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Enzyme and Microbial Technology 35 (2004) 339–354

Potential of extra cellular enzymes in remediation
of polluted soils: a review
Liliana Gianfreda∗ , Maria A. Rao
Dipartimento di Scienze del Suolo, della Pianta e dell’Ambiente, Università di Napoli Federico II, via Università 100, 80055 Portici, Napoli, Italy
Received 26 February 2004; accepted 31 May 2004

The use of microorganisms (bioremediation) and plants (phytoremediation) to remediate polluted environments is a promising and growing area of environmental biotechnology. The first productive step in cell-transformation of xenobiotic substances, including recalcitrant
and even plastic materials, is usually catalyzed by ecto and extra cellular enzymes, released by the cells in their nearby environment. Extra
cellular enzymes include a large range of oxidoreductases and hydrolases. Both these enzymes may explicate a degradative function and
transform polymeric substances into partially degraded or oxidized products that can be easily up-taken by cells. The presence of the whole
cell and its metabolic pathways to be completed are required.
The use of extra cellular and/or cell-free enzymes has been also proposed as an innovative remediation technique. They can offer some
advantages over the use of microbial cells. Several bottlenecks, however, still restrict their practical application in the recovery of polluted
A brief survey of many aspects dealing with the characteristics and potential abilities of both cell-present- and cell-free extra cellular
enzymes is provided. The feasibility of their application for bioremediation of polluted soils has been briefly addressed, as well.
© 2004 Elsevier Inc. All rights reserved.
Keywords: Extra cellular enzymes; Oxidoreductases; Hydrolases; Polluted soils; Remediation feasibility

1. Introduction
The environment is continuously polluted by a large array of hazardous chemicals with different structures and
different toxicity levels that are released from several anthropogenic sources. Three main sources of pollution can
be identified: industrial activities, munitions waste and agricultural practices (Fig. 1). The explosive development of
chemical industries has produced a large variety of chemical
compounds that include pesticides, fuels, solvents, alkanes,
polycyclic aromatic hydrocarbons (PAHs), explosives, dyes
and more.
Although these compounds have contributed to modernize our lifestyle, several of them may accumulate in soil,
water and air. They are highly persistent and adversely affect human and environmental health, because of their carcinogenic and mutagenic effects [1].
The environment has, however, a unique innate capability
to resist pollution and remediate itself. Indeed, several processes may occur in the contaminated place itself and natu∗ Corresponding author. Tel.: +39 081 2539179/2539166 (lab);
fax: +39 081 2539186.
E-mail address: (L. Gianfreda).

0141-0229/$ – see front matter © 2004 Elsevier Inc. All rights reserved.

rally attenuate the pollution [2]. Natural attenuation includes
chemical, physical and biological processes such as dispersion, sorption, volatilization, abiotic oxidation, hydrolysis
and biodegradation or intrinsic bioremediation.
Among these natural processes, abiotic oxidation, hydrolysis and biodegradation are the only effective attenuation
mechanisms, since they are capable to destroy the contaminants and transform them into innocuous end products.
When the migration rate of contaminants exceeds their
naturally occurring degradation rate, it is necessary to resort
to the use of engineered remediation processes that require
human intervention to enhance or accelerate the degradative
power of the selected remediating agents.
Several strategies have been devised to remediate and restore polluted environments: physical and chemical methods
and biological approaches, requiring the involvement of biological agents. These techniques may be utilized in situ, i.e.
in the contaminated place itself, offering numerous advantages over ex situ technologies. The first ones can be done
on site, eliminating transportation costs, are less expensive,
can be applied to diluted and widely diffused contaminants,
and minimize dangerous manipulations of the environment.
While in ex situ techniques, the treatments removing the
contaminants occur at a separate treatment facility [3].

alcohols. It indicates the susceptibility of the contaminant to be degraded into less toxic products. Oxidoreductases. enzymes carry out processes for which no efficient chemical transformations could have been devised. Furthermore.A. and is strongly influenced by the chemical structure. Gianfreda. not yet cell-accessible. The majority of these activities were exhibited by humic–enzyme complexes. showing properties often dissimilar from those of the free enzymes. 2. The molecular connectivity. These latter in turn provide to their complete mineralization. In order to be biodegraded. concentration and properties of the contaminant. All are effective agents in the transformation of organic pollutants because their enzymatic components are powerful catalysts. The main bioremediation agents are plants. being the two classes of enzymes involved in the transformation of both xenobiotic molecules and natural products. and types of atom-to-atom connections. including xenobiotics and even plastic materials. that can be determined by the structural formula of the compound. Later. Bioremediation and extra cellular enzymes Bioremediation. The most common contaminants can be classified on the basis of their biodegradability. Pollutants like simple hydrocarbons C1 –C15 . The authors underlined that enzyme-like activities rather than purified enzymatic proteins could be extracted from soil. amines. The process can be quite rapid for some natural compounds like cellulose or very slow for many xenobiotics.e. and by environmental conditions. either as a spontaneous or as a managed strategy. i. polycyclic aromatic hydrocarbons (PAHs) as well as pesticides are very difficult biodegradable. For instance. studies of the molecular structure of pollutants. Roles of extra cellular enzymes in cell metabolism. and amides are very easily biodegraded. Rao / Enzyme and Microbial Technology 35 (2004) 339–354 Fig. If soluble. [6] reviewed more extensively the same topic. they must be transformed into soluble or easily cell-available products. partial oxidation of recalcitrant pollutants such as PAHs by extra cellular oxidative enzymes give rise to products of increased polarity and water solubility and thus with a higher biodegradability [4]. products (Fig. which are deliberately released by the cells into their nearby environment. may also explicate a protective function by oxidizing toxic soluble products into insoluble. is the application of biological processes for the clean up of hazardous chemicals present in the environment. depends on the biodegradability of the contaminants. can result of particular significance in molecular topology studies [1]. contaminants must interact with enzymatic systems in the degrading organisms. phenols. By contrast. 2). acids. however. Nannipieri et al. the less is the likelihood for significant biodegradation” [2]. Theoretical predictions of compounds biodegradability can be usefully obtained by studies of molecular topology. The first effective step for cell-transformation of insoluble substances. 1. esters. The degradative efficiency of biological processes. Tabatabai and Fu [5] demonstrated that several oxidoreductases and hydrolases were extractable in a free form from soil. the less biodegradable is the compound. however. able to extensively modify structure and toxicological properties of contaminants or to completely mineralize the organic molecule into innocuous inorganic end products. Extra cellular enzymes include a large range of oxidoreductases and hydrolases. Fig. polychlorinated biphenyls (PCBs). microorganisms and plant–microorganisms associations.and extra cellular enzymes. occurrence of branching. . 2). they can easily enter cells. M. Pollution of the environment by inorganic and organic compounds.340 L. As claimed by Suthersan “A synthetic chemical that is not a product of biosynthesis will be degraded only if an enzyme or an enzyme system is able to catalyze the conversion of this compound to an intermediate or a substrate able to participate in existing metabolic pathways” and also “The greater the difference in structure of the xenobiotic form from the compounds produced in nature. 2. is usually the reaction catalyzed by ecto. Both these enzymes may explicate a degradative function and transform polymeric substances into partially degraded or oxidized products that can be easily up-taken by cells (Fig. if insoluble. Usually the most complex is the chemical structure.

The presence of PCBs. Microbial extra cellular enzymes 2. [8] showed that. Furthermore.3 0. however.1 48. Gramineae and Solanaceae efficiently and considerably released both oxidases and hydrolases in the root regions of the soil.034 1984. it served as redox mediator.3 0.0. Other non-ligninolytic enzymes. They can be summarized as follows: (1) White-rot fungi are ubiquitous organisms in natural environments. M.L. In another experiment.48 0. some cultures of plants of various species and morphology transformed polychlorinated biphenyls as well as polycyclic aromatic hydrocarbons.6 0. released by some of the investigated plants.4 0 0. Harvey et al. A great interest is growing for the use of fungi as bioremediating agents [10–13]. [7] followed the exudation of enzymes by the roots of 12 plant species in non-sterile soils for 56 days. In particular. which has broad substrate specificity and is able to oxidize several environmental pollutants [10. Microbial oxidoreductases In vivo microbial oxidoreductases are periplasmic enzymes associated with the cell surface of viable cells. The enzymes were able to transform several hydroxy benzoic acids. and actinomycetes.033 33.80 0. As a consequence.6 196. Chroma et al.A. expressed as fluorescein diacetate hydrolase. These enzymes are usually wall-associated enzymes and provide to partially transform substances in products more easily up-taken by plant roots or rhizosphere microorganisms.12]. in other cases was toxic to both the plant and its peroxidase production. in vitro. like cellobiose dehydrogenases.2. Cellobiose dehydrogenases are usually secreted under non-ligninolytic conditions when cellulose is the nutrient carbon.1. An important contribution can instead derive from the involvement of degradative enzymes released by plant roots in their surrounding environment.1.30 0 443. and allowed the peroxidase-dependent oxidation of NADH. laccase. SDS–PAGE and the isolectrofocusing of protein extracts from sterile and non-sterile cultures of alfalfa and from the unplanted sterile soil confirmed that the protein preparations from planted soils contained protein fractions with features characteristic of alfalfa peroxidase [7]. and either directly or indirectly they may oxide several contaminants [11. white-rot fungi appear unique and attractive organisms for the bioremediation of polluted sites for several reasons [10. ectomycorrizal fungi.12]. Extra cellular plant peroxidases may exhibit different functions depending on the pH values and the nature of the electron donors. [9] have demonstrated that cultures of Ipometa batada produced a wide range of peroxidases with isoelectric points between 3 and 341 9.004 15. a vast range of toxic environmental pollutants. 2. They demonstrated that several members of Fabaceae. Their main sources are fungi such as wood-degrading basidiomycetes.7 11.14]. may participate in the transformation of polluting substances [11. Gramms et al. (2) White-rot fungi are unique among eukaryotic or prokaryotic microorganisms. Table 1 reports the amounts of peroxidase.2. mM min−1 ) Laccase (ABTS.34 0. Rao / Enzyme and Microbial Technology 35 (2004) 339–354 2. At pH 7. whereas at pH 3. because they posses a very powerful extra cellular oxidative enzymatic system: the lignin-degrading enzyme system (LDS). such as NADH. and proteinase–lipase–esterase activity. .67 0 0.0 and in the presence of a secondary substrate.61 4. including low soluble com- Table 1 Extractable enzyme activities in non-sterile soil–root regions of plants grown for 56 days Plant species Peroxidase (guaiacol. and the enzyme activity depended on the pH and the nature of the electron donors. monophenol monoxygenase.3 2.13]. The enzymes were of plant origin because the results were confirmed in sterile soils. ␮M min−1 ) Control soil Fabaceae Alfaalfa Soybean Gramineae Grass mixture Maize Solanaceae Tobacco Tomato 0 0. Most fungi are robust organisms and may tolerate higher concentrations of pollutants than bacteria.1 0.074 79. soil-borne microfungi.046 0 As modified from [7]. ␮M min−1 ) FDA hydrolase (fluorescein diacetate. Recently. ␮M min−1 ) Monophenol monoxygenase (DL-DPOA. Plant root extra cellular enzymes Root-associated microorganisms often are assumed to be the only effectors of the increased xenobiotic biodegradation occurring in the rhizosphere. and that their activity was ascribable to the production of a constitutive cell-wall associated peroxidase.14 0017 0. the salicylhydroxamic acid was oxidized in its quinonic form. Gianfreda. terricolous basidiomycetes.14 0.

chrysosporium. 2. lignin peroxidase (LiP) and Mn-dependent peroxidase (MnP). the explosive of major environmental concern. and produce a large quantity of end-products. Together with an H2 O2 -generating system and cellulolytic and hemicellulolytic enzymes.6-dinitrotoluene. (5) LDS enzymes. and this latter mediates the oxidation of a variety of phenolic substrates [17]. benzo[a]pyrene.20]. have a very high oxidation–reduction potential.2 -azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid (ABTS). chrysosporium.4. As reviewed by Cameron et al. Various combinations of the two glycosylated hemecontaining peroxidases. LiP action is an H2 O2 (endogenously generated)-dependent one-electron oxidation of a variety of lignin-related aromatic structures. Inonatus dryophilus White-rot fungi 30–50%. 3-hydroxyanthranilate. Laccase LiP. RDX LiP. Most of the studies were performed with P. They are relatively non-specific.A. hydroxylation. benzo[a]phenanthrene.6-diamino-4-nitrotoluene. RDX: hexahydro-1. MnP. can be mineralized or degraded by white-rot fungi.5-trinitro-1. [12]. Table 2 summarizes several recent findings obtained in the biodegradation of a variety of pollutants by fungi.6-trinitrotoluene. 1-hydroxybenztriazole (HTB) and others are present in the reaction mixture [19.3. the capability of P. and naph- Table 2 Biodegradation of pollutants by fungi and their extra cellular oxidases Pollutant Enzyme Source Pollutant decrease References Azo dyes Biopolymers (kraft.4-diamino-6-nitrotoluene.4. and can potentially oxidize xenobiotics usually difficult to be affected by other peroxidases [15.3. Laccases can also oxidize non-phenolic aromatics when mediators such as 2.23] [11] [24. or carbon tetrachloride (CCl4 ) were significantly transformed by cellobiose dehydrogenase [11. they grow by hyphal extension and extend in the soil with growth. phenol dimerization and/or polymerization. high [12. redox mediators produced by the fungus render it capable of efficiently degrading several toxic compounds. Indeed. Pleurotus ostreatus.342 L. . This feature prevents their originating organisms to be adapted to the chemical being degraded.5-triazine. can reach the soil pollutants in ways that bacteria cannot. giving rise to the formation of aryl cation radicals. white-rot fungi and bacteria are capable to transform TNT and form dinitrotoluenes (DNTs) (e. MnP. For instance. MnPs catalyze the H2 O2 -dependent oxidation of Mn(II) to Mn(III). sanguineis Phanerochaete chrysosporium P. are mainly of constitutive nature. CDH: cellobiose dehydrogenase. The efficiency of pollutant biodegradation depended on both the type of pollutant and the fungus involved in the process. played the primary role in the process. the key enzymes to the process. and of a copper-containing phenoloxidase. some pollutants such as 2. LDSs: lignin-degrading enzyme systems. being filamentous fungi. 2. PCP: pentachlorphenol. PCBs: polychlorinated biphenyls. Very recalcitrant and toxic compounds like PAHs or PCBs with a high number of Cl substitutions were efficiently mineralized by various types of white-rot fungi (Table 2). chrysosporium. lignin) Bleach plant effluents CCl4 . non-specific. they may act synergistically during the decay of wood. TNT: 2.30] CCl4 : carbon tetrachloride. These can perform several non-enzymatic reactions like: benzylic alcohol oxidation.6-trinitrotoluene (TNT). PAHs: polycyclic aromatic hydrocarbons. T. LiP: lignin peroxidase. chrysosporium to degrade a number of PAHs (e. CDH Pycnoporus sanguineis White-rot fungi P. M. Furthermore. CHCl3 : carbon trichloride (chloroform). Rao / Enzyme and Microbial Technology 35 (2004) 339–354 pounds. MnP: Mn-dependent peroxidase. The lignin-degrading system.g. besides LDSs. and demethylation.6-dinitrotoluene) [31]. For example.29] TNT. P.g. Trametes versicolor Coriolopsis polyzona. several enzymes and highly reactive. T. constitute the lignin-degrading enzyme systems of white-rot fungi. MnP Up to 60% Very significant 85% Up to 50% 70–100%. On the contrary. carbon-carbon bond cleavage. DNTs are however quite recalcitrant to a further degradation. laccase (L). (4) White-rot fungi. In some cases. CDH LDSs.16].12]. high Very significant [21] [10. phenanthrene. 2-amino-4. and 4-amino-2. CHCl3 PAHs PCBs (1–6 Cl substitutions) Laccase LiP. as demonstrated in Phanaerochaete chrysosporium [12] they are usually expressed under nutrient-deficient conditions that are prevalently encountered in most contaminated soils. Molecular oxygen is the oxidant involved in the laccase-mediated oxidation of phenolic substrates to phenoxy radicals [18]. The LiPs are particularly attractive as pollutant degraders. That extra cellular oxidoreductases were involved in the biodegradation of several pollutants by fungi was conclusively demonstrated by studies performed with purified enzymes. chrysosporium was demonstrated to efficiently biodegrade and completely mineralize DNTs.12.25] [26.27] PCP LDSs 99% very high [28. versicolor. versicolor P. the involvement of other enzymes was recognized to be crucial to the mineralization process. or some of its enzymatic constituents.13] [22. chrysene. (3) White-rot fungi may grow using inexpensive substrates such as agricultural crop wastes that can be easily added as nutrients to the contaminated site. Gianfreda. The involvement of its lignolytic enzymes was proved by mineralization studies performed with purified Mn peroxidase [32]. MnP Laccase LiP.

esterases. may contribute largely to enrich the environment with solid wastes. Metcalfe et al. When the two fungal enzymes were tested in combination. White-rot fungi Curvularia senegalensis (not wrf). Corynebacterium. observed between the rate of keratin degradation and the enzyme production. Bacillus sp. their preferential substrates and main sources is reported in Table 3. The author concluded that the fungus and its proteolytic activity have a great potential for the in situ bioremediation of keratinous wastes. nylon. Pollutants of a great environmental concern are plastics. polyacrylates. keratinophilum fungus. phosphatase Amylase Trichoderma resei. the activity and diversity of these chitinases. phosphatases and phytases. [35] investigated an extra cellular group of bacterial hydrolases: the chitinases. alkaline protease Cellobiose dehydrogenase Esterase Un-named white-rot fungus (+Mn and lactate) Amycolatopsis. carbohydratases (e. Others.g. Polyurethanes. i.51] . that were not substrates of LiP action. In particular.e. thus indicating that other non-enzymatic factors were involved. automotive and industrial fields. Gianfreda. like phosphatases and phytases. that hydrolyze the ␤ 1–4 glycosidic bonds of chitin in soil.).g. These enzymes are physiologically necessary to living organisms. as well as used paper products discarded by human population. Microbial hydrolases The other microbial enzymes involved in the pollutant transformation are hydrolases.38] [39] [40] Non-natural materials Nylon Poly(l-lactic acid) Polyacrylate Polyurethane Nylon-degrading enzyme (MnP) Depolymerase.44] [45. to smaller molecules for subsequent absorption by cells. Several bacteria and fungi produce a group of extra or ecto cellular enzymes that include proteases.2. and newspaper and microcrystalline cellulose (MCC)). Pycnoporus cinnabarinus [43. xylanases. the authors observed that an increase of enzyme activity but a decrease of chitinase diversity occurred upon sludge application. 3). That a prominent role in the hydrolysis of phytin by fungi was explicated by an extra cellular phytase was demonstrated in comparative studies on the relative efficiency of intra and extra cellular phosphatases and phytases in six fungi [42]. no clear correlation was. M. filter. Some of them (e. amylases.L. As previously demonstrated by Kornillowicz-Kowalska [41]. was able to produce an extra cellular protease with an optimum proteolytic activity against keratin substrates. Both cellulases degraded all paper wastes though with a different efficiency. whereas most of the acid and alkaline phosphatase activity was found inside the cell. cellulases. synthetic. Penicillium funiculosum Actinobacteria Chysosporium keratinophilum Sreptomyces thermoviolaceus Sulphate reducing bacteria Bacillus licheniformis [34] [35] [36] [37. A list of microbial hydrolases involved in the transformation of natural and non-natural insoluble compounds. suggested that other enzymes (i. The extra cellular phytases were found 60% more efficient than their intracellular counterparts. who demonstrated that cellulases from Penicillum funicolosum and Tricoderma resei transformed paper materials of different origin (e. Keratinic wastes deriving from animal breeding. polylactides. monoxygenases) could be involved in the transformation of PAHs by the fungus [33].2. foolscap. Comamonas acidovarans Pseudomonas vesicularis. These materials are highly recalcitrant and when discarded in the environment. Due to their intrinsic low substrate specificity.46] [11] [47–49] Polyvinyl alcohol 2-4-Pentanedione esterase.e. the susceptibility of the cellulose substrates to their hydrolytic degradation depended on the different ratio of the two cellulases in the mixture (Fig. laccase [50. and mixed composites of different starting materials are widely applied in the medical. Singh [36] showed that the C. Table 3 Biodegradation of natural and non-natural insoluble materials by microbial hydrolases Material Enzyme Source References Natural materials Cellulose materials Chitin Keratin Kraft pulp Sewage sludge Starch materials Cellulase Chitinase Keratinase Xylanase. ␤-xylosidase Protease. contribute to the nutrition of plants and microbes by hydrolysis of organic P compounds into inorganic P. Rao / Enzyme and Microbial Technology 35 (2004) 339–354 thalene). 343 An interesting use of cellulases was reported by van Wyk [34]. processing and handling. office. however. etc. 2. proteases and carbohydratases) catalyze the hydrolysis of large molecules. starch polymers. the only form of phosphorous available to plants and microbial cells.g. hydrolases may play a pivotal role in the bioremediation of several pollutants including insoluble wastes. In a recent paper.A. Their results confirmed that actinobacteria are important effective agents in chitin degradation in soil and that amendment application such as sludge application may contribute to affect the presence. man-made materials widely used in modern society for several purposes. and contributed to the treatment of solid municipal wastes. such as proteins and carbohydrates. The authors reported the first molecular ecological assessment of chitinase diversity within a terrestrial environment. isolated by a waste site containing organic pollutants.

A solid-polyester-PUR-degrading enzyme. In several cases. The enzyme. Rao / Enzyme and Microbial Technology 35 (2004) 339–354 Fig. their persistence and high visibility may highly contribute to landfills with severe problems of environmental pollution. The results indicated that the enzyme has a special structure consisting of a hydrophobic PUR-surface-binding domain. Degradation of various paper materials by individual and mixtures of cellulases from Penicillum funicolosum and Tricoderma resei (from [34]).344 L. Further research led to identify a nylon-degrading enzyme in the extra cellular medium of a ligninolytic culture of the white rot fungus strain IZU-154 [44]. In spite of their intrinsic low vulnerability to microbial transformation. chrysosporium and Trametes versicolor under ligninolytic culture conditions [43]. different microbial mechanisms have been invoked to explain the capability of bacteria and fungi to efficiently degrade them ([52] and references therein). [43. Due to the molecular complexity of these materials. 3. Although the reaction system for nylon degradation significantly differed from that reported for Mn peroxidase. and its molecular and structural features were investigated. The potential applications of several enzymes in the solid. liquid. The molecular and enzymatic characteristics of the purified protein appeared the same as those of manganese peroxidase. intensive research was carried out to identify the microorganisms and their degrading-enzymes involved in the degradation of polyurethane (PUR) and its derivatives.A. largely used in several fields [52]. potentialities and restrictions of isolated enzymes in the transformation and detoxification of organic pollutants have been recently examined by Gianfreda and Bollag [54]. and although it showed similarities with horseradish peroxidase-catalyzed reactions. These particular characteristics were found also in other solid polyester-degrading enzymes and even in cellulases. was identified and purified from Comamonas acidovorans TB-35.53]. Bioremediation and cell-free enzymes Several of the above pollutants transformations rely on the action of extra cellular enzymes in the presence of their originating cells. Incubation of high-molecular-weight PLA with different amounts of en- zyme (65–1 mg/ml) at 37 ◦ C for 30 min produced only lactic acid thus suggesting an exo-type enzymatic action [45]. thus allowing hydrophobic solid substrates to be degraded [49. Nakamura et al. Gianfreda. The properties.44]. An appealing alternative to the bioremediation of polluted sites could be that of utilizing cell-free enzymes isolated from their originating cells. a Gram-negative bacterium capable of degrading polyester PUR [49]. The enzyme was isolated and purified to homogeneity. a linear polymer with amide bonds similar to those of proteins but not susceptible to the action of proteases. The depolymerising activity was due to the production of a unique extra cellular PLA-depolymerase. the primary effectors of plastic biodegradation are extra cellular enzymes. Interesting findings were reported by Deguchi et al. and hazardous . 3. essential for PUR degradation. In other words. The enzyme was isolated. an extra cellular esterase. strains K104-1 were able to depolymerise the polylactic acid (PLA) to its monomer lactic acid. most of these materials have been found to be biodegraded by soil microorganisms (Table 3). its encoding gene. MCC: microcrystalline cellulose. and a catalytic domain. like esterases and depolymerases. the biodegradation of the pollutant is started by an extra cellular enzyme/s but requires the presence of the whole cell and its metabolic pathways to be completed.52]. Nylon. For instance. one of the key enzymes of white-rot fungi LDS (see above). was significantly degraded by two fungi: P. M. purified and tested for its degradative efficiency towards synthetic PLA. the authors concluded that nylon degradation in the investigated fungus was catalyzed by Mn peroxidase [44]. Further studies were also dedicated to elucidate how to decrease or increase the microbial degradation of the PUR polymers by adding specific substances [49. [45] showed that Amycolatopsis sp.

60] [61. several causes concur to hamper and render difficult the use of isolated enzymes as tools in the detoxifications of polluted sites.. DDT. inhibitors of microbial metabolism. studies performed with PAH-derivatives. As pointed out by the authors.. In the presence of the mediating substances veratryl alcohol (for LiP).76–78] have demonstrated that laccases from different fungal origins were capable of removing a large variety of phenols and the efficiency was strictly dependent on the chemical structure of the phenol.A. chloroperoxidases. laccase.0 [63–66] Estrogenic chemicals Nitrile compounds Nematoloma forwardii Cladariomyces fumago Achromobacter sp. lindane Phenols. For other compounds and enzyme symbols see Table 2. pyrene Asphaltenes Carbofuran.5–10. reduced glutathione (GSH) (for MnP). relatively wide temperature. peroxidase White-rot fungi Agrobacterium. Fusarium solani Several microorganisms PCP. very recalcitrant compounds like asphaltenes and PCBs. LiP. Alcaligenes denitrificans. laccase Usually inducible. Furthermore.69] [57. and ABTS (for L. very stable in immobilized form 70–100% transformation High stability up to 60 ◦ C and pH 6.71. The three.L. parathion. Rao / Enzyme and Microbial Technology 35 (2004) 339–354 waste treatment were previously reviewed by Nannipieri and Bollag [55]. their capability to act in the presence of many toxic. whereas 31. PAHs was enhanced in most cases. Karam and Nicell [56] and Nicell [57]. pH and salinity ranges. have been obtained under laboratory conditions. substances. prepared from the white rot fungus Nematoloma frowardii and by mush-room tyrosinase (Tyr) and horseradish peroxidase (HRP) [58]..62] Cyanides Cyanidase.0 Sensitivity to SH-agents.. laccase Nitrilase. and/or under a wide range of environmental conditions. PCBs.4-DCP p-Tyrosol o-Tyrosol m-Tyrosol Catechol Resorcinol Methylcatechol Hydroxytyrosol Pyrogallol Gallic acid 1 1 1 1 1 1 2 2 2 2 3 3 −1Cl −1Cl −2Cl –CH2 CH2 OH –CH2 CH2 OH –CH2 CH2 OH – – –CH3 –CH2 CH2 OH – –COOH 18 20 66 73 28 11 100 40 76 86 100 98 From [70. M. cyanide hydratase MnP.71]. Flavobacterium sp. carbaryl LiP. coumaphos. often unfavourable to active microbial cells (i. Tyr..2% of pyrene were oxidized by MnP. known as intermediates or potential dead-end-products of microbial PAHs metabolism. Pesticides of different chemical nature. nitrile hydratase. diazinon MnP. whereas PAH-quinones and oxo-metabolites were not transformed. stereospecificity Very versatile under different operational conditions High stability at 50 ◦ C and pH 5. Pseudomonas sp. PAH Pyrethroids.e. cell-free enzymes can offer several advantages over the use of microbial cells. The most significant features of cell-free enzymes are their unique substrate-specificity and catalytic power.70–72] [73–75] . laccase Chloroperoxidase Carbamate hydrolase High (veratryl alcohol) High activity in organic solvents Cytosolic. The studies by Bollag and co-workers (for a detailed list see Table 2 and reported references in [18]) and Gianfreda and co-workers [70. Rhodococcus sp.0–11.6% of anthracene and 34. [67].. the transformation of 345 Table 5 Biodegradation of phenols by cell-free laccase from Cerrena unicolor Substrate –(OH) Substituent Substrate decrease (%) o-Cl-phenol p-Cl-phenol 2. manganese peroxidase and laccase.and four-ring polycyclic aromatic hydrocarbons anthracene. [67] [68] [54.2% of pyrene. the type and the number of substituents on the aromatic ring (Table 5). pyrene and fluoranthene were in vitro-oxidized by extra cellular lignin peroxidase. bacterial and plant cells. Nocardia sp. high and low concentrations of contaminants). HRP).5% of anthracene and 11. Like natural estrogens they can bind to the estrogen receptor and Table 4 Biodegradation of pollutants by cell-free enzymes Pollutant Enzymes Source Properties References Anthracene. Gianfreda. amidase Dehalogenases. even recalcitrant. An interesting application of fungal oxidases with estrogenic chemicals was shown by Tsutsumi et al. high specificity [58] [59. Estrogenic chemicals are man-made chemicals that mimic the effects of hormones (particularly estrogens). and their low sensitivity or susceptibility to the presence of predators. PAHs and others toxic pollutants were successfully transformed by oxidoreductases and hydrolases isolated by fungal. polychlorophenols. The majority of results summarized in Table 6. Bacillus cereus DDT: dichloro diphenyl tricholorethane. demonstrated that the hydroxylated PAHs metabolites were oxidized by all the oxidoreductases. and drastic changes in contaminant concentrations. As it will be addressed below. phenanthrene. several fungi Trametes versicolor Nocardia sp. Pseudomonas sp. Table 4 illustrates a wide range of cell-free enzymes applied to the biodegradation of different pollutants. MnP. however. LiP transformed 58.

4. esters. pharmaceuticals. mutagenic and carcinogenic in nature (Pollak et al. or as intermediates in the organic synthesis of a variety of different compounds (e. The overall results led the authors to explain the BPA and NP transformation mechanisms as due to the polymerization and partial degradation of the chemicals brought about by enzymatic oxidation [67]. ricinine. 5). 4 both BPA and NP disappeared within 1 h-treatment with a MnP. solvents. A laccase. Bisphenol A (2. Both Mn peroxidase and laccase removed the estro- genic activities of BPA and NP within 12 h. and are suspected of having estrogenic (endocrine-disrupting) activity.A. bromoxynil. such as cyanoglycosides. Decrease of bisphenol (BPA) and nonylphenol (NP) estrogenic activities by oxidoreductases from various origins (from [67]). efforts have been made to control their release in the environment. [80]). regulate the activity of estrogen responsive genes. Rao / Enzyme and Microbial Technology 35 (2004) 339–354 Fig. They comprise some of plant origins. drug intermediates (chiral synthons). chrysosporium ME-446. ketones and heterocyclics). 5.g. In vitro screening tests for chemicals with hormonal activities using yeasts [79] were utilized to evaluate the estrogenic activity of BPA or NP after the enzymatic action [67]. As shown in Fig. . carboxylic acids. phenylacetonitrile. Nitrile compounds are widespread in the environment. Similar results were obtained within 6 h with laccase in the presence of HBT (Fig. amides. Nitrile compounds are also extensively used in chemical industries to produce a variety of polymers and other chemicals. Gianfreda.346 L. amidines. extractants. and/or to remove them from it. Such effects have raised concern that prolonged exposure to environmentally relevant concentrations of these chemicals may adversely affect reproduction in wildlife and humans. 2-bis(4-hydroxyphenyl) propane. isolated and partially purified from the strain P. Other different nitrile compounds are used as feedstock. ioxynil. Fig. Consequently. Bisphenol (BPA) and nonylphenol (NP) disappearance by oxidoreductases from various origins (from [67]). Analysis by gel permeation chromatography indicated oligomers as the main products of BPA and NP enzymatic transformation [67].Most nitriles are however highly toxic. and the addition of the mediator 1-hydroxybenzentriazole (HBT) in the reaction system greatly improved the laccase potential. BPA) and nonylphenol (NP) are widely used in a variety of industrial and residential applications. If present in the environment at high concentrations they may cause severe diseases in humans. amines. pesticides (dichlobenil. Another interesting application of cell-free enzymes is the use of the so-called nitrile-degrading enzymes capable of degrading nitrile compounds [68]. partially purified from T. cyanolipids. M. etc. The greatest concern for the biodegradation of estrogenic chemicals is aimed at the removal of their estrogenic activity. also removed BPA by 70% and NP by 60%. versicolor IFO7043. aldehydes. buctril).

several investigations have demonstrated that white rot-fungi. 4. Cl-. or the unpredictable level of transformation and mineralization of pollutants. protocatechuic acid. • a stable biocatalyst. and their extra cellular oxidases. Thus. M. catechol. vanillic acid) ([18] and . may depend on both the pollutants and the enzymes. • a suitable enzyme must be identified. These are common contaminants of brown field sites. Wyatt and Knowles [81] reported that mixed cultures of bacteria containing different nitrile hydrolyzing enzymes (including nitrile hydratases. non-pathogenic strain. the application of these enzymes for the remediation of cyanide-polluted sites can be envisaged. or to extract the enzyme for cell-free applications.13] and references therein). the use of a readily cultivable microbial source. When pollutants transformation occurs in the polluted site.e.e. cell-associated or cell-free enzymes. involved in the bioremediation of a polluted site [84]. As a consequence possible negative. syringic acid. Rao / Enzyme and Microbial Technology 35 (2004) 339–354 As reviewed by Banerjee et al. Pollutant-derived disadvantages 4. guaiacol. nitrilase and amidase) biodegraded effluents from acrylonitrile manufacturing industries. as well as the production of transgenic plants resistant to the herbicide bromoxynil.e. i. The direct decontamination of acrylonitrile-containing polymer emulsions obtained by nitrile hydratase treatment [82]. stable under the environmental conditions at the polluted site. The enzymes may be of both constitutive and inducible nature and present some common features such as optimum activity at alkaline or sub-alkaline pH and a good stability in a large range of temperatures. The catalytic effectiveness of laccase and peroxidase of removing several toxic compounds (i. Another group of useful enzymes in the detoxification of toxic. it needs to be specific towards the desired chemical reaction substrates and products.55]. Feasibility of extra cellular enzymes application to polluted soils How feasible is the bioremediation of polluted soils with extra cellular enzymes? In general. and amenable to immobilization and/or stabilization procedures. rather than a single polluting substance are present. In a natural environment such as soil. synergistic effects on the enzyme efficiency may occur. [68]. A common feature in soil-degradation studies is however the generally low. additional requirements can be envisaged. are able to transform pollutants of different nature when acting directly in soil ([10. Gianfreda. several are the requirements for obtaining an ideal biotransformation. and extra cellular enzymes are required to start the process. The main requirements can be summarized as follows: • an efficient enzyme production system.e. syringic acid. A 99% decrease of detectable toxic components was measured. compared to laboratory studies of submerged liquid cultures. Br-phenols. and the consequent severe damages to natural fauna. • the enzyme-producing microorganism must be cultured to increase the enzyme yield.1. Based on their substrate specificity. especially old gasworks. nitrile hydratases and amidases. 347 • an efficient biocatalyst. however. One of their most common use is in the synthesis of several pharmaceuticals and useful chemicals [68]. Lynch [66] has reported that the introduction of a selected strain of Trichoderma in the rhizosphere soil rapidly catabolized phytotoxic concentrations (10 mM) of cyanide through the action of the two enzymes. nitrile-converting enzymes consist of three major categories: nitrilases. resulting from the introduction of microbial bromoxynil-specific nitrilase genes [83]. Fungi and bacteria may effectively catabolize cyanides by means of cyanide-catabolizing enzymes that include formamide hydrolase and rhodanase. The disadvantages of in situ application of either extra cellular. • the best source of such enzyme activity must be identified using selective screening by means of conventional microbiological approaches. a general lower efficiency of the biological agent is usually observed. the use of resting cells or purified enzymes that are highly active in the required reaction. • the enzyme must be stable under the operational conditions (see above). have flexible substrate selectivity.A. Large pollution of water by cyanide-wastes. These enzyme are. or of a genetically stable. confirm that this group of enzymes could be good potential candidates for the bioremediation of nitrile compound polluted sites. Cl-anilines. e.L. In a polluted site. i. several drawbacks or disadvantages may hinder or diminish the catalytic potential of these enzymatic catalysts [55]. For instance. versatile catalysts and could be potentially usable in several fields including bioremediation. fulvic acid. and may assure minimal side reactions. whichever the catalyst.g. When isolated enzymes are selected as the detoxifying agents. very few examples are available on the use of isolated enzymes for the detoxification of pollutants in soil [54. When cell-free enzymes are then considered. pollutant cocktails. or positive. may also occur. several bacteria and some fungi posses enzymatic capabilities to metabolize several natural and synthetic nitriles. naphthols. normally composed of combinations of many organic (and inorganic) contaminants. bentazon) has been extensively studied by Bollag and co-workers under laboratory conditions when acting in the presence of co-substrates or humic precursors (i. particularly soil. whole microbial cells or isolated enzymes. hazardous compounds is the one involved in the transformation of cyanides.

6 thriclorophenol (Table 7) [87]. that is its bioavailability [89]. several properties such as the amount of organic matter. The model system simulates a typical wastewater deriving from an olive oil factory. concentration. respectively). methylcatechol. Binary. whereas no effects were measured with glyphosate [88]. The 2. The main constituents are usually the compounds used in the model system.4-DCP +4-CP +2. ternary.4-DCP) decrease by laccases from different origins (i. by laccase or peroxidase may be completely annulled. In Mediterranean countries. M. enhancing or depressing effects of a laccase from Rhus vernicifera on catechol transformation were measured in a model system of four phenols (catechol. Incubation with 6 Units ml−1 for 24 h at 25 ◦ C.4D. These model system studies were aimed at achieving a basic understanding of the possible mechanisms for oxidative coupling reactions occurring in natural soils and sediments [85]. Gianfreda. In another study. and can limit the amount of pollutant available to microbial cells and their extra cellular enzymes. Kim et al. conditions with acid phosphatase from sweet potato a typical. Cerrena unicolor.4-DCP-laccase mediated decrease can also be affected by the presence of its parent precursor.4.4-dichlorophenol (2. by the co-presence of a variety of substances that are precursors of humic materials (Table 6).87]. b From Polyporus pinsitus. Their limiting effect can be greater toward .4-DCP Tyrosol Resorcinol Methylcatechol Hydroxytyrosol Pyrogallol Gallic acid 66 11 40 76 86 100 98 Cerrena unicolor 34 88 76 24 18 89 83 Phenol mixtures 2. In soil. the soil aggregation and sub-superficial heterogeneity can all influence the bioavailability of pollutants [90]. tyrosol and hydroxytytrosol) [78].4-D + Simazine +Catechol + Simazine +2.4D (Table 7) [71]. In our studies we have also demonstrated that for example the 2. Investigations performed under laboratory Substrate decrease (%)∗ Laccase activity (%) Single phenols 2.0) Peroxidasec (pH 3. From [70. and quaternary aqueous phenolic mixtures were investigated (Table 7).348 L. large quantities of wastewater with a high content of phenolic substances are usually produced as characteristic by-products of olive-oil production. Bioavailability is affected by both the inherent properties of the pollutants (i. 2. Differentiated effects on the enzyme activity were also observed by the presence of a polluting substance non-substrate of the enzyme. and glyphosate demonstrated that the activity of the enzyme was depressed by atrazine and carbaryl (40 and 34% of activity reduction. that is usually supplied to soil in combination with 2. Trametes villosa) may be differently affected by the co-presence of one or two other chlorophenols such as 4-chlorophenol or 2.4. c From horseradish. or greatly enhanced.e. Incubation with 4 Units ml−1 for 24 h at 25 ◦ C.78.e. the presence of dissolved organic matter. A key factor possibly affecting the pollutants transformation is the availability of the polluting substance to the detoxifying agent. atrazine. the thickness of silt and clay beds.4-D + Catechol Catechol +Methylcatechol (M) +Tyrosol (T) +Hydroxytyrosol (H) +M + T +T + H +M + H +M + T + H The substrate decrease values have been normalized by laccase units.6-TCP +2.4.6-TCP +2.A. another phenol such as catechol.71. [86] demonstrated that the transformation of bentazon. extra or ecto cellular enzyme. or simazine. Rao / Enzyme and Microbial Technology 35 (2004) 339–354 Table 6 Effects of co-substrates on bentazon transformation by oxidoreductases Table 7 Substrate removal and residual enzymatic activity after laccase action Humic monomers Laccaseb (pH 4. a very recalcitrant herbicide. Adsorption and/or entrapment of pollutants on/into organic and inorganic soil colloids and in soil matrix further complicate the whole system.0) Substrate Bentazona +Catechol +Ferulic acid +Guaiacol +Protocatechiuc acid +Pyrogallol +Resorcinol +Syringaldeyde +Vanillic acid +Caffeic acid 0 100 9 10 40 0 2 39 6 59 6 95 19 9 65 0 0 49 94 27 Modified from [86]. references therein). The nature of polymeric products as well as the possible mechanisms involved in the process was addressed. water or organic solubility) and the characteristics of the environment where they occur. a One mM for all the compounds. molecular structure.4-D 66 56 58 82 Trametes villosa 34 56 58 20 77 46 50 0 39 100 Cerrena unicolor 20 30 35 95 60 0 58 38 100 99 16 95 56 63 Rhus vernicifera 70 11 66 27 9 29 10 11 +Catechol +Simazine +4-CP + 2. and pesticides such as carbaryl. Increases and/or decreases of the pollutant transformation measured as both polymerization or mineralization of the target pollutant were detected.

atrazine.L. the enzymes alone. sand and soils. Measurements of residual laccase activities made in experiments performed with increasing concentrations (from 0 to 0. The results indicated that the enzyme partially. As respect to the control. When soils were used. and (iv) the influence of the microenvironment created by the support in the surrounding of the protein [88. a montmorillonite covered by different amounts of OH aluminum species (AM3 and AM18 . These processes may represent the main mechanism of inactivation because they hinder the interaction between the substrate and the enzyme [92]. that makes the whole system homogenous or heterogeneous. loading 3 and 18 mmol of Al per gram of montmorillonite). A montmorillonite (M). As a consequence. i.4-DCP by laccase in the presence of clays. Removal of 2. Strong depressing effects were measured in the presence of montmorillonite. immobilized. both not easily accessible to large molecules as enzymatic proteins [91]. soil components differently affected the 2. and glyphosate was tested on acid phosphatase immobilized on organic (tannic acid).4-DCP gave activity values similar to those obtained without sample filtration. The reduction of activity was quite dependent on the type of phenol. In the soil.4-DCP. . Rao / Enzyme and Microbial Technology 35 (2004) 339–354 349 isolated enzymes. or totally. The immobilization of enzymes on soil components affected the response of the enzyme to the presence of substances extraneous to the reaction substrates. Different causes were invoked by the authors to explain their results.76. a combination of HA and AM18 . the reduction of laccase activity increased by increasing the organic matter content of soils [97]. HA: a humic acid.4-DCP ruled out a possible inhibitory effect on laccase activity by 2. Enzymes may lose their activity upon pollutant transformation. loading 3 and 18 mmol of Al per gram of montmorillonite). clay-humic complexes. activity tests performed on samples filtered with filters that specifically adsorb 2. Fig. changes in their kinetics. M: Na-montmorillonite. They related to: (i) the “state” of the enzyme if free or immobilized on soil components. 6. AM18 and the combination AM18 +HA.93–95]. Indeed. Inactivation or degradation of enzymatic molecules may also take Fig. place. or entrapped in such matrices giving rise to the so-called “naturally immobilized enzymes” [94].e. 7). M. in turn.71. These latter. threedimensional assemblages of mineral and organic particles that will restrict their mobility and affect their activity (Fig. Aging processes can also lead to a significant reduction of pollutants bioavailability. Gianfreda. Soil bound enzymes. cell-associated or cell-free enzymes may also arise from the enzymes.97]. (iii) the conformational changes achieved by the enzymatic protein upon immobilization. sand and soils with different organic matter contents were utilized. stability and mobility will occur [96]. inorganic (montmorillonite) and organo-mineral (Al(OH)x-tannic acid-montmorillonite ) complexes a different behavior as respect to the free enzyme was observed [88]. Enzyme-derived disadvantages Disadvantages to the in situ application of extra cellular. as well. because of their sequestration from diffusion into sites within sorbing matrices or entry into nanopores. AM: montmorillonite covered by different amounts of OH aluminum species (AM3 and AM18 . Usually. In the experiments on phenols previously shown in Tables 5 and 7. 4. and the combination of the phenols in the mixture. 7. enzymes are present in complex.77. will determine the operating range of enzymes in soil around microorganisms and enzymes. The authors explained their results as due to the a progressive entrapment and/or adsorption of active enzyme molecules within/on phenol polymeric aggregates as they formed [70].4-DCP removal when in the presence of different soil components [70. (ii) the nature of interactions occurring between the enzyme and the immobilizing support. the more pronounced was the loss of laccase activity.4 mM) of 2. Enzyme molecules can be adsorbed. the entity of its transformation. the higher the phenol concentration.2. the residual activity of laccase was measured after phenol transformation under standard conditions [71]. When the influence of carbaryl.A. a humic acid (HA). Investigations were performed to evaluate the effectiveness of laccases from different origins towards the 2.95]. 6) [6.4-DCP-laccase activity (Fig. lost its catalytic activity (Table 7).

55. particularly stable to different denaturing agents. are among the most toxic pesticides used in agriculture. MnPs. The potential of enzymes for bioremediation purposes can greatly increase by the use of microorganisms and their enzymes from extreme environments. the use of whole-cells producing the enzyme can be more cost effective. [111]).109]. they may be reused and recovered at the end of the process. coli [108]. very low amounts of the purified protein are usually obtained thus rendering the whole process too costly for practical applications. The engineering of microbes and enzymes may also help to improve bioremediation. and with elevated catalytic activity. These advancements should lead to the successful genetic engineering of white-rot fungi and their enzyme systems. specialized enzymatic proteins. The regulation of expression of the key ligninolytic genes were studied. Organophosphate hydrolase is an enzyme produced and isolated by several soil microorganisms. chemical. particularly when the application on a large-scale of the selected biological catalyst is hampered by the low rate of pollutant degradation [105]. and malathion.13]. including many Pseudomonas strains. As a consequence. Thermophiles and psycrophiles have to adapt themselves to live and survive under extreme environmental conditions [112]. Parathion and paraoxon were efficiently detoxified with OPH anchored and displayed on the cell surface of Escherichia coli. and laccases were cloned and sequenced. Rao / Enzyme and Microbial Technology 35 (2004) 339–354 As regards to isolated enzymes. It is very effective in degrading these compounds but its practical application is hindered by the high cost of its purification. 8). For example. An interesting. Enzymes from both thermophilic and psycrophilic microorganisms usually display some unusual and particular features that may render them potential. powerful catalysts for the degradation of polluting chemicals ([110] and references therein. used for the successful transformation of phenolic pollutants. An example is that regarding the transformation of organophosphates by the so-called “live biocatalysts” [106]. organophosphorus hydrolase (OPH) and organophosphorus acid anydrase (OPA). an increasing knowledge of several aspects of the enzymology and molecular biology of the powerful extra cellular oxidative system (LDS) of fungi is now available [10. . the domain of the pore-forming protein OmpA and the enzyme. the production of a purified enzyme requires long and expensive isolation and purification procedures. are produced. may be adopted to increase the growth of the enzyme-producing microorganisms and to significantly reduce the cost of enzyme production [98]. including environmental applications [55–57. The range of sophisticated modern molecular technologies now available has provided the researcher with an immensely enhanced choice of potential enzyme sources.A. coumaphos. Organophosphates. their use in soil as free proteins may be hindered by their short life in an inhospitable environment such soil [54. paraoxon.104]. but their use is restricted by transport barrier of organophosphates across the cell membrane [107. By contrast. a drawback that greatly hampers their practical application is the cost of enzymes isolation and purification. The major genes encoding LiPs. and biological denaturing agents. Most advantageous bioremediation strategies for treating contaminated sites can thus possibly be planned and applied. at least to our knowledge. Even if the enzymes are available at very low costs. Improvements in the production of isolated enzymes may also derive from molecular technologies. Furthermore. Enzymes immobilized on natural and synthetic supports of different nature and through different immobilization mechanisms have been often proposed as efficient catalytic tools to overcome several disadvantages linked to the use of free enzymes [54. While the use of immobilized enzymes is widely diffused in several applicative fields. Furthermore. Alternative inexpensive technologies. Production of animal and plant enzymes could be performed by means of genetically modified microorganisms or plants [103.108]. As claimed by Chen and Mulchandani [106]. The system is made by the fusion of the N-terminal lipoprotein sequence (Lpp). Furthermore. using for instance agricultural wastes as the carbon source. and the X-ray crystallographic structures of the LiP and MnP isozymes are now known and available. M.102]. like parathion.55]. diazinon. The Lpp directs the Lpp–OmpA–OPH fusion to the outer membrane and the OmpA domain is able to pass through the lipid by-layer and to localize the enzyme on the cell surface (Fig. low-cost alternative to the use of purified enzymes can be the utilization of plant material loading enzymatic activities and proved to be effective in the detoxification of polluting substances. Given the best producer of the selected enzyme. coli wholecells expressing similar amounts of intracellular OPH. a by-product of olive oil production showing phenoloxidase activity [101. As it is the case of the results obtained in the investigations performed with minced horseradish roots [99.100] and olive husk. thus demonstrating the higher efficiency of the enzyme when acting outside the cell. recent advantages in genetic and molecular techniques have offered new chances to modify microbes or enzymes to function as “designer biocatalysts” in which certain required qualities from different organisms are brought together in a single catalyst to perform specific bioremediation.350 L. Immobilized enzymes have usually a long-term and operational stability.94]. These “live biocatalysts” displayed about seven times higher activity than E. their large-scale application in the bioremediation of polluted soils is not reported. They are easily degraded by two bacterial hydrolases. giving rise to less toxic compounds and further degradable by biological and chemical processes. The recombinant whole-cells with surfaceexpressed OPH were obtained using the same system used for the expression of ␤-lactamase on E. being very stable toward physical. Gianfreda. the enzyme showed a higher stability to both temperature (100% of activity upon 30 day-incubation at 37 ◦ C) and organic solvents.

This fascinating perspective has to resolve a still opened question: what should be the features of an enzyme to be suited for remediation of soils? Finally. exploration of extreme environments. Conclusions In conclusion. Project INCO-MED Contract no. 1999. References [1] Alexander M. Gianfreda. Boca Raton: CRC Press. effects on the enzyme efficiency. As mentioned by Burton [84] these achievements has lead to the point where rather than develop a purpose for an enzyme. fast-growing meshophiles. However. Luigi Pagano for his help in the preparation of figures and to Dr. The authors are grateful to Dr. the high costs associated with the isolation and purification of free enzymes. their large-scale application for remediation of polluted soils is still limited. coli (from [106]). exploitation of genome using advanced technologies. ICA3-2002-10021. structural. Biodegradation and bioremediation.L. This means to adopt appropriate cleanup criteria and determine what constitutes an environmental acceptable endpoint. 1999. Cloning and expression of genomic related information in heterologous. Rao / Enzyme and Microbial Technology 35 (2004) 339–354 351 Fig. has allowed an increased. a successful management or remediation of a contaminated site should make the site environmentally agreeable and usable for some acceptable purposes. either as cell-associated or cell-free enzymes. it is possible to design and develop an enzyme for a purpose. much less cheaper commercial production of thermophilic enzymes [113–115]. However. 5. the low stability of enzymes to the harsh conditions of soil all concur to restrict the wide use of enzymes as remediating agents of polluted soils. An increasing knowledge of cold-adapted enzymes properties is now being developing. DiSSPA Contribution no. In situ bioremediation. OmpA: pore forming protein. For instance. 0062. they are not widely applied in the remediation of polluted soils. as well [110]. [2] Suthersan SS. M. and protein engineering have opened new frontiers for the production and application of enzymes. The molecular. This may derive from several drawbacks and disadvantages depending on both the pollutants and the enzymes. Anna Maria Woods for her technical help in editing the paper in respect to the English language and style. 8. Many industrial applications have benefited from the use of these enzymes. coli. like E. Acknowledgments This research was partly supported by European Community. Parathion detoxification by organophosphate hydrolase (OPH) anchored and displayed on the cell surface of E. Lpp: lipoprotein. may behave as powerful catalysts in the biodegradation of harmful pollutants. often negative. San Diego: Academic Press. More challenges and potential advantages may also be envisaged for their application in biotechnology including bioremediation ([110] and references therein. Although immobilized enzymes may present a high stability under soil conditions.A. [111]). the simultaneous presence of several polluting substances in a contaminated site with synergistic. several extra cellular enzymes. kinetic and genetic properties of such enzymes have been in most cases elucidated. Remediation engineering: design concepts. .

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