Abdussamed URAS

Imobilization of Peroxidase on Carbon

Nanotubes

LA LSRE/LCM, DEQ, FEUP

Porto, September, 2013

Immobilization of Peroxidase

on Carbon Nanotubes

Erasmus Report

Abdussamed URAS

Supervisors

Doutora Ana Paula Tavares

Doutora Cláudia Silva

LSRE Departamento de Engenharia Química

Faculdade de Engenharia Universidade do Porto

Janeiro 2013

Abstract

Peroxidases catalyze a variety of reactions of toxic aromatic compounds including

phenols, biphenols, anilines, benzidines and related heteroaromatic compounds needing the

presence of perocides such as hydrogen peroxide (H2O2) to active them. Peroxidases can

contain a heme cofactor in their active sites, or alternately redox active cysteine or

selenocysteine residues.

Carbon nanotubes (CNTs) are allotropes of carbon with a cylindrical nanostructure.

Nanotubes have been constructed with length-to-diameter ratio of up to

132,000,000:1, significantly larger than for any other material. These cylindrical carbon

molecules have unusual properties, which are valuable for nanotechnology, electronics, optics

and other fields of materials science and technology. In particular, owing to their extraordinary

thermal conductivity and mechanical and electrical properties, carbon nanotubes find

applications as additives to various structural materials.

The objective of this work is the immobilization of peroxidase on modified carbon

nanotubes. New methodologies for enzyme immobilization on carbon supports were used to

optimize and maximize the efficiency of the immobilization.

The capacity for peroxidase immobilization was tested at a laboratory scale using

carbon nanotubes. Tests to determine the optimal conditions of immobilization of peroxidase

were performed using carbon nanotubes. Then the possible application of carbon nanotubes

with immobilized peroxidase in industrial processes was studied evaluating the following

parameters: thermal stability at high temperatures, and kinetic behavior.

Key Words: peroxidase immobilization; carbon nanotubes; thermal stability; kinetic

parameters

Immobilization of Peroxidase on MWNT | 2013

Laboratório Associado LSRE/LCM i

List of contents

List of figures ................................................................................................................................. ii

List of tables ................................................................................................................................. iii

1. Introduction ................................................................................................................... 1

1.1. Peroxidase: origin, function and applications ....................................................................... 1

1.2. Enzyme immobilization………………………………………………………………………………………………………3

1.2.1. Catalyses with immobilized enyzmes : general overview...................................................3

1.2.2. Methods and materials in carrier-based immobilization of enzymes……………………………..4

1.3. Objectives………………………………………………………………………………………………………………………….6

2. Experimental sections………………………………………………………………………………………………………8

2.1. Reagents……………………………………………………………………………………………………………………………8

2.2. Carbon nanotubes processing……………………………………………………………………………………………8

2.2.1. Functionalization and characterization of MWNT…………………………………………………………..8

2.3. Immobilization technique………………………………………………………………………………………………….9

2.4. Enzymatic activity measurement……………………………………………………………………………………….9

2.5. Experiments to determine the optimal conditions peroxidase immibilization………………….10

3. Results and discussion…………………………………………………………………………………………………….11

3.1. Optimal conditions for experimental peroxidase catalysis……………………………………………….11

3.1.1. Optimal peroxidase concentraiton to perform immobilization……………………………………..11

3.1.2. Optimal medium pH perform immobilization……………………………………………………………….12

3.1.3. Optimal contact time to perform immobilization………………………………………………………….13

4. Conclusion………………………………………………………………………………………………………………………14

5. Referances………………………………………………………………………………………………………………………15

Acknowledgements…………………………………………………………………………………………………………….16

Immobilization of Peroxidase on MWNT | 2013

Laboratório Associado LSRE/LCM ii

List of figures

Figure 1 Catalytic cycle of peroxidise-catalyzed substrate oxidation.......................................2

Figure 2 Different techniques of enzyme immobilization: (A) entrapment, (B) encapsulatoin, (C) support based and (D) self immobilization. Green circles represent anzymes........................4

Figure 3 Enzymatic activity registered in the catalysis using carbon nanotubes with immobilized peroxidase when immobilization was performed with peroxidase solutions of different concentrations…………………………………………………………………………………………………………………..…..11

Figure 4 Enzymatic activity registered in the catalysis using carbon nanotubes with immobilized peroxidase when immobilization was performed in medium with different pH value………………………………………………………………………………………………………………………………………12

Figure 5 Enzymatic activity registered in the catalysis using carbon nanotubes with immobilized

peroxidase when immobilization was performed for different periods of time……………………13

Immobilization of Peroxidase on MWNT | 2013

Laboratório Associado LSRE/LCM iii

List of tables

Table 1. Specific surface area (SBET), pore volume (Vp) and pore diameter (dp) for the

different MWNT samples..............................................................................................................9

1. Introduction

1.1. Peroxidase: origin, function and applications

Peroxidases are widely distributed in nature. Peroxidase activity has been identified in

plants, microorganisms, and animals, where they play important roles. They catalyze a variety

of reactions of toxic aromatic compounds including phenols, biphenols, anilines, benzidines

and related heteroaromatic compounds needing the presence of peroxides such as hydrogen

peroxide (H2O2) to activate them. Most peroxidases are heme-containing enzymes and are

known to catalyze the one-electron oxidation of a wide range of aromatic compounds and

contain iron (III) protoporphyrin IX (ferriprotoporphyrin IX) that is the prosthetic group (Karam

& Nicell, 1997). Peroxidases that have been used for the effluent treatment include

horseradish peroxidase (HRP, EC 1.11.1.7), lignin peroxidase (LiP; EC 1.11.1.14), manganese

peroxidase (MnP; EC 1.11.1.13) and chloroperoxidase (CPO; EC 1.11.1.10). The various types of

peroxidases differ in the nature of the reducing substrate, for example LiP catalyzes the

oxidation of a wide variety of aromatic nonphenolic lignin compounds, Manganese

peroxidases catalyse the Mn-mediated oxidation of and phenolic compounds,

chloroperoxidase catalyses the oxygen transfer reactions, H2O2 disproportionations and

oxidative chlorinations in which Cl− is transferred to an organic substrate (Conesa et al., 2002).

The peroxidase catalytic cycle, showed in Figure 1, involves distinct intermediate

enzyme forms and normally involves three reaction steps being hydrogen peroxide their

natural electron acceptor (Martinez, 2002). Initially, in the reaction 1, the native ferric enzyme

is oxidized by hydrogen peroxide to form an unstable intermediate called compound I (CI).

During the oxidation of the ferric enzyme by hydrogen peroxide, one electron is withdrawn

from the Fe3+, and concomitantly one other from the porphyrin ring, generating Fe4+ and

porphyrin cation radical, respectively, with reduction of peroxide to water. Then in reaction 2,

CI oxidises the substrate to give the compound II (CII), releasing a free radical. So, CII is

reduced by a second substrate molecule, regenerating the Fe3+ state and producing another

free radical (Martinez, 2002). Both compound I and compound II are powerful oxidants, with

redox potentials close to +1 V (Veitch, 2004). The first two reactions (ferric enzyme with H2O2

and compound I with the reducing substrates) are rapid, whereas the reaction of compound II

is typically 10-fold slower (Mester &Tien, 2000).

Hydrogen peroxide is their natural electron acceptor acting as a two-electron oxidizing

substrate for the ferric enzyme in reaction 1, which results in formation of compound I.

However, ligninolytic peroxidases differ in their reducing substrates that are one-electron

oxidized by compounds I and II in reactions 2 and 3 respectively.

Figure 1 - Catalytic cycle of peroxidase-catalyzed substrate oxidation

H2O

compound I

Fe IV

compound II

Fe IV

Fe III reaction I

reaction II

substrate substrateox

reaction III

substrate

substrateox + H2O H2O2

1.2. Enzyme immobilization

1.2.1. Catalysis with immobilized enzymes: general overview

When compared to chemical catalysts, enzymes (i) have higher reaction rates; (ii) need

milder reaction conditions – generally reactions are performed at temperatures below 100°C,

atmospheric pressure, pH values ranging from 4 to 8 and in the absence of aggressive

chemicals or solvents –; and (iii) allow higher reaction specificity (4), characteristics that make

them very attractive for use in many different kinds of industries. Nowadays, they are used as

free molecules in solution or immobilized, in this last case either as carrier-free complexes or

in appropriate carriers. Carrier-free enzyme immobilization consists on the binding of the

enzymes to each other spontaneously or, more frequently, through cross-linking agents,

without the need of a carrier (Figure 1 (D)) (5). On other hand, carrier-dependent enzyme

immobilization consists on the attachment of the enzymes to a carrier. The best one for the

catalysis depends on several factors of the particular reaction occurring.

Several advantages can be pointed out which favour the use of immobilized enzymes

over free enzymes (5):

• Retention and recovery: enzyme immobilization creates a two-phase reaction system

that facilitates the recovery of the enzymes/carriers from the reaction medium. This

makes easy to purify the products of reaction, thereby favouring continuous processes,

and, above all, allows the reuse of the enzymes;

• Stabilization: usually enzyme immobilization render them more stable because they

are protected from aggressive conditions of the reaction medium, such as

inappropriate medium pH and temperature, the presence of organic solvents, etc.;

• Enhanced activity: several cases have been reported where enzyme immobilization

enhances the activity of the enzymes. More than one cause can explain this behaviour:

(i) the higher density of enzymes in the aggregates may originate cooperative effects

that increase enzymes’ specific activity; (ii) enzymes have a tridimensional

conformation that can oscillate between slightly different organizations with distinct

activity, thus they will have an increased activity if they are immobilized on their most

active conformation;

• Modification of some characteristics of catalysis: immobilize enzymes can modify their

affinity for substrates as well as the optimal conditions of catalysis. Several

immobilization techniques are based on the interaction between specific chemical

groups on the enzymes and the carrier, which determines enzymes’ orientation on the

carrier. In consequence, the structure of the enzyme may change, influencing their

selectivity for substrates. In the same way, immobilization can improve or even reverse

the enantioselectivity of the enzymes. Moreover, enzyme immobilization frequently

increase enzymes’ thermostability and shift the optimal pH for catalysis;

• Multi-step reactions: more than one type of enzymes can be co-immobilized on the

same carrier, which makes possible the performance of multi-step and cascade

enzymatic reactions. It must be noted the great potential of this type of immobilization

because associations of enzymes can be created which doesn’t naturally occur, thus

opening the doors to the creation of artificial metabolic pathways;

• Reduced costs of downstream processes: because immobilized enzymes are easily

recovered from the reaction medium and can be widely reused, they eliminate the

need of some expensive processing downstream to the enzymatic catalysis step.

Despite all the advantages mentioned, it should be pointed out that enzyme

immobilization can also have a few negative points. The most important one may be the risk of

the enzymes to immobilize in an orientation that impairs the access of the substrate to the

active site, actually leading to a reduction of their activity.

1.2.2. Methods and materials in carrier-based immobilization of enzymes

The most used techniques of carrier-dependent immobilization are entrapment,

encapsulation and support based immobilization (Figure 1 (A) - (C)).

Entrapment of enzymes is usually performed in situ using a polymeric network (e.g.:

organic polymer; sol-gel). This technique of immobilization prevents direct contact of the

enzymes with the environment, thereby minimising the effects of gas bubbles, mechanical

sheer and hydrophobic solvents, but has as disadvantages mass transfer limitations and low

enzyme loading. Silica sol-gel matrices are a common example of a carrier used for enzyme

entrapment. However, more recently, another way to entrap enzymes emerged. First the

enzymes are linked to a polymer and then the polymers are cross-linked to each other,

creating a network within which enzymes are retained (5).

Figure 1 Different techniques of enzyme immobilization: (A) entrapment, (B) encapsulation, (C) support

based and (D) self immobilization. Green circles represent the enzymes.

Encapsulation works in a manner similar to entrapment, also protecting the enzyme

from the external environment but having limited application in processes using large

substrates, due to mass transfer limitations (5).

Support based immobilization consists on the attachment of the enzymes to the

surface of the carrier through different kinds of chemical interactions.

The simplest and cheapest method to perform the technique of support based

immobilization is adsorption, in which enzymes attach to the carrier surface mainly through

Van der Walls interactions and hydrogen bonding. In this way there is no need to perform any

chemical modification of the carrier, but enzymes tend to leach out along time, especially in

aqueous solvents. Networks synthesized from many different organic polymers as well as

silica-based materials are good examples of carriers used to perform this type of

immobilization.

Another way to achieve immobilization at the carrier surface is by ionic binding. Ionic

interactions are stronger than the interactions that govern adsorption, but protein leaching

can still occur.

Instead, covalent binding is a good solution to overcome leaching out of the enzymes.

This method takes advantage on functional chemical groups on enzymes and carriers surface,

as is the case of amine groups. In this case, supports with a high density of reactive groups

allow multi-point attachment of the enzyme, thereby contributing to its rigidity/stability.

Covalent attachment can also be achieved by the use of cross-linkers, such as glutaraldehyde,

which works like bridges between the carrier and the enzymes.

It should be highlighted that, in support based immobilization, a significant reduction

of the volumetric and specific activity of the enzymes occurs, since carriers can account for

more than 90% of the volume or mass of the catalyst (5).

The choice of the most suitable method to immobilize the desired enzyme depends on

the reaction conditions, both environmental conditions and the configuration of the reaction

apparatus, as well as on the carrier to be used.

Also, the choice of the most suitable carrier to perform immobilization, besides taking

in account the immobilization methods that can be performed to the desired purpose, is

influenced by: its chemical properties, (composition; hydrophilic or hydrophobic nature;

stability in the conditions of the reaction medium; etc.); its mechanical stability and geometric

properties (size, shape, thickness, porosity, etc.); the characteristics of the enzyme to be

immobilized (chemical composition, physical properties, structure, etc.); its ecological and

biological properties (non-toxic neither for the environment nor for human health; easily

disposable; etc.), among other factors (6).

A wide range of synthetic carriers, organic or inorganic, can be created to encounter

the optimal characteristics to perform immobilization of the desired enzyme, namely the

chemical composition, since this is a determinant property to the occurrence of successful

interactions between the enzyme and the carrier in many of the immobilization methods

described above. Another approach to guarantee the compatibility between enzyme/cross-

linker and the carrier consists in the chemical activation of the carrier. This modification is

very useful when using natural carriers or their derivatives – as is the case of agarose, dextran,

chitosan – since it makes possible to add the desired chemical groups to their structure,

rendering them useful materials for industrial applications. Besides, many of the natural

carriers used are by-products of several industries, so this is a way of reusing a material that

otherwise would be a waste.

1.3. Objectives

Considering the advantages of enzyme immobilization and the features of peroxidase,

the work developed has as purpose the evaluation of peroxidase catalytic behaviour under

different medium conditions, at laboratorial scale, when it is immobilized on carbon

nanotubes.

As so, to access the optimal conditions for peroxidase immobilization, the influence of

different factors were studied: the concentration of the peroxidase solution with which

immobilization is performed; the pH of the medium during immobilization; and the time of

contact between peroxidase and carrier.

It pertains to study the influence of these parameters in immobilization because they

generally affect the behaviour of all enzymes. In this case, attaching to the carrier strongly

depends on the surface available for enzyme immobilization and so the concentration of the

peroxidase solution used to perform immobilization should be optimized to the surface area

available. Usually the medium pH affects the ionization state of the amino acids composing

the protein and hence its overall structure. Consequently, this influences both the catalytic

capacity of the enzyme and its ability to immobilize. Finally, the immobilization on a carrier

surface depends on the establishment of an equilibrium between enzyme in solution and that

on the carrier and so the time of contact between the two components should be optimized to

achieve that state.

Then, to access the applicability of peroxidase immobilized on carbon nanotubes in

industrial processes, the thermostability at high temperatures, and the kinetic parameters of

the enzyme for catalysis of ABTS were studied.

It pertains to study peroxidase thermostability because all enzymes have maximal

activity at an optimal temperature, so, at the industrial scale, the reaction medium should be

fitted to the properties of the enzyme in use. Finally, the kinetic parameters for catalysis must

also be studied because the immobilization process can change the access of the substrate to

the enzyme, as well as the way they interact with each other. This could affect the rate of

catalysis and, in consequence, render an industrial process unviable.

2. Experimental section

2.1. Reagents

All studies were performed using commercial Peroxidase from Novozymes.

Additionally, all the tests with immobilized peroxidase were carried out using Multi-Walled

Carbon Nanotubes (MWNT).

2.2. Carbon Nanotubes processing

2.2.1. Functionalization and characterization of MWNT

Multi-walled carbon nanotubes were prepared from ethylene gas by chemical vapor

deposition using Fe nanoparticles supported on alumina as catalyst. Details on the reactor

configuration and preparation of the catalyst can be found elsewhere [1]. The catalyst was

removed by boiling with diluted sulfuric acid (50%) at 150 ºC for 3 h after which the nanotubes

were filtered when hot by a glass frit followed by washing with hot water and drying at 120 ºC.

This sample was designated as MWNT1.

MWNT2 were prepared by liquid-phase oxidation of MWNT1. For oxidative

functionalization, 1,5 g of purified nanotubes was mixed with 150mL of HNO3 7 M and

refluxed for 3 h at 125 ◦C under stirring. The nanotubes were filtered on a glass frit, washed

with water until the filtrate reached neutral pH, and dried overnight at 120 ◦C.

MWNT2 material was heat treated under inert atmosphere (N2) at 400 ºC for 1 h

(sample MWNT3) and at 900 ºC for 1 h (sample MWNT4), to selectively remove surface

groups. The obtained samples were characterized by adsorption of N2 at -196 ºC and

temperature programmed desorption (TPD). The total amounts of CO and CO2 evolved from

the samples were obtained by integration of the TPD spectra (not shonw).

Table 1. Specific surface area (SBET), pore volume (Vp) and pore diameter (dp) for the

different MWNT samples.

Sample SBET (m2/g) Vp (cm3/g) rp (nm)

MWNT1 255 0.944 3.2

MWNT2 276 1.073 15

MWNT3 291 1.154 20

MWNT4 301 1.247 25

2.3. Immobilization technique

In all experiments with carbon nanotubes, 1,2 mL of enzymatic solution (of adequate

concentration and pH according to the experiment being performed) was put in contact along

with 0,04 g of carbon nanotubes under stirring (during appropriate times according to the

experiment being performed). After immobilization, carbon nanotubes were washed 3 times

with phosphate buffer 0,1 M, pH 7,0.

2.4. Enzymatic activity measurement

To measure peroxidase activity when it was immobilized, carbon nanotubes were

mixed with 12 mL of ABTS 6 mM and 42 mL of citrate/phosphate buffer 0,05M/0,1M, pH 4,5

at 40 °C, and 3 mL of H2O2 under magnetic stirring at 100 rpm, for 4 minutes. Samples were

taken every minute. Their absorbance was measured spectrophotometrically (Thermo

Electron, model UV1 spectrophotometer) at 420 nm. After linear regression of the data

obtained, enzyme’s activity was determined as in Eq. 1:

𝑈

𝑔=

𝑎𝑏𝑠/𝑚𝑖𝑛 × 𝑓𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 × 𝑉𝑟𝑒𝑎𝑐𝑡𝑖𝑜𝑛 × 106

𝜀 × 𝑚𝑐𝑎𝑟𝑟𝑖𝑒𝑟 (Eq.1)

where:

U/g is the quantity of enzyme capable of breaking down 1 μmol of ABTS per minute

and per mass unit of carrier;

abs/min is the absorbance per minute determined by linear regression;

fdilution is the dilution factor of the sample;

Vreaction (L) is the volume of reaction;

106 is the conversion factor from M to μM;

ε is the molar extinction coefficient (36000 M-1cm-1 at 420 nm);

and mcarrier (g) is the mass of carbon nanotubes with enzyme immobilized.

On other hand, to measure the activity of free peroxidase, 100 μL of enzymatic

solution were mixed with 0,5 mL of ABTS 0,4 mM and 1,4 mL of citrate/phosphate buffer

0,05M/0,1M, pH 4,5 at 40 °C, and then abs/min was determined for 30 seconds in the

spectrophotometer at 420 nm. Peroxidase activity was calculated as in Eq. 2:

𝑈

𝐿=

𝑎𝑏𝑠/𝑚𝑖𝑛 × 𝑓𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 × 106

𝜀 (Eq.2)

where abs/min, fdilution, 106 and ε are defined as in Eq. 1, and U/L is the quantity of enzyme

capable of breaking down 1 μmol of ABTS per minute and per volume unit of enzymatic

solution.

2.5. Experiments to determine the optimal conditions for peroxidase immobilization

Experiences to find optimal peroxidase concentration were performed immobilizing

carbon nanotubes with peroxidase solutions of different concentrations – 1, 3, 4, 5, 6, 8, 9 and

10 µl/mL – for one hour. These solutions were prepared using phosphate buffer 0,05 M, pH

6,0. Duplicates were made.

To test the influence of pH on peroxidase immobilization, carbon nanotubes were

mixed, for one hour, with enzymatic solutions of optimal concentration prepared using buffers

at different pH values. Indeed, phosphate buffer 0,05 M, pH 4,0, 6,0 and 9,0. Duplicates were

made.

To determine the appropriate contact time between the carrier and the enzymatic

solution during immobilization, carbon nanotubes were mixed with peroxidase solution at

optimal concentration and pH for 30 min, 1 h, 1,5 h, 2 h, 2,5 h, 3 h, 3,5 h. Another experience

to test this parameter was carried out in the same conditions except for the concentration of

the enzyme solution used, which was half of the optimal one.

3. Results and discussion

3.1. Optimal conditions for experimental peroxidase catalysis

3.1.1. Optimal peroxidase concentration to perform immobilization

The results obtained for peroxidase solutions with concentrations under 5 µl/ml, the

enzymatic activity of the immobilized peroxidase rises gradually, while for concentrations

above it, enzymatic activity do not differ significantly.

0,00

10,00

20,00

30,00

40,00

50,00

60,00

0 5 10 15Per

oxi

das

e a

ctiv

ity

(U/g

car

rie

r)

Enzyme (µL/mL)

Series1

Figure 3. Enzymatic activity registered in the catalysis using carbon nanotubes with immobilized

peroxidase when immobilization was performed with peroxidase solutions of different concentrations.

3.1.2. Optimal medium pH to perform immobilization

As depicted in Figure 4, when immobilization is performed on different mediums,

peroxidase activity is stabile. So it can be said that the immobilized peroxidase is stable both at

pH range between 4 and 10.

0

500

1000

1500

2000

2500

3000

0 2 4 6 8 10 12

Pe

roxi

das

e a

ctiv

ity

(U/g

)

pH

Figure 4 Enzymatic activity registered in the catalysis using carbon nanotubes with immobilized

peroxidase when immobilization was performed in medium with different pH value.

3.1.3. Optimal contact time to perform immobilization

The time interval during which peroxidase solution and carbon nanotubes are in

contact influences how much enzyme adsorbs onto the carrier. For the time intervals tested,

immobilization with peroxidase solution of optimal concentration during 3,5 h seemed to be

the most appropriate time to obtain maximal activity (Figure 5).

For shorter time intervals, enzymatic activity registered is lower because there wasn’t

enough time to enzymes stably adsorb onto the carrier.

In this case, the maximal recovered activity was registered for contact periods near 3,5

h. So, it was concluded that it isn’t worth to perform immobilization for periods earlier than 3,5

h, since it has no practical effects on the recovered activity of the immobilized enzyme.

0,0000

2,0000

4,0000

6,0000

8,0000

10,0000

12,0000

0 50 100 150 200 250

Pe

roxi

dse

act

ivit

y (U

/g c

arr

ier)

Contact time (min)

Series1

Figure 5 Enzymatic activity registered in the catalysis using carbon nanotubes with immobilized

peroxidase when immobilization was performed for different periods of time.

4. Conclusion

In this work it was possible to determine the optimal conditions to perform

experiments, at a laboratorial scale, with peroxidase immobilized by carbon nanotubes. It was

verified that the use of a peroxidase solution with a concentration of 8µl / mL is sufficient to

obtain the maximal enzymatic activity after immobilization. Additionally, this process must be

undertaken in media of neutral or milder acid pH for at least 3,5 h to obtain the best results.

5. References

1. Cooper, V.A. and Nicell J.A. 1996. Removal of phenols from a foundry from wastewater using

Horseradish peroxidase. Water Res. 30: 954-964.

2. Karam, J. and Nicell J.A. 1997. Potential applications of enzymes in waste treatment. J.

Chem. Technol. Biotechnol. 69: 141.

3. Conesa, A., Punt, P.J. and van den Hondel C.A.M.J.J. 2002. Fungal peroxidases: molecular

aspects and applications. J. Biotechnol. 93: 143–158.

4. Martınez A.T. 2002. Molecular biology and structure-function of lignin-degrading heme

peroxidases. Enzyme Microb. Technol. 30: 425–444.

5. Veitch, N.C. 2004. Horseradish peroxidase: a modern view of a classic

enzyme, Phytochemistry 65: 249-259.

6.Mester, T. and Tien M. 2000. Oxidation mechanism of ligninolytic enzymes involved in the

degradation of environmental pollutants. Int. Biodeter. Biodegr. 46: 51-59

Acknowledgements

All the work I have been developing in the last months is only possible due to the

contribution of many people around me to whom I am sincerely grateful.

They are:

My supervisors Ana Paula Tavares and Cláudia Silva, for all their help and teaching on

the experiments with peroxidase, from practical issues of the laboratorial work to the data

processing, and all the opportunities they are providing me. These are my first steps in the

world of research work and they both are the parents teaching the baby learning how to walk.

All my colleagues of laboratory E301, for receiving me so well and share their

friendship with me, but specially Ricardo Segundo, for helping me all with all I needed and

taking care of me.

All my family, for supporting me above everything else, but specially to my parents, to

whom I owe everything that I am and that I can achieve.