Liliana Gianfreda

Applications of laccases and tyrosinases (phenoloxidases) immobilized on different supports: a review

This review summarizes all the research efforts that have been spent to immobilize laccase and tyrosinase for various applications, including synthetic and analytical purposes, bioremediation, wastewater treatment, and must and wine stabilization. All immobilization procedures used in these areas are discussed. Considerations on the efficacy of immobilized copper oxidases and products, in addition to their kinetic parameters are also discussed. The available data indicate that the immobilization of laccase into cationic polymer cross-linked with epichlorohydrin appears to be a promising procedure for industrial applications. The development of laccase and tyrosinase-based biosensors to monitor a wide range of compounds appears to be at a mature stage of technology.

Bioremediation and monitoring of aromatic-polluted habitats

Bioremediation may restore contaminated soils through the broad biodegradative capabilities evolved by microorganisms towards undesirable organic compounds. Understanding bioremediation and its effectiveness is rapidly advancing, bringing available molecular approaches for examining the presence and expression of the key genes involved in microbial processes. These methods are continuously improving and require further development and validation of primer- and probe-based analyses and expansion of databases for alternative microbial markers. Phylogenetic marker approaches provide tools to determine which organisms are present or generally active in a community; functional gene markers provide only information concerning the distribution or transcript levels (deoxyribonucleic acid [DNA]- or messenger ribonucleic acid [mRNA]-based approaches) of specific gene populations across environmental gradients. Stable isotope probing methods offer great potential to identify microorganisms that metabolize and assimilate specific substrates in environmental samples, incorporating usually a rare isotope (i.e., 13C) into their DNA and RNA. DNA and RNA in situ characterization allows the determination of the species actually involved in the processes being measured. DNA microarrays may analyze the expression of thousands of genes in a soil simultaneously. A global analysis of which genes are being expressed under various conditions in contaminated soils will reveal the metabolic status of microorganisms and indicate environmental modifications accelerating bioremediation.

Enzyme stabilization: state of the art

Abstract‘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.

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.

Dephenolisation of olive mill waste-waters by olive husk

Olive mill waste-waters (OMWW) are a by-product of olive-oil production and a major environmental problem in the Mediterranean area. The use of a polyphenol-oxidase from olive husk is proposed for the enzymatic removal of low-molecular phenolics in OMWW. For comparison purposes, a purified microbial polyphenol-oxidase (Trametes versicolor laccase) was also employed. Though both enzymatic systems show relevant activity towards phenol polymerization, olive husk is much more appealing in view of possible applications because of its availability and extremely low cost, associated with excellent enzymatic activity and specificity.