain polymer that forms a polarization layer over the membrane. The results provide significant information on the kinetics and thermodynamics of the complex phenomena taking place during deactivation. Deactivation by urea is also compared with thermal deactivation.

Series mechanism of enzyme deactivation. Characterization of intermediate forms.

Acid phosphatase (E.C. 3.1.3.2) undergoes complex thermal deactivation phenomena, as revealed by the two-slope pattern of the enzyme logarithmic-specific-activity versus time curves. The native enzyme first decays toward an equilibrium distribution of less, but still active, intermediate structures and these, in turn, undergo a final degradation to a completely inactive form. The effect of the experimental conditions at which the enzyme is kept during the deactivation process on the characteristics of these intermediate enzymatic structures has been investigated. The kinetic parameters of p-nitro-phenyl phosphate hydrolysis, as catalyzed by some of these intermediate forms, have been determined and the results compared to those obtained with the native enzyme.

Acid phosphatase deactivation by a series mechanism.

Acid phosphatase (E.C.3.1.3.2.) thermal deactivation at pH 3.77 has been investigated by monitoring the enzyme activity as a function of time in the hydrolysis of p-nitrophenyl phosphate. The experimental curves obtained show a two-slope behavior in a log (activity)versus-time plot, which indicates that deactivation occurs via a complex mechanism. From the dependence of the kinetic parameters on both deactivation and hydrolysis temperatures, it is inferred that the deactivation mechanism involves intermediate, temperature-dependent, less-active forms of the enzyme. This interpretation is confirmed by the results of additional tests in which the temperature was suddenly changed during the deactivation process.

Enzyme immobilization by means of ultrafiltration techniques.

Unstirred, plane membrane, ultrafiltration cells have been used as enzymatic reactor units. Because of the concentration polarization phenomena which take place in the system, at steady-state the enzyme is confined (dynamically immobilized) within an extremely narrow region upstream the ultrafiltration membrane. Correspondingly its concentration attains fairly high values. Kinetic studies have been therefore performed under quite unusual experimental conditions in order to better approximate local enzyme concentration levels in immobilized enzyme systems. Studies have been also carried out on the kinetics of enzyme deactivation in the continuous presence of substrate and reaction products. Once the enzyme concentration profile is completely developed, further injection into the system of suitable amounts of an inert proteic macromolecule (albumin polymers) gives rise to the formation of a gel layer onto the ultrafiltration membrane within which the enzyme is entrapped (statically immobilized). The effect of this immobilization technique has been studied as far as the kinetics of the main reaction, the substrate mass transfer resistances and the enzyme stability are concerned. The rejective properties of such gel layers towards enzymatic molecules have been exploited in producing multilayer, multi-enzymatic reactors.

Theoretical and experimental analysis of a soluble enzyme membrane reactor.

Recently enzyme immobilization techniques have been proposed that are mainly founded on the formation of an enzyme-gel layer onto the active surface of an ultrafiltration membrane within an unstirred ultrafiltration cell. If the membrane molecular-weight cutoff is less than the enzyme molecular weight and hence such as to completely prevent enzyme permeation (once the enzyme solution has been charged into the test cell and pressure applied to the system), a time progressive increase in enzyme concentration takes place at the upstream membrane surface that can eventually lead to gelation and hence to enzyme immobilization. However, depending on the total enzyme amount fed, the maximum enzyme concentration achieved in the unsteady state could be less than the gelation level. In this situation, no immobilization occurs and the enzyme still remains in the soluble form although it is practically confined within a limited region immediately upstream the membrane and at fairly high concentrations. In this paper, the experimental conditions that allow gelling to occur are discussed together with a theoretical analysis of the soluble enzyme membrane reactor which is obtained when no gelling takes place. Such a system could be usefully employed in performing kinetic analyses at high enzyme concentration levels that are still in the soluble form.