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340 L. Gianfreda, M.A. Rao / Enzyme and Microbial Technology 35 (2004) 339–354

Fig. 1. Pollution of the environment by inorganic and organic compounds.

2. Bioremediation and extra cellular enzymes

Bioremediation, either as a spontaneous or as a managed

strategy, is the application of biological processes for the

clean up of hazardous chemicals present in the environment.

The main bioremediation agents are plants, microor-

ganisms and plant–microorganisms associations. All are

effective agents in the transformation of organic pollutants

because their enzymatic components are powerful cata-

lysts, able to extensively modify structure and toxicological

properties of contaminants or to completely mineralize the

organic molecule into innocuous inorganic end products.

Furthermore, enzymes carry out processes for which no

efficient chemical transformations could have been devised.The degradative efficiency of biological processes, how-

ever, depends on the biodegradability of the contaminants.

It indicates the susceptibility of the contaminant to be de-

graded into less toxic products, and is strongly influenced by

the chemical structure, concentration and properties of the

contaminant, and by environmental conditions. 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 partici-

pate in existing metabolic pathways” and also “The greater

the difference in structure of the xenobiotic form from the

compounds produced in nature, the less is the likelihood for

significant biodegradation” [2].

The most common contaminants can be classified on

the basis of their biodegradability. Pollutants like simple

hydrocarbons C1–C15, alcohols, phenols, amines, acids, es-

ters, and amides are very easily biodegraded. By contrast,

polychlorinated biphenyls (PCBs), polycyclic aromatic hy-

drocarbons (PAHs) as well as pesticides are very difficult

biodegradable. Usually the most complex is the chemical

structure, the less biodegradable is the compound. Theo-

retical predictions of compounds biodegradability can be

usefully obtained by studies of molecular topology, i.e.

studies of the molecular structure of pollutants, occurrence

of branching, and types of atom-to-atom connections. The

molecular connectivity, that can be determined by the struc-

tural formula of the compound, can result of particular

significance in molecular topology studies [1].

In order to be biodegraded, contaminants must interact

with enzymatic systems in the degrading organisms. If sol-uble, they can easily enter cells, if insoluble, they must be

transformed into soluble or easily cell-available products.

The first effective step for cell-transformation of insoluble

substances, including xenobiotics and even plastic materials,

is usually the reaction catalyzed by ecto- and extra cellular

enzymes, which are deliberately released by the cells into

their nearby environment. The process can be quite rapid

for some natural compounds like cellulose or very slow for

many xenobiotics.

Extra cellular enzymes include a large range of oxidore-

ductases and hydrolases. Both these enzymes may explicate

a degradative function and transform polymeric substances

into partially degraded or oxidized products that can be eas-ily up-taken by cells (Fig. 2). These latter in turn provide to

their complete mineralization. For instance, partial oxidation

of recalcitrant pollutants such as PAHs by extra cellular ox-

idative enzymes give rise to products of increased polarity

and water solubility and thus with a higher biodegradability

[4].

Oxidoreductases, however, may also explicate a protective

function by oxidizing toxic soluble products into insoluble,

not yet cell-accessible, products (Fig. 2).

Tabatabai and Fu [5] demonstrated that several oxidore-

ductases and hydrolases were extractable in a free form from

soil, being the two classes of enzymes involved in the trans-formation of both xenobiotic molecules and natural prod-

ucts. Later, Nannipieri et al. [6] reviewed more extensively

the same topic. The authors underlined that enzyme-like

activities rather than purified enzymatic proteins could be

extracted from soil. The majority of these activities were

exhibited by humic–enzyme complexes, showing properties

often dissimilar from those of the free enzymes.

Fig. 2. Roles of extra cellular enzymes in cell metabolism.


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L. Gianfreda, M.A. Rao / Enzyme and Microbial Technology 35 (2004) 339–354 341

2.1. Plant root extra cellular enzymes

Root-associated microorganisms often are assumed to be

the only effectors of the increased xenobiotic biodegrada-

tion occurring in the rhizosphere. An important contribution

can instead derive from the involvement of degradative en-

zymes released by plant roots in their surrounding environ-ment. These enzymes are usually wall-associated enzymes

and provide to partially transform substances in products

more easily up-taken by plant roots or rhizosphere microor-

ganisms.

Gramms et al. [7] followed the exudation of enzymes

by the roots of 12 plant species in non-sterile soils for 56

days. They demonstrated that several members of Fabaceae,

Gramineae and Solanaceae efficiently and considerably re-

leased both oxidases and hydrolases in the root regions of

the soil. Table 1 reports the amounts of peroxidase, laccase,

monophenol monoxygenase, and proteinase–lipase–esterase

activity, expressed as fluorescein diacetate hydrolase, re-

leased by some of the investigated plants. The enzymes were

of plant origin because the results were confirmed in ster-

ile soils. Furthermore, SDS–PAGE and the isolectrofocus-

ing 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].

In another experiment, Chroma et al. [8] showed that, in

vitro, some cultures of plants of various species and mor-

phology transformed polychlorinated biphenyls as well as

polycyclic aromatic hydrocarbons, and that their activity was

ascribable to the production of a constitutive cell-wall asso-ciated peroxidase. The presence of PCBs, however, in other

cases was toxic to both the plant and its peroxidase produc-

tion.

Extra cellular plant peroxidases may exhibit different

functions depending on the pH values and the nature of the

electron donors. Recently, Harvey et al. [9] have demon-

strated that cultures of Ipometa batada produced a wide

range of peroxidases with isoelectric points between 3 and

Table 1

Extractable enzyme activities in non-sterile soil–root regions of plants grown for 56 days

Plant species Peroxidase (guaiacol,

mM min−1)

Laccase (ABTS,

M min−1)

Monophenol monoxygenase

(DL-DPOA, M min−1)

FDA hydrolase (fluorescein

diacetate, M min−1)

Control soil 0 0.14 0.48 0.034

Fabaceae

Alfaalfa 1984.7 48.6 33.1 0.30

Soybean 11.1 0.033 0.80 0

Gramineae

Grass mixture 443.3 196.3 79.3 4.34

Maize 2.6 0.074 0.61 0.004

Solanaceae

Tobacco 15.4 0.14 0.67 0.046

Tomato 0 0017 0 0

As modified from [7].

9. The enzymes were able to transform several hydroxy

benzoic acids, and the enzyme activity depended on the

pH and the nature of the electron donors. At pH 7.0, the

salicylhydroxamic acid was oxidized in its quinonic form,

whereas at pH 3.0 and in the presence of a secondary sub-

strate, such as NADH, it served as redox mediator, and

allowed the peroxidase-dependent oxidation of NADH.

2.2. Microbial extra cellular enzymes

2.2.1. Microbial oxidoreductases

In vivo microbial oxidoreductases are periplasmic en-

zymes associated with the cell surface of viable cells.

Their main sources are fungi such as wood-degrading ba-

sidiomycetes, terricolous basidiomycetes, ectomycorrizal

fungi, soil-borne microfungi, and actinomycetes.

A great interest is growing for the use of fungi as biore-

mediating agents [10–13]. Most fungi are robust organisms

and may tolerate higher concentrations of pollutants than

bacteria. In particular, white-rot fungi appear unique and at-

tractive organisms for the bioremediation of polluted sites

for several reasons [10,14]. They can be summarized as fol-

lows:

(1) White-rot fungi are ubiquitous organisms in natural

environments.

(2) White-rot fungi are unique among eukaryotic or

prokaryotic microorganisms, because they posses

a very powerful extra cellular oxidative enzymatic

system: the lignin-degrading enzyme system (LDS),

which has broad substrate specificity and is able to ox-

idize several environmental pollutants [10,13]. Othernon-ligninolytic enzymes, like cellobiose dehydroge-

nases, may participate in the transformation of pol-

luting substances [11,12]. Cellobiose dehydrogenases

are usually secreted under non-ligninolytic condi-

tions when cellulose is the nutrient carbon, and either

directly or indirectly they may oxide several contami-

nants [11,12]. As a consequence, a vast range of toxic

environmental pollutants, including low soluble com-


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thalene), that were not substrates of LiP action, suggested

that other enzymes (i.e. monoxygenases) could be involved

in the transformation of PAHs by the fungus [33].

2.2.2. Microbial hydrolases

The other microbial enzymes involved in the pollutant

transformation are hydrolases. Several bacteria and fungiproduce a group of extra or ecto cellular enzymes that in-

clude proteases, carbohydratases (e.g. cellulases, amylases,

xylanases, etc.), esterases, phosphatases and phytases. These

enzymes are physiologically necessary to living organisms.

Some of them (e.g. proteases and carbohydratases) catalyze

the hydrolysis of large molecules, such as proteins and car-

bohydrates, to smaller molecules for subsequent absorption

by cells. Others, like phosphatases and phytases, contribute

to the nutrition of plants and microbes by hydrolysis of or-

ganic P compounds into inorganic P, the only form of phos-

phorous available to plants and microbial cells.

Due to their intrinsic low substrate specificity, hydrolases

may play a pivotal role in the bioremediation of severalpollutants including insoluble wastes.

A list of microbial hydrolases involved in the transfor-

mation of natural and non-natural insoluble compounds,

their preferential substrates and main sources is reported

in Table 3. Keratinic wastes deriving from animal breed-

ing, processing and handling, as well as used paper prod-

ucts discarded by human population, may contribute largely

to enrich the environment with solid wastes. Singh [36]

showed that the C. keratinophilum fungus, isolated by a

waste site containing organic pollutants, was able to pro-

duce an extra cellular protease with an optimum proteolytic

activity against keratin substrates. As previously demon-strated by Kornillowicz-Kowalska [41], no clear correlation

was, however, observed between the rate of keratin degra-

dation and the enzyme production, thus indicating that other

non-enzymatic factors were involved. The author concluded

that the fungus and its proteolytic activity have a great po-

tential for the in situ bioremediation of keratinous wastes.

Table 3

Biodegradation of natural and non-natural insoluble materials by microbial hydrolases

Material Enzyme Source References

Natural materialsCellulose materials Cellulase Trichoderma resei, Penicillium funiculosum [34]

Chitin Chitinase Actinobacteria [35]

Keratin Keratinase Chysosporium keratinophilum [36]

Kraft pulp Xylanase, -xylosidase Sreptomyces thermoviolaceus [37,38]

Sewage sludge Protease, phosphatase Sulphate reducing bacteria [39]

Starch materials Amylase Bacillus licheniformis [40]

Non-natural materials

Nylon Nylon-degrading enzyme (MnP) Un-named white-rot fungus (+Mn and lactate) [43,44]

Poly(l-lactic acid) Depolymerase, alkaline protease Amycolatopsis, Bacillus sp. [45,46]

Polyacrylate Cellobiose dehydrogenase White-rot fungi [11]

Polyurethane Esterase Curvularia senegalensis (not wrf),

Corynebacterium, Comamonas acidovarans

[47–49]

Polyvinyl alcohol 2-4-Pentanedione esterase, laccase Pseudomonas vesicularis, Pycnoporus cinnabarinus [50,51]

An interesting use of cellulases was reported by van Wyk

[34], who demonstrated that cellulases from Penicillum funi-

colosum and Tricoderma resei transformed paper materials

of different origin (e.g. foolscap, filter, office, and newspa-

per and microcrystalline cellulose (MCC)), and contributed

to the treatment of solid municipal wastes. Both cellulases

degraded all paper wastes though with a different efficiency.When the two fungal enzymes were tested in combination,

the susceptibility of the cellulose substrates to their hy-

drolytic degradation depended on the different ratio of the

two cellulases in the mixture (Fig. 3).

In a recent paper, Metcalfe et al. [35] investigated an ex-

tra cellular group of bacterial hydrolases: the chitinases, that

hydrolyze the 1–4 glycosidic bonds of chitin in soil. The

authors reported the first molecular ecological assessment

of chitinase diversity within a terrestrial environment. Their

results confirmed that actinobacteria are important effective

agents in chitin degradation in soil and that amendment ap-

plication such as sludge application may contribute to affect

the presence, the activity and diversity of these chitinases. Inparticular, the authors observed that an increase of enzyme

activity but a decrease of chitinase diversity occurred upon

sludge application.

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].

The extra cellular phytases were found 60% more efficient

than their intracellular counterparts; whereas most of the

acid and alkaline phosphatase activity was found inside the

cell.

Pollutants of a great environmental concern are plastics,i.e. synthetic, man-made materials widely used in modern

society for several purposes. Polyurethanes, polyacrylates,

polylactides, nylon, starch polymers, and mixed composites

of different starting materials are widely applied in the med-

ical, automotive and industrial fields. These materials are

highly recalcitrant and when discarded in the environment,


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waste treatment were previously reviewed by Nannipieri and

Bollag [55], Karam and Nicell [56] and Nicell [57].

As pointed out by the authors, 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; their capa-

bility to act in the presence of many toxic, even recalcitrant,substances, and/or under a wide range of environmental

conditions, often unfavourable to active microbial cells (i.e.

relatively wide temperature, pH and salinity ranges, high

and low concentrations of contaminants); and their low

sensitivity or susceptibility to the presence of predators,

inhibitors of microbial metabolism, and drastic changes in

contaminant concentrations.

Table 4 illustrates a wide range of cell-free enzymes ap-

plied to the biodegradation of different pollutants.

Pesticides of different chemical nature, very recalcitrant

compounds like asphaltenes and PCBs, polychlorophe-

nols, PAHs and others toxic pollutants were successfully

transformed by oxidoreductases and hydrolases isolated byfungal, bacterial and plant cells. The majority of results

summarized in Table 6, however, have been obtained under

laboratory conditions. As it will be addressed below, several

causes concur to hamper and render difficult the use of iso-

lated enzymes as tools in the detoxifications of polluted sites.

The three- and four-ring polycyclic aromatic hydrocar-

bons anthracene, phenanthrene, pyrene and fluoranthene

were in vitro-oxidized by extra cellular lignin peroxidase,

manganese peroxidase and laccase, prepared from the white

rot fungus Nematoloma frowardii and by mush-room ty-

rosinase (Tyr) and horseradish peroxidase (HRP) [58]. LiP

transformed 58.6% of anthracene and 34.2% of pyrene,whereas 31.5% of anthracene and 11.2% of pyrene were ox-

idized by MnP. In the presence of the mediating substances

veratryl alcohol (for LiP), reduced glutathione (GSH) (for

MnP), and ABTS (for L, Tyr, HRP), the transformation of

Table 4

Biodegradation of pollutants by cell-free enzymes

Pollutant Enzymes Source Properties References

Anthracene, pyrene LiP, MnP, laccase Nematoloma forwardii High (veratryl alcohol) [58]

Asphaltenes Chloroperoxidase Cladariomyces fumago High activity in organic solvents [59,60]

Carbofuran, carbaryl Carbamate hydrolase Achromobacter sp.,

Pseudomonas sp.

Cytosolic, high specificity [61,62]

Cyanides Cyanidase, cyanide hydratase Alcaligenes denitrificans, several

fungi

Usually inducible, very stable in

immobilized form

[63–66]

Estrogenic chemicals MnP, laccase Trametes versicolor 70–100% transformation [67]

Nitrile compounds Nitrilase, nitrile hydratase,

amidase

Nocardia sp., Rhodococcus sp.,

Fusarium solani

High stability up to 60 ◦C and

pH 6.0–11.0

[68]

PCP, DDT, PCBs, lindane Dehalogenases, laccase Several microorganisms Sensitivity to SH-agents,

stereospecificity

[54,69]

Phenols, PAH MnP, LiP, laccase,

chloroperoxidases, peroxidase

White-rot fungi Very versatile under different

operational conditions

[57,70–72]

Pyrethroids, parathion,

coumaphos, diazinon

Agrobacterium, Pseudomonas sp.,

Flavobacterium sp., Nocardia sp.,

Bacillus cereus

High stability at 50 ◦C and pH

5.5–10.0

[73–75]

DDT: dichloro diphenyl tricholorethane. For other compounds and enzyme symbols see Table 2.

Table 5

Biodegradation of phenols by cell-free laccase from Cerrena unicolor

Substrate –(OH) Substituent Substrate

decrease (%)

o-Cl-phenol 1 −1Cl 18

p-Cl-phenol 1 −1Cl 20

2,4-DCP 1 −2Cl 66

p-Tyrosol 1 –CH2CH2OH 73

o-Tyrosol 1 –CH2CH2OH 28

m-Tyrosol 1 –CH2CH2OH 11

Catechol 2 – 100

Resorcinol 2 – 40

Methylcatechol 2 –CH3 76

Hydroxytyrosol 2 –CH2CH2OH 86

Pyrogallol 3 – 100

Gallic acid 3 –COOH 98

From [70,71].

PAHs was enhanced in most cases. Furthermore, studies

performed with PAH-derivatives, known as intermediates or

potential dead-end-products of microbial PAHs metabolism,

demonstrated that the hydroxylated PAHs metabolites were

oxidized by all the oxidoreductases, whereas PAH-quinones

and oxo-metabolites were not transformed.

The studies by Bollag and co-workers (for a detailed list

see Table 2 and reported references in [18]) and Gianfreda

and co-workers [70,71,76–78] have demonstrated that lac-

cases from different fungal origins were capable of removing

a large variety of phenols and the efficiency was strictly de-

pendent on the chemical structure of the phenol, the type and

the number of substituents on the aromatic ring (Table 5).

An interesting application of fungal oxidases with es-trogenic chemicals was shown by Tsutsumi et al. [67].

Estrogenic chemicals are man-made chemicals that mimic

the effects of hormones (particularly estrogens). Like nat-

ural estrogens they can bind to the estrogen receptor and


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Fig. 4. Bisphenol (BPA) and nonylphenol (NP) disappearance by oxidoreductases from various origins (from [67]).

regulate the activity of estrogen responsive genes. Such

effects have raised concern that prolonged exposure to

environmentally relevant concentrations of these chem-

icals may adversely affect reproduction in wildlife and

humans.

Bisphenol A (2, 2-bis(4-hydroxyphenyl) propane; BPA)

and nonylphenol (NP) are widely used in a variety of

industrial and residential applications, and are suspected

of having estrogenic (endocrine-disrupting) activity. As

shown in Fig. 4 both BPA and NP disappeared within

1 h-treatment with a MnP, isolated and partially purified

from the strain P. chrysosporium ME-446. A laccase, par-

tially purified from T. versicolor IFO7043, also removed

BPA by 70% and NP by 60%, and the addition of the

mediator 1-hydroxybenzentriazole (HBT) in the reaction

system greatly improved the laccase potential. Analysis by

gel permeation chromatography indicated oligomers as themain products of BPA and NP enzymatic transformation

[67].

The greatest concern for the biodegradation of estrogenic

chemicals is aimed at the removal of their estrogenic ac-

tivity. In vitro screening tests for chemicals with hormonal

activities using yeasts [79] were utilized to evaluate the es-

trogenic activity of BPA or NP after the enzymatic action

[67]. Both Mn peroxidase and laccase removed the estro-

Fig. 5. Decrease of bisphenol (BPA) and nonylphenol (NP) estrogenic activities by oxidoreductases from various origins (from [67]).

genic activities of BPA and NP within 12 h. Similar results

were obtained within 6 h with laccase in the presence of HBT

(Fig. 5). The overall results led the authors to explain the

BPA and NP transformation mechanisms as due to the poly-

merization and partial degradation of the chemicals brought

about by enzymatic oxidation [67].

Another interesting application of cell-free enzymes is

the use of the so-called nitrile-degrading enzymes capable

of degrading nitrile compounds [68]. Nitrile compounds

are widespread in the environment. They comprise some

of plant origins, such as cyanoglycosides, cyanolipids, rici-

nine, phenylacetonitrile, etc. Nitrile compounds are also

extensively used in chemical industries to produce a variety

of polymers and other chemicals. Other different nitrile

compounds are used as feedstock, solvents, extractants,

pharmaceuticals, drug intermediates (chiral synthons), pes-

ticides (dichlobenil, bromoxynil, ioxynil, buctril), or asintermediates in the organic synthesis of a variety of differ-

ent compounds (e.g. amines, amides, amidines, carboxylic

acids, esters, aldehydes, ketones and heterocyclics).Most ni-

triles are however highly toxic, mutagenic and carcinogenic

in nature (Pollak et al. [80]). If present in the environment at

high concentrations they may cause severe diseases in hu-

mans. Consequently, efforts have been made to control their

release in the environment, and/or to remove them from it.


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Table 6

Effects of co-substrates on bentazon transformation by oxidoreductases

Humic monomers Laccaseb

(pH 4.0)

Peroxidasec

(pH 3.0)

Bentazona 0 6

+Catechol 100 95

+Ferulic acid 9 19

+Guaiacol 10 9

+Protocatechiuc acid 40 65

+Pyrogallol 0 0

+Resorcinol 2 0

+Syringaldeyde 39 49

+Vanillic acid 6 94

+Caffeic acid 59 27

Modified from [86].a One mM for all the compounds.b From Polyporus pinsitus. Incubation with 4 Units ml−1 for 24h at

25 ◦C.c From horseradish. Incubation with 6 Units ml−1 for 24 h at 25 ◦C.

references therein). The nature of polymeric products as wellas the possible mechanisms involved in the process was ad-

dressed. These model system studies were aimed at achiev-

ing a basic understanding of the possible mechanisms for

oxidative coupling reactions occurring in natural soils and

sediments [85].

Increases and/or decreases of the pollutant transformation

measured as both polymerization or mineralization of the

target pollutant were detected. Kim et al. [86] demonstrated

that the transformation of bentazon, a very recalcitrant herbi-

cide, by laccase or peroxidase may be completely annulled,

or greatly enhanced, by the co-presence of a variety of sub-

stances that are precursors of humic materials (Table 6).In our studies we have also demonstrated that for exam-

ple the 2,4-dichlorophenol (2,4-DCP) decrease by laccases

from different origins (i.e. Cerrena unicolor , Trametes vil-

losa) may be differently affected by the co-presence of

one or two other chlorophenols such as 4-chlorophenol or

2,4,6 thriclorophenol (Table 7) [87]. The 2,4-DCP-laccase

mediated decrease can also be affected by the presence of

its parent precursor, 2,4D, another phenol such as catechol,

or simazine, that is usually supplied to soil in combination

with 2,4D (Table 7) [71]. In another study, enhancing or

depressing effects of a laccase from Rhus vernicifera on

catechol transformation were measured in a model system

of four phenols (catechol, methylcatechol, tyrosol and hy-

droxytytrosol) [78]. Binary, ternary, and quaternary aqueous

phenolic mixtures were investigated (Table 7). The model

system simulates a typical wastewater deriving from an

olive oil factory. In Mediterranean countries, large quan-

tities of wastewater with a high content of phenolic sub-

stances are usually produced as characteristic by-products

of olive-oil production. The main constituents are usually

the compounds used in the model system.

Differentiated effects on the enzyme activity were also ob-

served by the presence of a polluting substance non-substrate

of the enzyme. Investigations performed under laboratory

Table 7

Substrate removal and residual enzymatic activity after laccase action

Substrate Substrate

decrease (%)∗Laccase

activity (%)

Single phenols Cerrena unicolor

2,4-DCP 66 34

Tyrosol 11 88

Resorcinol 40 76Methylcatechol 76 24

Hydroxytyrosol 86 18

Pyrogallol 100 89

Gallic acid 98 83

Phenol mixtures Trametes villosa

2,4-DCP 66 34

+4-CP 56 56

+2,4,6-TCP 58 58

+2,4-D 82 20

Cerrena unicolor

+Catechol 77 20

+Simazine 46 30

+4-CP + 2,4,6-TCP 50 35

+2,4-D + Simazine 0 95+Catechol + Simazine 39 60

+2,4-D + Catechol 100 0

Rhus vernicifera

Catechol 58 70

+Methylcatechol (M) 38 11

+Tyrosol (T) 100 66

+Hydroxytyrosol (H) 99 27

+M + T 16 9

+T + H 95 29

+M + H 56 10

+M + T + H 63 11

The substrate decrease values have been normalized by laccase units.

From [70,71,78,87].

conditions with acid phosphatase from sweet potato a typ-

ical, extra or ecto cellular enzyme, and pesticides such as

carbaryl, atrazine, and glyphosate demonstrated that the ac-

tivity of the enzyme was depressed by atrazine and carbaryl

(40 and 34% of activity reduction, respectively), whereas no

effects were measured with glyphosate [88].

A key factor possibly affecting the pollutants transfor-

mation is the availability of the polluting substance to the

detoxifying agent, that is its bioavailability [89]. Bioavail-

ability is affected by both the inherent properties of the

pollutants (i.e. concentration, molecular structure, water or

organic solubility) and the characteristics of the environ-

ment where they occur.

In soil, several properties such as the amount of or-

ganic matter, the thickness of silt and clay beds, the pres-

ence of dissolved organic matter, the soil aggregation and

sub-superficial heterogeneity can all influence the bioavail-

ability of pollutants [90].

Adsorption and/or entrapment of pollutants on/into or-

ganic and inorganic soil colloids and in soil matrix further

complicate the whole system, and can limit the amount of

pollutant available to microbial cells and their extra cel-

lular enzymes. Their limiting effect can be greater toward


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isolated enzymes. Aging processes can also lead to a sig-

nificant reduction of pollutants bioavailability, because of

their sequestration from diffusion into sites within sorbing

matrices or entry into nanopores, both not easily accessible

to large molecules as enzymatic proteins [91].

4.2. Enzyme-derived disadvantages

Disadvantages to the in situ application of extra cellular,

cell-associated or cell-free enzymes may also arise from the

enzymes.

Enzymes may lose their activity upon pollutant transfor-

mation. In the experiments on phenols previously shown in

Tables 5 and 7, the residual activity of laccase was measured

after phenol transformation under standard conditions [71].

The results indicated that the enzyme partially, or totally,

lost its catalytic activity (Table 7). The reduction of activ-

ity was quite dependent on the type of phenol, the entity

of its transformation, and the combination of the phenolsin the mixture. Usually, the higher the phenol concentra-

tion, the more pronounced was the loss of laccase activity.

Measurements of residual laccase activities made in experi-

ments performed with increasing concentrations (from 0 to

0.4 mM) of 2,4-DCP ruled out a possible inhibitory effect

on laccase activity by 2,4-DCP. Indeed, activity tests per-

formed on samples filtered with filters that specifically ad-

sorb 2,4-DCP gave activity values similar to those obtained

without sample filtration. The authors explained their results

as due to the a progressive entrapment and/or adsorption of

active enzyme molecules within/on phenol polymeric aggre-

gates as they formed [70]. These processes may represent

the main mechanism of inactivation because they hinder the

interaction between the substrate and the enzyme [92].

In the soil, enzymes are present in complex, three-

dimensional assemblages of mineral and organic particles

that will restrict their mobility and affect their activity

(Fig. 6) [6,93–95]. Enzyme molecules can be adsorbed, im-

mobilized, or entrapped in such matrices giving rise to the

so-called “naturally immobilized enzymes” [94]. Inactiva-

tion or degradation of enzymatic molecules may also take

Fig. 6. Soil bound enzymes.

Fig. 7. Removal of 2,4-DCP by laccase in the presence of clays, clay-humic

complexes, sand and soils. M: Na-montmorillonite; AM: montmorillonite

covered by different amounts of OH aluminum species (AM3 and AM18,

loading 3 and 18 mmol of Al per gram of montmorillonite); HA: a humic

acid.

place. As a consequence, changes in their kinetics, stabil-

ity and mobility will occur [96]. These latter, in turn, will

determine the operating range of enzymes in soil around

microorganisms and enzymes.

Investigations were performed to evaluate the effective-

ness of laccases from different origins towards the 2,4-DCP

removal when in the presence of different soil components

[70,71,76,77,97]. A montmorillonite (M), a montmorillonite

covered by different amounts of OH aluminum species

(AM3 and AM18, loading 3 and 18 mmol of Al per gram

of montmorillonite), a humic acid (HA), a combination of

HA and AM18, sand and soils with different organic mattercontents were utilized. As respect to the control, i.e. the

enzymes alone, soil components differently affected the

2,4-DCP-laccase activity (Fig. 7). Strong depressing effects

were measured in the presence of montmorillonite, AM18and the combination AM18 +HA. When soils were used,

the reduction of laccase activity increased by increasing the

organic matter content of soils [97].

The immobilization of enzymes on soil components

affected the response of the enzyme to the presence of sub-

stances extraneous to the reaction substrates, as well. When

the influence of carbaryl, atrazine, and glyphosate was

tested on acid phosphatase immobilized on organic (tan-

nic acid), inorganic (montmorillonite) and organo-mineral

(Al(OH) x-tannic acid-montmorillonite ) complexes a dif-

ferent behavior as respect to the free enzyme was observed

[88]. Different causes were invoked by the authors to ex-

plain their results. They related to: (i) the “state” of the

enzyme if free or immobilized on soil components, that

makes the whole system homogenous or heterogeneous; (ii)

the nature of interactions occurring between the enzyme and

the immobilizing support; (iii) the conformational changes

achieved by the enzymatic protein upon immobilization;

and (iv) the influence of the microenvironment created by

the support in the surrounding of the protein [88,95].


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As regards to isolated enzymes, a drawback that greatly

hampers their practical application is the cost of enzymes

isolation and purification. Given the best producer of the se-

lected enzyme, the production of a purified enzyme requires

long and expensive isolation and purification procedures.

Furthermore, very low amounts of the purified protein

are usually obtained thus rendering the whole process toocostly for practical applications. Alternative inexpensive

technologies, using for instance agricultural wastes as the

carbon source, may be adopted to increase the growth of

the enzyme-producing microorganisms and to significantly

reduce the cost of enzyme production [98]. Even if the

enzymes are available at very low costs, their use in soil

as free proteins may be hindered by their short life in an

inhospitable environment such soil [54,55,94].

An interesting, low-cost alternative to the use of pu-

rified enzymes can be the utilization of plant material

loading enzymatic activities and proved to be effective

in the detoxification of polluting substances. As it is the

case of the results obtained in the investigations performedwith minced horseradish roots [99,100] and olive husk, a

by-product of olive oil production showing phenoloxidase

activity [101,102], used for the successful transformation

of phenolic pollutants.

Improvements in the production of isolated enzymes may

also derive from molecular technologies. Production of an-

imal and plant enzymes could be performed by means of

genetically modified microorganisms or plants [103,104].

The range of sophisticated modern molecular technolo-

gies now available has provided the researcher with an im-

mensely enhanced choice of potential enzyme sources. For

example, an increasing knowledge of several aspects of theenzymology and molecular biology of the powerful extra

cellular oxidative system (LDS) of fungi is now available

[10,13]. The major genes encoding LiPs, MnPs, and laccases

were cloned and sequenced. The regulation of expression

of the key ligninolytic genes were studied, and the X-ray

crystallographic structures of the LiP and MnP isozymes are

now known and available. These advancements should lead

to the successful genetic engineering of white-rot fungi and

their enzyme systems. Most advantageous bioremediation

strategies for treating contaminated sites can thus possibly

be planned and applied.

The engineering of microbes and enzymes may also help

to improve bioremediation, particularly when the applica-

tion on a large-scale of the selected biological catalyst is

hampered by the low rate of pollutant degradation [105].

As claimed by Chen and Mulchandani [106], recent advan-

tages in genetic and molecular techniques have offered new

chances to modify microbes or enzymes to function as “de-

signer biocatalysts” in which certain required qualities from

different organisms are brought together in a single catalyst

to perform specific bioremediation.

An example is that regarding the transformation of

organophosphates by the so-called “live biocatalysts” [106].

Organophosphates, like parathion, paraoxon, diazinon,

coumaphos, and malathion, are among the most toxic pesti-

cides used in agriculture. They are easily degraded by two

bacterial hydrolases, organophosphorus hydrolase (OPH)

and organophosphorus acid anydrase (OPA), giving rise to

less toxic compounds and further degradable by biological

and chemical processes.

Organophosphate hydrolase is an enzyme produced andisolated by several soil microorganisms, including many

Pseudomonas strains. It is very effective in degrading

these compounds but its practical application is hindered

by the high cost of its purification. By contrast, the use

of whole-cells producing the enzyme can be more cost

effective, but their use is restricted by transport barrier of

organophosphates across the cell membrane [107,108].

Parathion and paraoxon were efficiently detoxified with

OPH anchored and displayed on the cell surface of Es-

cherichia coli. The recombinant whole-cells with surface-

expressed OPH were obtained using the same system used

for the expression of -lactamase on E. coli [108]. The sys-

tem is made by the fusion of the N-terminal lipoprotein se-quence (Lpp), the domain of the pore-forming protein OmpA

and the enzyme. The Lpp directs the Lpp–OmpA–OPH fu-

sion 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. 8). These “live biocatalysts” dis-

played about seven times higher activity than E. coli whole-

cells expressing similar amounts of intracellular OPH, thus

demonstrating the higher efficiency of the enzyme when

acting outside the cell. Furthermore, the enzyme showed a

higher stability to both temperature (100% of activity upon

30 day-incubation at 37 ◦C) and organic solvents.

Enzymes immobilized on natural and synthetic supportsof 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 [54,55]. Immobilized enzymes have usually

a long-term and operational stability, being very stable to-

ward physical, chemical, and biological denaturing agents.

Furthermore, they may be reused and recovered at the end

of the process. While the use of immobilized enzymes

is widely diffused in several applicative fields, including

environmental applications [55–57,109], their large-scale

application in the bioremediation of polluted soils is not

reported, at least to our knowledge.

The potential of enzymes for bioremediation purposes can

greatly increase by the use of microorganisms and their en-

zymes from extreme environments. Enzymes from both ther-

mophilic and psycrophilic microorganisms usually display

some unusual and particular features that may render them

potential, powerful catalysts for the degradation of pollut-

ing chemicals ([110] and references therein, [111]). Ther-

mophiles and psycrophiles have to adapt themselves to live

and survive under extreme environmental conditions [112].

As a consequence, specialized enzymatic proteins, particu-

larly stable to different denaturing agents, and with elevated

catalytic activity, are produced.


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Fig. 8. Parathion detoxification by organophosphate hydrolase (OPH) anchored and displayed on the cell surface of E. coli (from [106]). Lpp: lipoprotein;OmpA: pore forming protein.

The molecular, structural, kinetic and genetic properties of

such enzymes have been in most cases elucidated. Cloning

and expression of genomic related information in heterol-

ogous, fast-growing meshophiles, like E. coli, has allowed

an increased, much less cheaper commercial production of

thermophilic enzymes [113–115]. An increasing knowledge

of cold-adapted enzymes properties is now being develop-

ing, as well [110].

Many industrial applications have benefited from the useof these enzymes. More challenges and potential advantages

may also be envisaged for their application in biotechnol-

ogy including bioremediation ([110] and references therein,

[111]).

5. Conclusions

In conclusion, several extra cellular enzymes, either as

cell-associated or cell-free enzymes, may behave as powerful

catalysts in the biodegradation of harmful pollutants. How-

ever, their large-scale application for remediation of polluted

soils is still limited. This may derive from several drawbacks

and disadvantages depending on both the pollutants and the

enzymes. For instance, the simultaneous presence of several

polluting substances in a contaminated site with synergistic,

often negative, effects on the enzyme efficiency, the high

costs associated with the isolation and purification of free

enzymes, the low stability of enzymes to the harsh condi-

tions of soil all concur to restrict the wide use of enzymes as

remediating agents of polluted soils. Although immobilized

enzymes may present a high stability under soil conditions,

they are not widely applied in the remediation of polluted

soils.

However, exploration of extreme environments, exploita-

tion of genome using advanced technologies, and protein

engineering have opened new frontiers for the production

and application of enzymes. As mentioned by Burton [84]

these achievements has lead to the point where rather than

develop a purpose for an enzyme, it is possible to design

and develop an enzyme for a purpose. This fascinating per-

spective has to resolve a still opened question: what should

be the features of an enzyme to be suited for remediation of soils?

Finally, a successful management or remediation of a con-

taminated site should make the site environmentally agree-

able and usable for some acceptable purposes. This means to

adopt appropriate cleanup criteria and determine what con-

stitutes an environmental acceptable endpoint.

Acknowledgments

This research was partly supported by European Commu-

nity, Project INCO-MED Contract no. ICA3-2002-10021.The authors are grateful to Dr. Luigi Pagano for his help

in the preparation of figures and to Dr. Anna Maria Woods

for her technical help in editing the paper in respect to the

English language and style. DiSSPA Contribution no. 0062.

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