The prospects for peroxidase-based biorefining of petroleum fuels

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ORIGINAL ARTICLE

The prospects for peroxidase-based biorefining of petroleum fuels

MARCELA AYALA, JORGE VERDIN, & RAFAEL VAZQUEZ-DUHALT

Departamento de Ingenierıa Celular y Biocatalisis, Instituto de Biotecnologıa, Universidad Nacional Autonoma de Mexico,

UNAM. Apartado Postal 510-3 Cuernavaca, Mor. 62250 Mexico

AbstractPeroxidases have many potential uses for biotechnological processes. In this review, peroxidase-catalyzed reactionspotentially applicable to the petroleum industry are described. Although peroxidases are attractive catalysts fordesulfurization, aromatic oxidation and asphaltene transformation, there are important issues that must be overcomebefore any industrial application can be considered. The opportunities and challenges of enzymatic petroleum biorefiningare documented and discussed, with emphasis on the available tools to design a biocatalyst with appropriate performancefor the oil and other industries.

Keywords: Petroleum biorefining, peroxidase, biocatalysis, desulfurization, biocracking, oxidative inactivation

Introduction

The exploitation of petroleum as a primary source

of both energy and raw material began a century

ago. Our society is highly dependent on oil for

energy, transportation and general industrial pro-

duction. Doubtless, history will describe our time

as the oil-based society. The world’s oil took

500 millions years to accumulate by nature, and it

will be consumed in only two centuries. The

production peak is estimated to occur sometime

between 2010 and 2020, and, by the end of this

century, oil resources will be drastically reduced

(Hall et al. 2003). When the world’s oil reserves

become scarce, more expensive fuel sources, such

as hard-to-extract oil deposits, tar sands, and

synfuels from coal, will come to the fore of

production (Figure 1).

The US Geological Survey recently estimated that

one trillion barrels have already been harvested, and

that about three trillion barrels of oil remain to be

recovered worldwide, half from proven reserves and

half from undeveloped or undiscovered sources

(Hall et al. 2003). On the other hand, the Institute

for the Analysis of Global Security (IAGS 2005)

estimated that since the shift from coal to oil,

the world has consumed over 875 billion barrels,

and less optimistically estimates that another

1000 billion barrels of proved and probable reserves

remain to be recovered. Finally, the Oil and Gas

Journal estimates that at the beginning of 2004,

worldwide reserves were 1.28 trillion barrels (Radler

2004). Thus, we can conclude from these estimates

that at least an equivalent amount of the total oil

consumed during the past 100 years is still to be

produced, but may require new methods of refining

during the next century.

Environment is a key issue for the petroleum

industry. Serious adverse environmental impact

arises from the emission of reactive hydrocarbons,

carbon monoxide, sulfur oxides and nitrogen oxides

when fossil fuels are burned. From the onset of the

Industrial Revolution in the eighteenth century up to

the mid-1970s, the emission of acidifying com-

pounds to the atmosphere increased steadily. These

pollutants are transported through the atmosphere

and deposited as ‘acid rain’, causing acidification of

soils and surface waters, with adverse effects on

terrestrial and freshwater biota. Increasing public

pressure to limit or reduce these emissions will

severely constrain the use of fossil fuels as a source

of energy supply. Unless this lost fossil fuel output is

replaced on a large scale by acceptable alternatives,

the cost, in terms of economic development fore-

gone, will be very high.

Correspondence: M. Ayala, Departamento de Ingenierıa Celular y Biocatalisis, Instituto de Biotecnologıa, Universidad Nacional

Autonoma de Mexico. A.P. 510-3. Cuernavaca, Morelos 62250, Mexico. Fax: 52 777 3172388. E-mail: [email protected]

Biocatalysis and Biotransformation, March�August 2007; 25(2�4): 114�129

ISSN 1024-2422 print/ISSN 1029-2446 online # 2007 Informa UK Ltd

DOI: 10.1080/10242420701379015

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In recent decades, there have been concerted

attempts to reduce the emission of the problematic

sulfur dioxide and nitrogen oxides, and it is encoura-

ging to see new evidence that these measures are

beginning to take effect. Emissions of sulfur dioxide

(SO2), declined in the UK and the EU by 71 and

72%, respectively, between 1986 and 2001, while

nitrous oxide emissions declined by about 40%

(Fowler et al. 2005). The success of the policy of

low-sulfur fossil fuels is clear. There is evidence that

reduced sulfur deposition has led to significant

improvements in the chemical status of acidified

surface water throughout Europe and North Amer-

ica (Skjelkvale et al. 2005).

On the other hand, limits on the aromatics

content in fuels have also been prescribed. Since

1993, the aromatic hydrocarbon content in diesel

fuel has been set to 10% in California and 11% in

Europe. A low content of aromatic hydrocarbons in

fuels reduces the emission of carcinogenic sub-

stances, such as benzene and polycyclic aromatic

hydrocarbons (PAH).

Heavy oil deposits, tar-sands and synfuels as fuel

sources imply a high environmental impact because

of their high sulfur, nitrogen, aromatic and heavy

metal contents. These components should be re-

moved in order to obtain cleaner fuels. New

technologies should improve the refining efficiency

in terms of the consumed energy and the environ-

mental impact of the processes. Development and

implementation of new technologies for conven-

tional processes, such as cracking, hydrogenation,

isomerization, alkylation, polymerization and hydro-

desulfurization, may be expected. There is also

scope for the introduction of non-conventional

technologies, representing potential substitutes or

complementary processes to traditional oil refining.

The introduction of non-conventional biotechnolo-

gies in the petroleum industry may improve its

energetic efficiency and, overall, reduce its environ-

mental impact.

It is important to note that, so far, no enzymatic or

biochemical processes exist in the oil refining in-

dustry. Thus, the goal of this work is to assess the

prospects for enzymatic transformations of petro-

leum products and their derivatives, in order to

evaluate the possible introduction of biotechnologi-

cal processes in the petroleum refining industry.

Enzymatic petroleum biorefining

The application of biotechnology to fuel refining has

been investigated over the last two decades (Ayala &

Vazquez-Duhalt 2004). The main driving force has

been the demand for environmentally benign fossil

fuels, accentuated by the fact that such fuels

are increasingly difficult to obtain as oil with low

impurities is becoming scarce. The main objective is

obtaining a robust biocatalyst, efficient enough to

become commercially successful, as well as design-

ing a suitable bioprocess, compatible with refinery

facilities. There are some examples of biotechnolo-

gies potentially applicable to fuel refining (Vazquez-

Duhalt et al. 2002b; Le Borgne & Quintero 2003).

One of the more advanced lines of research is the

reduction of the sulfur content in diesel by applying

whole bacterial cells to oil�water emulsions. In this

schema, the role of bacteria is to degrade sulfur-

containing compounds to produce sulfur-free hydro-

xylated hydrocarbons and water-soluble sulfur salts.

The most studied bacteria, Rhodococcus erythropolis

IGTS8, was first isolated at the Institute of Gas

Technology in 1990 (Kilbane 1992). After more

than 10 years of research by Energy BioSystems

Corporation, an intensively engineered bacteria was

obtained, two orders of magnitude more active than

the original strain (McFarland 1999). Nevertheless,

further biocatalyst rate improvement is still needed,

and a low water process is still to be developed in

order to make the process commercially attractive to

the refineries. Although the project reached pilot-

plant scale, no additional progress has been re-

ported.

Besides whole-cell catalysts, another plausible

option offered by biotechnology is enzyme-based

catalysis. Enzyme-based catalysts have several ad-

vantages over whole-cell catalysts. In terms of

robustness, an enzyme may function in very low

water content environments; in contrast with cells,

an enzyme may even be less labile to thermal and

organic solvent denaturation if the environment is

hydrophobic; in terms of design, it is usually more

straightforward to modulate the kinetic and stability

properties of a single protein than the properties of

an enzymatic cascade, intrinsically constrained by

cell metabolism; finally, multiple strategies, such as

immobilization, solvent engineering and protein

200019501900 2050 2100

Conventionalpetroleum sources

Heavy oils andnon conventional

petroleum sources

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Figure 1. Petroleum availability estimation from the World

Resource Institute (1996).

The prospects for peroxidase-based biorefining of petroleum fuels 115

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engineering, may be combined in order to enhance

the desired characteristics of the enzyme.

Although sulfur content is one of the main

concerns of refineries around the world in terms of

fuel quality and environmental impact, other issues,

such as nitrogen content, aromatic and other heavy

molecule content, are also being actively studied.

These issues will acquire significance as heavier

oils become the common refinery feed, and refi-

neries will have to address the problem in order

to comply with environmental regulations. In the

next section, we analyze the potential of heme-

peroxidases, for the production of clean fossil fuels.

Heme-peroxidases as multifunctional catalysts

Peroxidases are enzymes able to catalyze oxido-

reduction reactions, using peroxide as electron

acceptor. Iron-peroxidases contain a prosthetic

heme group that functions as the active redox site.

Welinder (1992) proposed a classification of heme-

peroxidases according to their structural properties.

Three superfamilies of peroxidases have been de-

scribed: plant peroxidases, animal peroxidases, and

catalases. Due to their versatility, the enzymes

belonging to the plant superfamily are probably the

best candidates for industrial applications. The plant

peroxidase superfamily comprises three classes:

prokaryotic (class I), fungal (class II) and plant

(class III) peroxidases. All of these enzymes are

monomeric proteins with a non-covalently attached

ferriprotoporphyrin IX as prosthetic group, which is

co-ordinated to the enzyme via the imidazole side

chain of a His residue in the proximal pocket. On

the opposite, distal face of the heme, His and

Arg catalytic residues participate during the per-

oxide cleavage. Unlike prokaryotic peroxidases from

class I, fungal and plant peroxidases are glycosylated

(0�25%) and contain two calcium ions and

four disulfide bridges in their structure. Some

exceptions to this general classification can be

found, such as the heme-chloroperoxidase (CPO)

from Caldariomyces fumago (Sundaramoorthy et al.

1995). Representative members of this super-

family are cytochrome c peroxidase (CcP; class I),

lignin and manganese peroxidase (LiP and MnP;

class II) and horseradish peroxidase (HRP; class III).

The former and the latter have been extensively

studied and represent the best models for heme-

peroxidases. A comprehensive review on these en-

zymes is provided elsewhere (Erman & Vitello 2002;

Veitch 2004).

During catalysis, the oxidation of substrate mole-

cules results from the interaction with peroxide-

activated enzyme species (Figure 2). The cycle

begins when the two oxidative equivalents from the

peroxide are transferred to the enzyme to form

Compound I, a ferryl species (FeIV�O) coupled to

a free radical, located either on the porphyrin ring

or a nearby residue (Svistunenko 2005). Depending

on the substrate and the characteristics of the

enzyme’s active site, the two-electron reduction of

peroxide is coupled either to the one-electron (A,

Figure 2) or two-electron (B, Figure 2) oxidation of

substrate molecules. In pathway A, one-electron is

transferred from the substrate to the protein to form

Compound II, through interaction with the d-meso

heme edge (Ator & Ortiz de Montellano 1987;

DePillis et al. 1990; Gilfoyle et al. 1996) or through

long-range pathways, usually involving solvent-

exposed residues (Blodig et al. 2001; Perez-Boada

et al. 2005). The second enzyme intermediate,

Compound II, accepts a second electron to return

to the ground ferric state. In some enzymes, access

to the ferryl oxygen in Compound I results in two-

electron oxidation of substrates (pathway B); this

Figure 2. Peroxidase cycle showing classical peroxidase pathway (A) and peroxygenase pathway (B).

116 M. Ayala et al.

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peroxygenase activity might proceed in an enantio-

selective way (van Deurzen et al. 1997). The redox

potential, and hence the ability, of the enzyme

intermediates to oxidize certain substrates depends

on the heme environment, such as the residues

forming the heme cavity, as well as the topology of

the active site.

Peroxidases do not possess a highly substrate-

specific active site, where binding of a narrow set of

molecules take place, as in the classical key-lock

mechanism usually described for enzymes. On the

contrary, as most substrates interact with the rela-

tively exposed d-meso heme edge and with solvent-

exposed residues, peroxidases usually display low

specificity, and, thus, are able to catalyze the

oxidation of a variety of molecules (Smith & Veitch

1998). Table I lists a number of different reactions

catalyzed by peroxidases with different substrates.

Thus, the most attractive features for the potential

development of peroxidase-based industrial biocata-

lysts, are the strong oxidant character and low

specificity of these enzymes.

Peroxidase-catalyzed reactions applicable

for petroleum refining

Oxidation of aromatic compounds

The content of aromatic and PAHs in refined fuels

may be as high as 30%. Aromatic content is

undesirable, as incomplete combustion of these

molecules leads to the formation of particulate

matter, recognized as a health problem. Moreover,

some of the PAH species are highly recalcitrant and

mutagenic, so their release into the environment is

detrimental. The selective oxidation of aromatic

molecules could either facilitate their removal or

reduce their environmental impact.

Class II peroxidases are able to catalyze the

oxidation of PAH to produce quinones or hydro-

xylated species, in aqueous systems containing mis-

cible organic solvents (Vazquez-Duhalt et al. 1994;

Bogan et al. 1996; Wang et al. 2003). The products

have been found to be more water-soluble and

significantly less mutagenic than the parental com-

pounds, or not mutagenic at all. An inverse correla-

tion between the peroxidase specific activity and

the ionization energy of the substrates has been

observed. This correlation suggests that the mech-

anism involves the formation of free radical species

via oxidative dehydrogenation, followed by reaction

with oxygen or water to yield the final product. The

maximum substrate ionization energy varies depend-

ing on the enzyme, as shown in Table II. Some

significant issues, such as the ability of peroxidases to

catalyze this reaction in non-aqueous systems, as well

their efficiency in transforming polyaromatic com-

plex mixtures, remain to be studied.

Desulfurization

Current refining processes are able to reduce the

sulfur content of diesel fuels to B50 ppm (Song

2003). However, this technology is rather expensive

as it is based on metal catalysts that are prone to

inactivation in the presence of common fuel impu-

rities (such as nitrogen-containing compounds) and

it consumes hydrogen for sulfur elimination. The

technology is less efficient with chemically complex

species, such as large or sterically hindered com-

pounds. Thus, a complementary technology may

be useful to polish fuels that are not efficiently

and economically desulfurized by conventional

technologies.

Peroxidases have been shown to catalyze the

oxidation of sulfur-containing molecules. This reac-

tion proceeds through the peroxygenase pathway

(see Figure 2, pathway B). Particularly, CPO has

the highest sulfoxidation activity reported to date

for a heme-peroxidase (van Deurzen et al. 1997).

This enzyme has been described as an unusual

Table I. Examples of the diversity of reactions catalyzed by

peroxidases.

Activity Reaction catalyzed Substrates

Peroxidase Oxidative

dehydrogenation

Phenols, anilines, indoles

N- and O-

demethylation

Methoxylated aromatics,

n-alkylated anilines,

carbazoles

Oxidative

dehalogenation

Halogenated phenols

Peroxygenase N- and S-

oxidation

Aromatic sulfides, anilines

C�H bond

oxidation

Benzyl, allyl and propargyl

derivatives, indoles

Epoxidation Aromatic and aliphatic

olefins

Catalase Peroxide

dismutation

Hydrogen peroxide,

organic peroxides

Haloperoxidase Oxidative

halogenation

Alicyclic ketones, PAHs,

phenols, anilines, indoles,

flavonoids

Table II. Ionization energy limit for the peroxidase-catalyzed

oxidation of polycyclic aromatic compounds.

Peroxidase

Ionization energy

limit (eV) Reference

Chloroperoxidase 8.18 Ayala et al. (2000)

Manganese peroxidase 8.03 Bogan et al. (1996)

Lignin peroxidase 7.96 Vazquez-Duhalt et al.

(1994)

Versatile peroxidase 7.42 Wang et al. (2003)

Horseradish peroxidase 7.35 Cavalieri et al. (1983)

The prospects for peroxidase-based biorefining of petroleum fuels 117

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heme-peroxidase as it resembles enzymes from the

cytochrome P450 family, where substrates may

access the iron of the heme group (Sundaramoorthy

et al. 1995). The transfer of oxygen from the ferryl

iron of Compound I to certain substrates is catalyzed

by CPO, and it has been enhanced in some HRP

mutants that lack aromatic residues blocking access

to the iron (Newmyer & Ortiz de Montellano 1995).

Sulfoxidation has been shown to proceed in an

enantioselective way, which suggests more specific

binding of the substrate when the enzyme catalyzes

peroxygenation reactions instead of classical perox-

idatic reactions. Apparently, there is no correlation

between the ionization energy of the substrates and

the specific activity of the enzyme, which suggests

that the sulfoxidation reaction might also be influ-

enced by other substrate properties (Ayala et al.

2000).

CPO treatment combined with a distillation step

has been used to reduce the sulfur content of straight

run diesel fuel from 1.6 to 0.27% (Ayala et al. 1998;

Vazquez-Duhalt et al. 2002a). The reaction system

was an aqueous medium with a low percentage of

water-miscible organic solvent. Although the sulfox-

idation reaction is considered to involve a rather

specific substrate binding, it is noteworthy that the

hydrocarbon profile of sulfur-containing compounds

shows that most of the compounds are oxidized in the

presence of CPO, highlighting the relevance of low

specificity for this kind of applications (Figure 3).

Some simple estimates of the cost of this technol-

ogy may be performed. The total turnover number

of CPO (number of substrate molecules that can be

converted per molecule of enzyme before inactiva-

tion) for the oxidation of indole has been calculated

to be as high as 860 000 (Seelbach et al. 1997).

Using this value as reference, 1 g of enzyme could

desulfurize 1.4 ton of fuel from 500 to 30 ppm. In

our hands, and using the sulfur-containing com-

pound thianthrene, CPO showed a turnover number

of 500 000. Thus, 1 g of enzyme could reduce the

sulfur content from 500 to 30 ppm of 0.81 ton of

fuel. Considering the current cost of enzyme, the

desulfurization of one barrel of fuel would be too

expensive to be implemented on a large scale. It is

estimated that the enzyme stability should be in-

creased by two orders of magnitude to be economic-

ally attractive. Nevertheless, it should be kept in

mind that a biotechnological process would have

lower costs in terms of capital investment and energy

consumption (Linguist & Pacheco 1999).

Asphaltene transformation

The asphaltenic and viscous heavy oils from bitu-

minous deposits are huge energy reserves, to be

exploited over the coming decades. More than 70

countries possess bituminous deposits. In Canada

alone, the oil reserves considered to be technically

recoverable are estimated to be 280�300 Gb (bil-

lion of barrels), larger than the Saudi Arabia

oil reserves, that are estimated at 240 Gb (Govern-

ment of Alberta Canada 2002). These highly

asphaltenic resources must be rigorously treated in

order to convert them into an upgraded crude oil

before they can be refined to produce gasoline and

other fuels.

Asphaltene is a dark amorphous solid, especially

rich in heteroatoms (S, O, N) and metals (Fe, Ni,

V), and represents the highest molecular weight

Figure 3. GC hydrocarbon profile of straight run diesel fuel

before (A) and after (B) CPO treatment. GC was monitored by a

general flame ionization detector (FID) and a sulfur-specific flame

photometric detector (FPD).

118 M. Ayala et al.

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fraction of petroleum (Strausz et al. 1992). The

presence of a high concentration of asphaltenes is

the origin of many problems associated with either

recovery, separation or processing of heavy oils and

bitumen. This fraction is thought to be largely

responsible for other adverse oil properties, such as

high viscosity and the propensity to form emulsions,

polymers and coke. Considerable research has been

performed to elucidate the chemical nature of

asphaltenes, and their molecular structure has been

an enigma for several decades (Strausz et al. 1992;

Groenzin & Mullins 2000). There are indications

that asphaltenes are chemically diverse and, in

general, contain condensed aromatic cores substi-

tuted with alkyl and alicyclic moieties. Nitrogen,

oxygen and sulfur heteroatoms are present as non-

and heterocyclic groups. A significant amount of

porphyrins (petroporphyrins) can be found contain-

ing mainly nickel and vanadium. The proposed

chemical nature of asphaltene molecules has evolved

from a very complex, high molecular weight species,

comprising several functional groups in the same

molecule, to a collection of lower molecular weight

species, each bearing different functionalities. To

illustrate this evolution, the structures of asphaltene

molecules, proposed by Strausz et al. (1992)

and Groenzin and Mullins (2000), are shown in

Figure 4.

So far, there is no clear evidence that asphaltenes

are degraded by microbial activity. Thus, the asphal-

tenic fraction is recognized as the most recalcitrant

fraction of oil. Although microorganisms have been

found associated with bitumen containing high

amounts of asphaltenes (Wyndham & Costerton

1981), no changes in asphaltene content could be

found after bioconversion of heavy oils; furthermore,

the asphaltenic fraction did not support bacterial

growth (Premuzic et al. 1999; Thouand et al. 1999).

Nevertheless, the first clear experimental evidence

that enzymes are able to modify asphaltene mole-

cules was reported 13 years ago (Fedorak et al.

1993). CPO from the fungus C. fumago was able to

transform petroporphyrins and asphaltenes prefer-

ably in systems containing organic solvent (Fedorak

et al. 1993; Mogollon et al. 1998). Mass transfer

limitations are expected in aqueous reactions for

highly hydrophobic materials, such as asphaltenes

and petroporphyrins. CPO catalyzed the oxidation

of a petroporphyrin rich-fraction of asphaltenes in a

ternary solvent system and in the presence of

hydrogen peroxide, producing notable spectral

changes (Figure 5A).

Cytochrome c is a heme-protein that has perox-

idase-like activity in vitro. As CPO, a doubly

modified cytochrome c (PEG-Cyt-Met) was also

able to catalyze the oxidation of a petroporphyrin

rich-fraction of asphaltenes in a ternary solvent

system and in the presence of tert-butyl hydroper-

oxide (Garcia-Arellano et al. 2004). Both CPO and

PEG-Cyt-Met reactions produced spectral changes

in this petroporphyrin rich-fraction, as shown in

Figure 5(A) (Fedorak et al. 1993). Fourier trans-

form infrared spectroscopy (FTIR) analysis, be-

fore and after biocatalytic treatment, showed

significant differences, as illustrated in Figure 5(B)

(Garcia-Arellano et al. 2004). An increased pro-

portion of oxygen-containing groups, such as hy-

droxyl (3310 cm�1), carboxyl (1300, 1770 and

1710 cm�1), aldehydes (1730 cm�1), sulfoxides

(1040 cm�1), sulfones (1130 cm�1), and sulfonates

(1160 and 1260 cm�1) was detected. Sulfur is, after

carbon, the most important element in asphaltene

Figure 4. Proposed asphaltene molecules from (A) Strausz et al. (1992) and (B) Groenzin and Mullins (2000).

The prospects for peroxidase-based biorefining of petroleum fuels 119

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molecules, and most of it is contained in thiophenes

and organic sulfides moieties. Most of the sulfur

compounds seem to be oxidized due to the enzy-

matic reaction. According to the FTIR spectrum,

both CPO and chemically modified cytochrome c

also catalyze the oxidation of carbon atoms in

asphaltenes.

The enzyme-mediated destruction of petropor-

phyrins leads to the removal of Ni and V from

asphaltenes. The biocatalytic process with CPO

removed up to 57% of Ni and 52% of V, while

PEG-Cyt-Met mediated transformation removed

95% of V and 74% of Ni (Table III). The destruction

of the petroporphyrin molecules resulted in the loss

of the Soret band and metal removal.

Biocatalytic cracking, or biocracking, is probably

the most interesting biotechnology target for heavy

oil upgrading. Asphaltenes are a very complex

mixture, and are defined only by their solubility

properties: the asphaltenic fraction is insoluble in

short chain n-alkanes, especially pentane. The

asphaltene polymolecular structure is governed by

a balance between the propensity of fused aromatic

ring system to stack via p-bonding (reducing solubi-

lity) versus the steric disruption of stacking due to

alkane groups (increasing solubility) (Buenrostro-

Gonzalez et al. 2001). As mentioned above, due to

their complexity, the molecular weight of asphal-

tenes is still controversial (Strausz et al. 1992;

Groenzin & Mullins 1999, 2000). Size exclusion

chromatography has yielded average molecular

weights as high as 10 000 Da; vapor pressure osmo-

metry showed values of 4000 Da; mass spectroscopy

techniques measured values of 700 Da (field ioniza-

tion), ranging from 200 to 1200 Da (laser deso-

rption); while a molecular weight distribution of

500�1000 Da was obtained with the technique of

fluorescence depolarization. The larger molecular

weights obtained with some techniques could be

explained by the evident aggregation of asphaltenes.

This aggregation defines the solution-dispersion

process of asphaltenes in oil. Nevertheless, the

possibility of asphaltene cracking by a biocatalytic

process is a potential alternative to the energy-

expensive and inefficient physicochemical processes.

The enormous amount of energetic resource

found as asphaltene-rich deposits justify the explora-

tion of alternative upgrading technologies. The

enzymatic treatment of asphaltenes is an alternative

process for the removal of heavy metals, in order to

reduce catalyst poisoning found in the conventional

hydrotreatment and cracking processes. On the

other hand, a direct enzymatic cracking of asphal-

tenes molecules should not be excluded.

Challenging issues for peroxidase utilization

Substrate range

In the preceding section, we pointed out that for the

peroxidase-catalyzed oxidation of aromatic and sul-

fur-containing compounds, the substrates must

have a certain ionization energy value and be able

to access the heme iron, respectively. It is very likely

that the oxidation of asphaltene molecules also

depends on one or both of these properties. Given

50

55

60

65

70

75

80

85

90

95

100

5001000150020002500300035004000

(cm–1)

OH, R-NH-R

3310C-H ar

3060

1730R-CHO,

1770 -COOH

-COOH1710

C=C conj.1650

CH3,CH 2

14601380

1300

12601160

1130

1040R-S=O, V=O ?

7354 H ar adj.

8143 H ar adj.

870H ar single

R-SO2-OH

R-SO-O-R'

-CO-O- ?

Control

PEG-Cyt-Met treated

0

0.2

0.4

0.6

0.8

1

300 350 400 450 500 550 600

Wavelength (nm)

d

a

b

c

A B

Figure 5. (A) Absorption spectra of the petroporphyrin rich-fraction of asphaltenes after biocatalytic treatment. Control without treatment

(a); control without biocatalyst and in the presence of hydroperoxide (b); reaction with one addition of biocatalyst (c); and reaction after a

second addition of biocatalyst (d). (B) FTIR spectra of untreated and biocatalytically treated porphyrin-rich fractions from asphaltenes.

FTIR was performed using the film-spreading technique.

Table III. Nickel and vanadium removal from petroporphyrin rich fractions of asphaltenes by peroxidase-mediated reaction.

Enzyme/heavy metal Nickel (%) Vanadium (%) Reference

Chloroperoxidase 20 19 Fedorak et al. (1993)

57 52 Mogollon et al. (1998)

Chemically modified cytochrome c 74 95 Garcia-Arellano et al. (2004)

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the complexity of the refinery streams, it would be

desirable that the enzymatic catalyst could recognize

a broad spectrum of substrates. In order to further

broaden the substrate range for peroxidases, we can

envisage two strategies.

Modulation of the redox potential of the enzyme. It is

known that the heme environment influences the

electronic states of Compound I and Compound II,

which ultimately determine the enzyme’s reactivity.

Table IV lists the experimental redox potential data

of the species involved in peroxidase catalysis. The

redox potential of the Fe�3/Fe�2 couple has been

interpreted to reflect the redox potential of Com-

pound I and Compound II (Millis et al. 1989).

Thus, one of the more oxidant enzymes in Table IV

would be manganese peroxidase, which catalyzes the

oxidation of Mn(II) to Mn(III) (Em�1500 mV).

There are a number of residues that are conserved

throughout the peroxidase plant superfamily which

surround the heme group, and form the distal and

proximal cavities. Following HRP numbering, Arg

38, Phe 41, His 42 and Asn 70 are present in the

distal cavity, while His 170, Phe 221 and Asp 247

comprise the proximal side. Distal Arg and His are

catalytic residues involved in peroxide cleavage,

while proximal His pentaco-ordinates the heme

group. Exceptions are found, such as CcP, that

bears Trp instead of Phe residues in both proximal

and distal sides; and ascorbate peroxidase (APX)

and Coprinus cinereus peroxidase (CiP), that bear a

Trp and Leu residue in the proximal side, respec-

tively, instead of a Phe residue.

Experimental evidence and theoretical studies

support the generalization that an anionic environ-

ment and a nearby electron-donating residue on the

proximal side, favor the transfer of the free radical

originally formed in the porphyrin ring. This is the

case for CcP and some F221W and F221Y HRP

mutants mimicking CcP, where proximal Trp or

Tyr residues provide an oxidizable site for the

formation of a protein-based free radical (Miller

et al. 1995; Morimoto et al. 1998; Wirstam et al.

1999). The redox potential of peroxidases depends

on the location of the free radical, as observed for the

F221W HRP mutant, which showed a more positive

redox potential of �178 mV for the Fe�3/Fe�2

couple, closer to that of CcP (Morimoto et al. 1998).

Thus, the introduction of cationic sites and removal

of the electron-donating residues, as well as a

diminished polarity of the heme proximal environ-

ment may favor the retention of the porphyrin-based

radical, as happens for HRP, and as theoretically and

experimentally determined for CcP and for APX

(Bonagura et al. 1996; Jensen et al. 1998; Wirstam

et al. 1999; de Visser et al. 2003; Barrows et al.

2004). Similar studies with other peroxidases

bearing protein-based radicals, such as LiP (Blodig

et al. 2001), versatile peroxidase (Perez-Boada et al.

2005), and turnip peroxidase (Ivancich et al. 2001),

would further test this hypothesis.

Two structural factors, on the proximal side, have

been proposed to determine the redox potential of

peroxidases. The first is related to the bond length

between the heme iron and the protein, which

depends on the relative position of the proximal

His. A weaker and longer bond between the His

residue and the heme iron would make the latter

more electron deficient, and, thus, destabilize the

higher oxidation states; this would lead to a more

positive redox potential for the Fe�3/Fe�2 couple,

and, thus, an increased oxidative capability of the

enzyme. Such a correlation has been observed for

CcP, LiP and MnP, but not for HRP (Banci 1997;

Table IV. Redox potential of different electronic states of peroxidases from the plant superfamily.

Peroxidase pH Redox couple Redox potential (mV) Reference

Horseradish peroxidase 7 Fe�3/Fe�2 �278* Yamada et al. (1975)

Compound I/Compound II 880 Farhangrazi et al. (1995)

898 Hayashi and Yamazaki (1979)

Compound II/Fe�3 870 Farhangrazi et al. (1995)

900 Hayashi and Yamazaki (1979)

Turnip peroxidase 8 Fe�3/Fe�2 �233 to 160* Ricard et al. (1972)

Cytochrome c peroxidase 6.1 Fe�3/Fe�2 �194* Conroy et al. (1978)

Compound I/Compound II 740* Mondal et al. (1996)

Compound II/Fe�3 1080 Purcell and Erman (1976)

C. cinereus peroxidase 7 Fe�3/Fe�2 �183* Battistuzzi et al. (2006)

Compound I/Compound II 915 Farhangrazi et al. (1994)

Compound II/Fe�3 982 Farhangrazi et al. (1994)

Chloroperoxidase 6.9 Fe�3/Fe�2 �140* Makino et al. (1976)

Lignin peroxidase 7 Fe�3/Fe�2 �120 to �140* Millis et al. (1989)

Manganese peroxidase 7 Fe�3/Fe�2 �88 to �93* Millis et al. (1989)

*Voltammetric data.

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Choinowski et al. 1999). The second structural

factor is related to the hydrogen bonding of the

proximal His. It has been suggested that strong

hydrogen bonding to neighboring residues increases

its basicity. The stronger imidazolate character

would stabilize the higher oxidations states, which

would result in a reduced redox potential value of

the Fe�3/Fe�2 couple (Poulos & Kraut 1981; Banci

et al. 1991). In this sense, the most important

residue is the proximal Asp, which forms a hydrogen

bond with the proximal His. Its substitution with

residues that modify the hydrogen bond network

would have an effect on the redox potential of the

Fe�3/Fe�2 couple. However, mixed results have

been observed. In CcP, substitution with a Glu

residue led to an increase of 70 mV in the redox

potential (Goodin & McRee 1993), whereas in MnP,

the same substitution resulted in a decreased redox

potential (Santucci et al. 2000). Moreover, substitu-

tion with the poor hydrogen-bonding Asn in CiP

decreased the redox potential of the enzyme, which

is in clear contradiction with the hypothesis men-

tioned above (Ciaccio et al. 2003). Clearly, the

modulation of the redox potential in peroxidases is

multifactorial and there are probably other contri-

buting factors.

One of these may be the two calcium-binding sites

in class II and III peroxidases. For instance, Verdin

et al. (2006) measured the redox potential of the Fe�3/

Fe�2 couple of calcium-depleted versatile peroxidase,

and noted a drastic reduction of 200 mV at catalytic

pH. Moreover, Howes et al. (2005) have shown that

the T171S HRP mutant retained the functional and

structural properties of the native enzyme. However, in

the mutant, the redox potential of the Fe�3/Fe�2

couple was increased by about 100 mV. Apparently,

the Thr 171 regulates the hydrogen bonding between

the structural calcium ion and the proximal His. It

would be interesting to test the ability of this mutant to

oxidize a broader range of substrates, with higher

redox potential values.

It has been noted that the environment in the distal

side mainly directs the peroxide activation process,

whereas the environment in the proximal side reg-

ulates the stability of enzyme intermediates and the

electron transfer process. However, Smulevich et al.

(1991) proved that changes in the distal side also

exert long-distance perturbations in the proximal

side. The substitution of His 181, Arg 48 and Trp

51 in the distal cavity of CcP disrupted the hydrogen

bond between the proximal Asp and His. Nagano

et al. (1996) substituted the distal Asn 70 in HRP for

Val or Asp, and noted that although the redox

potential of the Compound I/Compound II couple

was similar to the native enzyme, in the mutants the

Compound II was very unstable and the redox

potential of the Compound II/ferric enzyme couple

was about 100 mV higher. Finally, modification of the

heme itself also provides a means to manipulate the

redox potential of the enzyme. The role of heme

propionates has been discussed as modulators of the

nature and stability of Compound I (Barrows &

Poulos 2005; Guallar & Olsen 2006). For instance,

He et al. (1996) showed that introduction of electron-

withdrawing groups in the pyrrole ring of HRP

increased, by 100 mV, the redox potential of the

Compound I/Compound II couple.

Unfortunately, the redox potential data are not

available for most of the constructed peroxidase

mutants. Although the kinetic characterization pro-

vides some information on the substitution effects, it

does not reflect the effect on redox potential values.

Thus, better voltammetric or indirect methods to

estimate the redox potential values of the higher

oxidation states (Compound I and Compound II)

would greatly benefit experimental data interpreta-

tion, and the elucidation of other factors modulating

the oxidizing strength of peroxidases.

Topology of the active site. A second strategy to

broaden the substrate range of peroxidases has to

do with the topology found in the heme cavity.

Enhanced access to the heme iron or enlarged heme

cavity would improve the kinetics of the peroxygen-

ase activity, as demonstrated for plant peroxidases.

Fungal and plant peroxidases have an aromatic

residue (either Phe or Trp) adjacent to the distal

His. Apparently, the removal of this aromatic residue

unblocks the access to the ferryl oxygen, and, thus,

enhances peroxygenase activity. In a study by

Newmyer and Ortiz de Montellano (1995), the

sulfoxidation activity of HRP was increased 100-

fold by substituting the bulky Phe 41 by Ala.

Moreover, the mutants catalyzed styrene epoxida-

tion, an activity absent in the native enzyme.

Similarly, Celik et al. (2001) removed the distal

Trp in APX, increasing the sulfoxidation activity of

the mutant from 10- to 100-fold, as well as the

enantioselectivity of the reaction.

To increase heme cavity size, Tanaka et al. (1997)

constructed a H42E HRP mutant, inspired by the

unusual distal Glu residue found in CPO. They

confirmed that a Glu residue performs as an efficient

acid�base catalyst. Although the redox potential of

the Fe�3/Fe�2 couple remained practically un-

changed, the enlarged heme cavity led to a 50- to

1000-fold increase in the peroxygenase activity of

mutant HRP. Furthermore, during crystallographic

studies of recombinant soybean peroxidase (SBP),

Henriksen et al. (2001) observed that the Ile 74

caused displacement of the distal Phe 41 and His 42,

thus rendering a more open active site compared to

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HRP, which bears an Ala residue in the analogous

position. Indeed, the sulfoxidation of thioanisole

was faster when catalyzed with SBP than with

HRP (Dai & Klibanov 2000). However, the hypoth-

esis that this enlarged heme cavity enhances perox-

ygenase activity by increasing the transformation

rates, has not been explored with fungal peroxidases.

Activity in organic media

One of the characteristics of an enzyme-based

process for the oil industry would be the ability to

function in organic media, with low water content.

Several features must be addressed in this regard.

Enzyme stabilization may be achieved by means

of chemical modification and immobilization, as

already demonstrated for several heme-proteins

(Takahashi et al. 1984; Miland et al. 1996; Ayala

et al. 2002; Garcia-Arellano et al. 2002; Wang et al.

2003; Bruns & Tiller 2005; Song et al. 2005; Liu

et al. 2006). The preparation of the enzyme also

influences the activity (Lee & Dordick 2002; Cao

et al. 2003); in the case of peroxidases, some studies

are available on the influence of the enzyme micro-

environment and biocatalyst preparation (Dai &

Klibanov 1999; Kimura et al. 2004; Trevisan et al.

2004; Bruns & Tiller 2005; Park & Clark 2006). In

this regard, it is known that the hydration level in

protein molecules dramatically influences enzymatic

activity in organic solvents (Yang et al. 2004).

Although hydration level is difficult to estimate,

some studies have shown good correlation between

the thermodynamic water activity of the system and

the enzyme’s activity, rendering aw as a reliable

indicator of hydration water. The optimum value

of aw depends on the enzyme and the reaction

system. Few studies are available for peroxidases

(Dai & Klibanov 1999; van de Velde et al. 2001a;

Michizoe et al. 2003), therefore further research is

still needed in order to overcome this drawback.

However, the most limiting factor for the use of

peroxidases in organic solvents is substrate desolva-

tion (Ryu & Dordick 1992). In organic systems, the

ground state stabilization of hydrophobic substrates

reduces their availability to the enzyme, which is

reflected by increased affinity constants and lower

catalytic activity. Some strategies, such as manipula-

tion of the microenvironment and solvent engineer-

ing, may be applied to alleviate this constraint.

Thermodynamic approaches to estimate substrate

desolvation in certain systems have been described

(Ryu & Dordick 1992; Schmitke et al. 1996). An

application for the oil industry will certainly require

more sophisticated modeling in order to lessen the

impact of substrate desolvation on the enzymatic

reaction efficiency. However, to date, no studies are

available regarding the protein or solvent engineer-

ing of peroxidases in order to favor substrate binding

in hydrophobic media.

Oxidative inactivation

Oxidative inactivation is the most troublesome issue

hampering the commercial application of heme

peroxidases (van de Velde et al. 2001b). It occurs

when the enzyme reacts with excess H2O2 or in the

absence of reducing substrate (Jenzer et al. 1986;

Arnao et al. 1990; Weinryb 1996). The oxidative

inactivation mechanism for peroxidases has been

recently reviewed (Valderrama et al. 2002). Oxida-

tively inactivated peroxidases characteristically show

heme degradation, although the oxidation of redox

active amino acids has also been observed (Jenzer

et al. 1986; Wariishi & Gold 1990; Villegas et al.

2000; Pfister et al. 2001). Normally, peroxidases

react with H2O2 to form Compound I, which is

sequentially reduced by an exogenous substrate, first

to Compound II and, afterwards, to native ferric

enzyme, as shown in Figure 2 (Renganathan &

Gold 1986). Under inactivation conditions, Com-

pound II reacts with H2O2 to yield Compound III,

a Fe3��O2� complex (Wariishi & Gold 1990). The

decay of this intermediate produces superoxide

radicals that may react with an additional H2O2

molecule to produce hydroxyl radicals (Jenzer et al.

1986; Wariishi & Gold 1990; Chen & Schopfer

1999). These very reactive species attack the por-

phyrin ring leading to irreversible peroxidase inacti-

vation (Jenzer et al. 1986; Wariishi & Gold 1990).

Besides Compound III, Compound I also acts as an

oxidizing agent during the inactivation process. In

the absence of an exogenous substrate, Compound I

decays into Compound II by substracting electrons

from the apoprotein (Jenzer et al. 1986; Hiner et al.

2001; Pfister et al. 2001). The oxidation of Trp and

Tyr residues coupled to Compound I reduction has

been observed in several peroxidases (Choinowski

et al. 1999; Hiner et al. 2001; Pfister et al. 2001;

Perez-Boada et al. 2005; Pogni et al. 2005). In yeast

CcP, Tyr oxidation leads to protein cross-linking

(Pfister et al. 2001), while Trp oxidation in LiP and

APX produces hydroxylated adducts (Choinowski et

al. 1999; Hiner et al. 2001). To date, the deleterious

effect of Compound I reduction during oxidative

inactivation has not been clearly elucidated, but

could be expected to be important in peroxidases

that contain functionally compromised oxidizable

amino acids, such as LiP and versatile peroxidase

(Choinowski et al. 1999; Perez-Boada et al. 2005;

Pogni et al. 2005).

An alternative inactivation mechanism, mainly

supported by the formation of the inactive compound

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P670 at the level of Compound I, has been proposed

(Arnao et al. 1990; Hiner et al. 2002). In this model,

Compound I reacts with H2O2 to form the complex

Compound I�H2O2, from which three catalytic

pathways originate: catalase activity pathway, Com-

pound III-forming pathway, and the inactivation

pathway, that, afterwards, leads to compound

P670. Both catalase activity and Compound III-

forming pathways are proposed to be peroxidase

protection mechanisms that avoid the accumula-

tion of Compound I and, hence, irreversible inactiva-

tion. Unlike the Compound III-centered inactivation

model, this mechanism rules out any deleterious role

for such species.

Several strategies have been implemented in order

to circumvent oxidative inactivation. They include

the fine regulation (feed-on-demand) of hydrogen

peroxide concentration during catalyzed reactions

(Seelbach et al. 1997), as well as the construction of

random and site-directed peroxidase mutants with

improved oxidative stability (see Table V). Substitu-

tion of Met and Tyr residues for less oxidizable

amino acids has been the rationale for producing

site-directed peroxidase mutants with improved

oxidative stability. MnP was stabilized 6.3-fold after

substituting methionine residues in positions 67, 237

and 273 with leucine residues (Miyasaki & Takaha-

shi 2001). MnP was also stabilized by engineering

the H2O2-binding pocket (Miyasaki-Imamura et al.

2003). In this case, Ala 79, Asn 81 and Ile 83

residues were saturation mutated in order to con-

struct a library from which a variant (A79S, N81L,

I83L) 4.4-times more stable than the wild-type MnP

was selected. The most successful stabilization case,

so far, is Coprinus cinereus peroxidase (CiP). This

enzyme was submitted to a combination of site-

directed mutagenesis, random mutagenesis and in

vivo shuffling, followed by screening for higher

thermal and oxidative stability. This strategy yielded

a multiple mutant (I49S, V53A, T121A, M166F,

E239G, M242I and Y272F) 100-times more stable

than wild-type CiP in the presence of hydrogen

peroxide (Cherry et al. 1999). Except for the

obvious and modest role of mutations M166F,

M242I and Y272F, the molecular characterization

of stabilized CiP does not clearly explain the

important contribution of mutations I49S, V53A,

T121A and E239G to oxidative stability (Houborg

et al. 2003). Rationalization of the molecular causes

of CiP oxidative stabilization would contribute to

understanding the oxidative inactivation process.

In this sense, iso-1-cytochrome c has been ration-

ally stabilized against peroxide inactivation following

a redox-inspired approach (Valderrama et al. 2006).

Although not an enzyme, the in vitro peroxidase

activity of cytochrome c is well characterized

(Vazquez-Duhalt 1999). By analyzing simulta-

neous events during oxidative inactivation (i.e.

cross-linking, heme bleaching, activity loss, iron

co-ordination), it was possible to alter them inde-

pendently by protein engineering, and, thus, mod-

ulate the electron transfer pathways (Valderrama &

Vazquez-Duhalt 2005). As a result, a cytochrome c

variant with a 15-fold improved total turnover was

obtained. This approach serves as an example of the

strategies that could produce more stable peroxidase

variants.

Molecular tools for the generation of

tailor-made heme peroxidases

Before the arrival of recombinant DNA technolo-

gies, covalent chemical modification was the only

molecular tool available to modify and/or enhance

peroxidase properties. Increased thermal stability

and tolerance to non-aqueous media have been

the main goals pursued with this approach. Inter-

and intra-crosslinking of o-amino lysine groups

with bifunctional reagents, such as glutaraldehyde

and ethylene-glycol-bis(N-hydroxysuccinimidylsuc-

cinate) have been successfully utilized to obtain

more thermostable peroxidases, in both soluble

and crystalline forms (O’Brien et al. 2001; Ayala

et al. 2002). Modification of heme proteins with

polyethylene glycol has improved not only thermo-

stability, but also tolerance to organic solvents

(Garcia-Arellano et al. 2002; Wang et al. 2002).

Indeed, cytochrome c, a non-enzymatic heme pro-

tein with in vitro peroxidase activity, shows a wider

substrate range after double chemical modification

with polyethylene glycol and methyl ester (Tinoco &

Vazquez-Duhalt 1998). Chemical modification, a

non-site-specific tool, often leads to activity reduc-

tion due to the reaction of the modifying agent with

residues close to, or inside the catalytic site. More-

over, the yield of heterogeneous and irreproducible

Table V. Site-directed and random heme peroxidase mutants with improved H2O2 stability.

Enzyme Improving factor Mutations Reference

Manganese peroxidase 6.3 M67L, M237L, M273L Miyasaki and Takahashi (2001)

4.4 A79S, N81L, I83L Miyasaki-Imamura et al. (2003)

C. cinereus peroxidase 100 I49S, V53A, T121A, M166F, E239G,

M242I, Y272F

Cherry et al. (1999)

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chemically modified mixtures is an important draw-

back. Nevertheless, when structural information is

available, chemical modification combined with site-

directed mutagenesis is a powerful tool suited for

introducing non-natural side chains in specific sites

(DeSantis & Jones 1999). Introduction of non-

codified functionalities into proteins, by this mean

or by modifying the protein synthesis machinery to

achieve the in vivo incorporation of non-natural

amino acids, could provide novel approaches to

enhance peroxidase properties.

Site-directed mutagenesis, in addition to an in-

creasing knowledge of the structure�function rela-

tionships in peroxidases, has yielded enzymes with

improved stability and broader substrate range with-

out the drawbacks of chemical modification. Intro-

duction of disulfide bonds to improve thermal

stability, as well as the substitution of bulky amino

acids around the active site to allow substrates access

to the heme edge, have become common strategies

(Reading & Aust 2000; Celik et al. 2001). In spite of

those successes, engineering of peroxidases for im-

proving oxidative stability remains very challenging.

A general strategy consisting of the substitution of

Met and/or Tyr residues for less oxidizable amino

acids has been applied to several peroxidases

(Cherry et al. 1999; Miyasaki & Takahashi 2001;

Morawski et al. 2001). However, this strategy gave

modest results in MnP (Miyasaki & Takahashi 2001)

and it was completely ineffective for HRP (Morawski

et al. 2001).

Directed evolution has emerged as an alternative

to rational protein engineering. It has been shown

very useful when structural data is limited and/or

when the protein-engineering problem has a diffuse

solution. Low levels of peroxidase heterologous

expression is representative of such kind of problem.

When homologous expression is not possible or

convenient, heme peroxidases have been heterolo-

gously expressed in Aspergillus sp. (Steward et al.

1996; Conesa et al. 2001), tobacco plants (Pelle-

grineschi et al. 1995), recombinant baculovirus

(Johnson & Li 1991; Pease et al. 1991) and

Escherichia coli followed by in vitro folding of purified

inclusion bodies (Smith et al. 1990; Whitwam et al.

1995; Doyle & Smith 1996), always with very low

yields. By using directed evolution techniques, it was

possible to express functional HRP in E. coli without

further in vitro folding (Lin et al. 1999). In addition

to heterologous expression and thermal and oxida-

tive stabilization cases, directed evolution has also

been utilized to produce specificity changes, such as

that of CcP evolved for increased activity against

guaiacol (Iffland et al. 2000).

Although genetic tools have displaced traditional

covalent chemical modification, the latter has

recently reappeared, enhancing the capabilities of

site-directed mutagenesis. Heme peroxidases are so

complex that the application of only one of the

molecular tools available today is unlikely to give rise

to more commercially suited enzymes. Successful

cases clearly show that complex problems may only

be attacked with powerful eclectic strategies.

Conclusions

The introduction of new technologies to the refining

industry will ensure the supply of clean fuels during

the next decades. The systems described here may

serve as models for future process development,

either enzyme-based or enzyme-inspired. Peroxi-

dases are enzymes with great potential, but with

important disadvantages, impeding industrial appli-

cation. Some of the structural features affecting the

redox properties, substrate recognition and oxidative

stability of peroxidases are already identified. Ther-

modynamic analysis of substrate�enzyme binding in

hydrophobic media combined with protein and

medium engineering are essential to the successful

development of a peroxidase-based process. The

synergistic combination of theoretical and molecular

tools will certainly provide a better understanding of

peroxidase functioning and better grounds to design

the desired thermodynamic and kinetic properties

into the enzyme.

Acknowledgements

This work was supported by CONACYT 2004-

C01-42 grant.

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