Maria A. Rao

Interaction of acid phosphatase with clays, organic molecules and organo-mineral complexes: kinetics and stability

Abstract The properties of synthetic active enzymatic complexes, simulating those usually present in soil environment, were investigated. Complexes were formed by the interaction of acid phosphatase with clays (montmorillonite and Al hydroxide), tannic acid and organo-mineral aggregates, obtained by mixing tannate, OH-Al species and/or montmorillonite. Immobilized acid phosphatase showed catalytic features quite different from those of the free enzyme. The presence of OH-Al species in the matrix generally resulted in an improvement of some enzymatic properties. A gain in activity of about 45 and 55% was observed for the complexes acid phosphatase–tannate–OH-Al species after thermal deactivation at 60°C and 2 h of exposure to proteinase K. High residual activities ranging from 17 to 61% and from 28 to 57% of the initial one were measured for complexes of the enzyme with inorganic and organic/organo-mineral matrices, respectively. In contrast, the association with a pure constituent such as montmorillonite and/or tannic acid gave rise to an immobilized enzyme, displaying a completely different catalytic behaviour. Compared to the free enzyme, acid phosphatase–montmorillonite and acid phosphatase–tannate complexes had a different pH-activity dependence and a higher and lower sensitivity to temperature and proteolysis, respectively.

Interactions Of Acid Phosphatase With Clays, Organic Molecules And Organo-mineral Complexes1

Synthetic active enzymatic complexes have been formed by the interaction of acid phosphatase with clays (montmorillonite and chlorites), organic molecules, and organo-mineral associations. The inhibition of each component on the activity of the free enzyme and the residual activity of the enzymatic complexes have both been determined. Clays and tannic acid displayed various inhibitory effects on acid phosphatase activity. Montmorillonite and aluminum hydrous oxide showed the highest (70%) and the lowest (<5%) inhibitory influence, respectively. The covering of montmorillonite surfaces with OH-Al species increased not only the amount but also the residual activity of adsorbed acid phosphatase molecules. When 18 meq of Al g -1 clay were adsorbed on montmorillonite surfaces, the residual activity of adsorbed enzyme increased up to 54% of that added initially. The activity retained by tannate-acid phosphatase complexes depended on incubation time and tannic acid/enzyme ratios. This suggests that a complexation between tannic acid and enzyme molecules, rather than a polymerization process of tannic acid involving acid phosphatase, took place. The presence of OH-Al species during the interaction between the two components favored the entrapment of more enzymatic molecules at higher activity levels.

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.

ROLE OF ENZYMES IN THE REMEDIATION OF POLLUTED ENVIRONMENTS

Environmental pollution is growing more and more due to the indiscriminate and frequently deliberate release of hazardous, harmful substances. Research efforts have been devoted to develop new, low-cost, low-technology, eco-friendly treatments capable of reducing and even eliminating pollution in the atmosphere, the hydrosphere and soil environments. Among biological agents, enzymes have a great potentiality to effectively transform and detoxify polluting substances because they have been recognized to be able to transform pollutants at a detectable rate and are potentially suitable to restore polluted environments. This brief review will examine some classes of pollutants and enzymes capable of transforming them effectively into innocuous products. Particular attention will be devoted to pollutants with a high polluting potential such as polyphenols, nitriles, PAHs, cyanides and heavy metals. The enzymatic processes developed and implemented in some of these detoxification treatments will be examined in details. The main advantages as well as the main drawbacks that are still present in the extensive application of enzymes in the in situ restoration of polluted environments will be discussed.

Interactions Between Xenobiotics and Microbial and Enzymatic Soil Activity

In the second half of the twentieth century, the indiscriminate release of xenobiotic chemicals of different chemical and structural complexity into the environment provoked serious and most often irreversible alterations of the natural environmental balance. Indeed, soil contamination by highly toxic compounds has greatly increased, with negative, irreversible effects on soil quality and health. Several chemical, biological, and biochemical soil properties have been profoundly altered, and their main effect has been the continuous loss of soil functions in sustaining the survival of living organisms. Among chemical pollutants, compounds like pesticides and polycyclic aromatic hydrocarbons arrive to the soil from different anthropic sources and have high toxicity toward humans, plants, and animals. Assessing the soil quality is a basic requirement for sustainable land use. Soil microbial and biochemical activities are sensitive to several natural and human-induced changes and may provide a helpful tool to assess soil status, its quality, and its productivity. This article is a survey of the mutual interactions establishing in a soil among xenobiotic substances with particular reference to pesticides and polycyclic aromatic hydrocarbons and microbial and enzymatic soil activities.

Oxidative catalysts for the transformation of phenolic pollutants: a brief review

Phenolics are often produced as wastes by several industrial and agricultural activities. Many of these compounds and their derivatives are extremely dangerous to living organisms, because they are highly toxic and thus represent a serious environmental concern. Conventional remediation methods of phenol-polluted systems have some disadvantages due to high cost, time-consuming procedures and formation of toxic residues. Conversely, the use of oxidative catalysts, both enzymatic or inorganic, is a promising alternative technology to address the clean up of such wastes. Oxidative enzymes and inorganic compounds, both naturally occurring in soil, behave as biotic and abiotic catalysts and support the transformation of phenolic compounds. The complete mineralization of phenolic pollutants as well as the formation of polymeric products, often less toxic than their precursors, may occur. The present paper gives a brief review of many aspects concerning the properties of biotic and abiotic catalytic agents effective in the transformation of phenolic compounds. The main mechanisms of the processes as well as their feasibility for catalytic practical applications will be addressed. Examples of their potentiality in the detoxification of phenol-polluted systems will be provided, as well.

Adsorption, activity and kinetic properties of urease on montmorillonite, aluminium hydroxide and AL(OH)x-montmorillonite complexes

Abstract The adsorption of urease on a montmorillonite (M), a non-crystalline aluminium hydroxide (AL) and an Al(OH) x -montmorillonite complex (AM) as well as the activity, the kinetics and the stability of the enzyme-clay mineral complexes were studied. The equilibrium adsorption isotherms of urease on clay minerals fitted both the Langmuir or the Freundlich equations. The Langmuir adsorption isotherm of the enzyme on M was of H type (“high affinity”) whereas the isotherms on AL and AM were of L type (“Langmuir”). On adding up to 21.2mg of enzyme g −1 clay, the amount of urease held on the clay minerals followed the order M > AM > AL throughout the pH range explored (4.0–9.0). The adsorption of urease on M, AM and AL was differently affected by pH. The specific activity of enzyme immobilized on M and AM was relatively high (71 and 64% respectively) as compared to that of the free enzyme; in contrast the specific activity of urease adsorbed on AL was considerably reduced (15%). The free and immobilized urease showed similar pH- and temperature-activity profiles and both states obeyed Michaelis-Menten kinetics. The V max and K m parameters, as well as the thermal stability of adsorbed urease were always lower than those of the free urease, whereas the proteolytic stability of urease held on AL was higher than that of the enzyme free or adsorbed on M and AM. Finally, it was ascertained that the covering of the surfaces of montmorillonite with different amounts of OH-A1 species reduced the quantity as well as the activity of adsorbed enzyme.

Invertase β-fructosidase): Effects of montmorillonite, AL-hydroxide and AL(OH)x-montmorillonite complex on activity and kinetic properties

Abstract The effects of a montmorillonite (M), a non-crystalline aluminium hydroxide (AL) and an Al(OH)2-montmorillonite complex (a montmorillonite partially coated with OH-Al ions; AM) on the adsorption, residual activity and kinetic properties of invertase were determined. In the range of pH 3.0–9.0 the ability of the clay minerals to adsorb protein molecules was in the order M > AM > AL and was influenced by the nature of the buffer solution. Adsorption resulted in a significant reduction of invertase activity (more than 85% at pH 4.65). the extent of which depended on clay mineral (AL > AM > M) and on pH. The free and immobilized enzyme showed maximum activity in the range of pH 4.5–5.5 and both states obeyed Michaelis-Menten kinetics. The Vmax, and Km values indicated a partial non-competitive inhibition of the clay minerals. Invertase adsorbed on AM and AL was more stable to thermal deactivation than was free enzyme, whereas invertase held on M was much less stable. In contrast, invertase adsorbed on M was more stable to the action of protease than free enzyme or that adsorbed on AM or AL.

Oxidative Transformation of Natural and Synthetic Phenolic Mixtures by Trametes versicolor Laccase

The efficiency of Trametes versicolor laccase in the transformation of phenols (caffeic acid, catechol, hydroxytyrosol, methylcatechol, protocatechuic acid, syringic acid, m-tyrosol, 3-hydroxybenzoic acid, 3-hydroxyphenylacetic acid, 2,6-dihydroxybenzoic acid, 4-hydroxybenzaldehyde) usually present in waste water, such as that derived from an olive oil factory, was investigated. According to their response to 24 h laccase action the 11 phenolic compounds were classified in three groups: reactive (88-100% transformation), intermediate reactive (transformation lower than 50%), and recalcitrant (not transformed at all). The enzyme was able to transform the 11 substrates even when they were present in a mixture and also toward a phenolic extract from a Moroccan olive oil mill waste water (OMW) sample. The disappearance of protocatechuic, 3-hydroxyphenylacetic, and 2,6-dihydroxybenzoic acids, and 4-hydroxybenzaldehyde was enhanced whereas that of caffeic acid and m-tyrosol was depressed when the phenols were present in the mixture. A reduction of enzyme activity occurred in single and/or complex phenolic mixtures after enzymatic oxidation. No correspondence between phenol transformation and disappearance of enzymatic activity was, however, observed. The overall results suggest that laccases are effective in the transformation of simple and complex phenolic mixtures.

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.