1

lr

Prof. Dr. Magdy Aly Amin

Professor of Microbiology and Immunology

Faculty of Pharmacy, Cairo University

Prof. Dr. Ahmed Ibrahim El-Batal

Professor of Applied Microbiology and Biotechnology

National Center of Radiation Research and Technology

(NCRRT)

Department of Microbiology and Immunology

Faculty of Pharmacy

Cairo University

2013

Studies on Possible Activation of Microbial

Laccase Production Using Gamma

Irradiation

Thesis submitted by

Nora Mohamed Ahmad ElKenawy

B.Sc. of Pharmaceutical Sciences

(Microbiology and Immunology)

Cairo University 2008

In partial fulfillment for Master Degree

in

Pharmaceutical Sciences

(Microbiology and Immunology)

Under Supervision

of

Assis. Prof. Dr. Aymen Samir Yassin

Assistant Professor of Microbiology and Immunology

Faculty of Pharmacy, Cairo University

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“The roots of education are

bitter, but the fruit is sweet”

Aristotle

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Acknowledgment

Thank you Allah for giving me the will, strength and

patience to do that work. I will never be able to count your

blessings.

I would have not been able to complete this journey

without the aid and support of many people that I would

like to take this opportunity to thank.

I must express my gratitude and deepest thanks towards my

supervisor, Professor Dr. Magdy Amin. Your leadership,

support and understanding, have set an example I hope to

match some day.

Also, I would like to thank my superviser Professor Dr.

Ahmed El-Batal for introducing me to the topic and for his

continuous support and guidance during the practical work,

offering all the facilities needed.

Furthermore, I would like to express my gratitude to my

supervisor Dr. Aymen Yassin for the useful comments,

remarks and engagement through the learning process of

this master thesis.Thank you for giving me much of your

effort and time, you have taught me a lot.

A big thanks to my parents for instilling in me confidence

and a drive for pursuing my master. Thank you for your

endless love and support, I will be forever grateful.

A special thanks for the one who kept me going during my

thesis with his love, kindness and support, without him I

would have given up long time ago. I am blessed that I

have a husband like you. Thank you, Ahmad.

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Abstract

Enzyme production is an essential discipline in

biotechnology. Laccase enzyme is an oxidoreductase that

catalyzes the oxidation of various aromatic compounds,

with the simultaneous reduction of oxygen into water.

Although the enzyme is present in plants, insects and

bacteria, the most important source is fungi and particularly

the Basidiomycetes. In fungi, the enzyme plays a role in the

removal of potentially toxic phenols arising during fungal

morphogenesis, sporulation, phytopathogensis and

virulence.

In this work, the production of fungal laccase was

optimized from a local isolate of Pleurotus ostreatus using

solid state fermentation. Factorial design was used to study

the effect of several nutrients and inducers on enzyme

activity.

Purification, characterization of the enzyme, the effect of

temperature and pH were studied. The effect of gamma

radiation on fungal growth and enzyme production was

investigated.

The optimization of the production conditions yielded an

enzyme with activity over 32,054 IU/gram of fermented

substrate. Factorial design was capable of establishing the

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conditions that multiplied the activity of the enzyme several

folds and consequently, reducing the cost of production.

The enzyme was capable of decolorizing several dyes with

over 80 % reduction in color in case of methyl orange and

trypan blue. The decolorization of dyes is a simple method

to assess the aromatic degrading capability of laccase. The

enzyme was also used in the synthesis of gold

nanoparticles, proving that laccase from Pleurotus

ostreatus has a strong potential in several industrial

applications, which opens a door towards using of fungal

laccase in further biotechnological processes.

Keywords: Laccase; Pleurotus ostreatus; Gamma

radiation; Gold nanoparticles (GNPs).

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I. Introduction

Enzymes are natural catalysts. They are produced by living

organisms to increase the rate of an immense and diverse

set of chemical reactions required for life. They are

involved in all processes essential for life such as DNA

replication and transcription, protein synthesis, metabolism

and signal transduction. Their ability to perform very

specific chemical transformations and reactions makes

them an integral part in almost all industrial products (Li et

al., 2012).

Without enzymes modern life would be unthinkable.

Enzymes are present in almost every detergent package

catalyzing the removal of stains from our clothes. Enzymes

improve dairy products and give bread the right crumb

structure and mouth feel. They find wide use in the

catalysis of synthetic organic reactions, in the production of

bio-ethanol and the bulk production of glucose and

fructose. In the textile, pulp, paper and animal feed

industries enzymes were also present (Bloom et al., 2005

and Clouthier and Pelletier, 2012).

In the medical and health sector enzymes have their

contribution as well. They are used in clinical diagnosis,

7

therapy, in genetic engineering and drug production (Ran

et al., 2008).

The role of enzymes in keeping the environment clean is

immense; being implemented in the biodegradation of

various environmental pollutants and biocatalyzing various

reactions by substituting the environmentally hazardous

and toxic chemical catalysts offering an environment

friendly alternative (Bird et al., 2008).

At present, almost 4000 enzymes are known, and of these,

approximately 200 microbial original types are used

commercially. However, only about 20 enzymes are

produced on truly industrial scale. With the improved

understanding of the enzyme production biochemistry,

fermentation processes, and recovery methods, an

increasing number of industrial enzymes can be foreseeable

(Li et al., 2012).

Among the enzymes that have a huge potential in industry:

is laccase an important member of the lignolytic family,

with broad substrate specificity.

Laccase, is blue copper oxidase (E.C. 1.10.3.2

benzenediol: oxygen oxidoreductase) and can catalyze the

one-electron oxidation of phenolics, aromatic amines, and

other electron-rich substrates with the concomitant

8

reduction of O2 to H2O. Most of the laccases studied are of

fungal origin especially from the white-rot fungi class.

Fungal laccases play an important role in plant

pathogenesis, pigment production, and degradation of

lignocellulosic materials (Gianfreda et al., 1999 and

Thurston, 1994).

The ability of laccase producing microorganisms or

purified laccase to eliminate a wide range of pollutants is

currently one of the most interesting subjects for

researchers in environmental biotechnology (Baldrian,

2006).

Potential application of laccase includes textile dye

decolourization, pulp bleaching, effluent detoxification,

biosensors, and bioremediation.

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Aim of Work

The present study aims to investigate the enhancement of

microbial production of the laccase enzyme and some of its

beneficial applications.

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II. Literature Review

1. Biocatalysis and green technology

Considerable emphasis has been placed on developing

environmentally benign or “green” technology to replace

existing technologies (Bermek et al., 2002). A major

component of the green technology revolution is

biocatalysis, which is finding increasing interest in the last

decades especially in the biotechnology field.

Biocatalysis can be broadly defined as the use of biological

molecules (usually enzymes) to catalyze specific chemical

reactions.

2. Enzymes as biocatalysts

Enzymes are complex protein molecules serving as

biological catalysts. They are produced by living organisms

to catalyze the biochemical reactions required for life

(Kumar et al., 2010).

The most cited advantages of biocatalysis are as follows:

A. Enzymes relative selectivity and specificity (Kumar

et al., 2010):

• Substrate selectivity – ability to distinguish a

particular compound among a mixture of chemically

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related compounds e.g proteases acting according to a

specific amino acid sequence.

• Stereoselectivity – ability to act on a substrate or

produce a product of one enantiomeric or diastereomeric

form as in enzymes involved in resolution of racemic

mixtures.

• Regioselectivity – ability to act on one location in a

molecule where preference of one direction of chemical

bond making or breaking over all other possible

directions.

• Functional group selectivity – ability to selectively

act on one functional group even when other groups

may be more chemically reactive.

B. Mild reaction conditions

Most enzymes operate in aqueous solution at mild

temperature, pH and at atmospheric pressure. Chemical

catalysts often require organic solvents, high temperatures,

extremes of pH and high pressure. Enzymes can therefore

result in lower energy and materials cost.

C. Environment friendly

Proteins are naturally biodegradable, hence not causing a

problem in their disposal as they operate in aqueous

solution compared to chemical catalysts that often require

12

organic solvents that could be hazardous to the

environment.

D. High catalytic efficiency

Enzymes naturally have high turnover numbers, i.e. high

number of substrate molecules catalyzed per molecule of

enzyme, resulting in less catalyst required to complete the

conversion.

3. Enzyme nomenclature

a. E.C numbers

Enzymes are classified into six different groups

according to the reaction being catalyzed. The

nomenclature was determined by the Enzyme

Commission in 1961 (with the latest update having

occurred in 1992); hence all enzymes are assigned an

“EC” number. The classification does not take into

account amino acid sequence (i.e., homology), protein

structure, or chemical mechanism. “EC” number are

four digits, for example a.b.c.d, where “a” is the class,

“b” is the subclass, “c” is the sub-subclass, and “d” is

the sub-sub-subclass. The “b” and “c” digits describe

the reaction, while the “d” digit is used to distinguish

between different enzymes of the same function based

on the actual substrate in the reaction.

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Example: for Alcohol: NAD+ oxidoreductase EC

number is 1.1.1.1 (Boyce and Tipton, 2001).

b. Recommended name:

The most commonly used name for the enzyme is usually

used, provided that it is unambiguous. A number of generic

words indicating reaction types may be used in

recommended names, but not in the systematic names, e.g.

dehydrogenase, reductase, oxidase, peroxidase, kinase,

tautomerase, deaminase and dehydratase etc. Where

additional information is needed to make the reaction clear,

a phrase indicating the reaction or a product may be added

in parentheses after the second part of the name, e.g. (ADP-

forming), (dimerizing) and (CoA acylating) ( Boyce and

Tipton, 2001).

4. Enzymes classification

Enzymes are classified into six different groups according

to the reaction being catalyzed. The pie chart shown in

Figure (1) represents the distribution of the six known

classes of enzymes (Faber, 1997):

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Fig 1: Distribution of classes of enzymes

It is clear that the most popular group is the hydrolases,

followed by oxidoreductases and transferases.

The six classes of enzymes are described below briefly

with examples for every class (Horton et al., 2005; Becker

et al., 2008):

Class: 1 Oxidoreductases

Oxidoreductases catalyze oxidation or reduction reactions,

where electrons are transferred from one molecule (the

reductant) to another molecule (the oxidant). They are

commonly known as dehydrogenases or oxidases that

catalyze the removal of hydrogen atoms and electrons from

the compounds on which they act. They include manganese

peroxidase, laccase and alcohol dehydrogenases.

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Class: 2 Transferases

Transferases are enzymes that catalyze the movement of a

functional group from one molecule to another. These

functional groups are very diverse and can include

phosphate, methyl and amino groups. They include acetate

kinase, and alanine deaminase.

Class: 3 Hydrolases

Hydrolase enzymes are simply enzymes that catalyze

hydrolysis of molecules; by breaking of single bonds

through the addition of water. There are a huge variety of

hydrolase enzymes. For example, the digestive enzymes

that are classified based on their target:

*Proteases/ peptidases cleave peptide bonds between amino

acids in protein in order to breakdown proteins.

*Lipases break down lipids into fatty acids and glycerol by

cleaving ester bonds.

*Amylases break down starch molecules into glucose

sugar.

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Class: 4 Lyases

Lyases catalyze lysis reactions that generate a double bond.

These are a type of elimination reaction but are not

hydrolytic or oxidative. For example L-aspartate

decarboxylase which converts aspartate into alanine and

CO2.

Class: 5 Isomerases

Isomerases are enzymes that can catalyze structural

changes within a molecule. There is only one substrate and

one product with nothing gained or lost, so they represent

only a change in shape. Isomers have the same molecular

formula but differ in their structural formula. These

differences can change the chemical properties of the

molecule. There are multiple classes of isomerases, for

example geometric, structural, enantiomers and

stereoisomers examples: alanine racemase, which converts

L- alanine into D-alanine.

Class: 6 Ligases

Ligases are responsible for the catalysis of ligation; the

joining of two substrates. Usually chemical potential

energy is required e.g DNA ligase.

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5. Use of enzymes in industry

Although enzymes are formed within living cells, they can

continue to function in vitro (in the test-tube) and their

ability to perform very specific chemical transformations is

making them increasingly useful in industrial processes

Table (1).

Table 1: The possible applications of enzymes in industry (Bloom et al.,

2005; Clouthier and Pelletier, 2012)

Industry Enzyme Function

Laundry

Detergents

Protease (91%)

Lipase (6%)

Amylase (2%)

Cellulase (1%)

Used in pre-soaks to remove protein -based

stains

Now commonly included to digest oils and

fats

Removes resistant starch residues

Digests the cotton fuzz, which accumulates

with excessive washing

Dairy

Industry

Rennin ( from the

stomachs of

young ruminant

animals)

Lipases

Lactases

Manufacture of cheese

Enhances ripening of blue-mold cheeses

Break down lactose to glucose and

galactose

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Textiles

Industry

Amylase

Now widely used to remove starch from

woven fabrics

Starch is used as an adhesive on the threads

of many fabrics to prevent damage during

weaving

Brewing

Industry

Amylases

Glucanases

Proteases

Splits polysaccharides and proteins in the

malt

Reduces clouding of juices

Improves filtration characteristics

Pulp and

Paper

Industry

ß-xylanases

Lipases

Emerging technology for enhancing pulp-

bleaching

Reduces pitch, which causes paper to stick

to rollers and tear

6. Use of synthetic dyes in textile industry

The discovery of synthetic dyes overwhelmed the role of

natural dyes in the industry due to its low production cost,

brighter colors, and better resistance towards environmental

factors i.e. heat, sun light and humidity. This has led to a

higher consumption of synthetic dyes over natural dyes for

most types of industrial applications (Moussavi and

Mahmoudi, 2009).

Dyes consist of two main groups of compounds

chromophores and auxochromes. Chromophores determine

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the color of the dye while the auxochromes determine the

intensity of the color (Moussavi and Mahmoudi, 2009).

7. The hazards of dyes in the environment

Dyes have become one of the main sources of severe water

pollution as a result of the rapid development of the textile

industries. The release of the colorant effluent has triggered

a major concern on the human health as well as marine

lives.

Once discharged, the dyes can confer toxic and

carcinogenic properties to the water and can contribute to

the total organic loading (Dos Santos et al., 2004;

Cristovao et al., 2009). In addition, the coloring of the

water will affect the normal absorption of sunlight for

many aquatic organisms.

Color is usually the first contaminant to be recognized in

wastewater. A very small amount of dye in water of (10-50

mg/L) is highly visible and affects the quality and

transparency of water and gas solubility of water bodies

(Cristovao et al., 2009). Many azo dyes (constituting the

largest dye group) may be decomposed into potential

carcinogenic amines under anaerobic conditions in the

environment (Chung and Stevens, 1993). That is why

there are tremendous efforts to help overcome this problem

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8. Current methods for dye treatment

The currently used methods include physical methods,

chemical and biological methods (Zilly et al., 2002).

A. Physical methods for treatment of dye containing

waters:

1. Adsorption

This technique involves the concentration of dye molecules

on a solid surface. Among the supports used for adsorption,

carbon based is the oldest (Forgacs et al., 2004; Gupta &

Suhas 2009). However, this method is expensive since

large amounts of material are required for the treatment of

dye wastewaters (Forgacs et al., 2004; Bukallah et al.,

2007). Organic supports as chitosan, chitin, orange peels,

banana pith, lignin, sawdust, etc. are relatively cheap as

they usually originate from renewable sources or from

wastes of industrial processes (Saravanabhavan et al.,

2007; Singh et al., 2009).

2. Sedimentation

It is a basic form of a primary treatment. It can be

significantly improved with the addition of certain

chemicals, such as aluminum, calcium or ferric ions, to the

process stream (Gupta and Suhas, 2009).

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3. Filtration

Filtration techniques include microfiltration, ultrafiltration,

nanofiltration, and reverse osmosis. This method is based

on the use of a coated membrane that will retain the dyes

thus producing colorless water. Although the method was

shown to be efficient for the removal of dyes (Mo et al.,

2008), its application is still limited due to problems of

membrane clogging, generation of high operating

pressures, significant energy consumption, high membrane

costs, relatively short membrane lifetimes (Gupta and

Suhas, 2009) and the required pretreatment of some dyes

with coagulants (Mo et al., 2008).

B. Chemical methods for treatment of dye containing

waters:

1. Electrochemical methods

They include electro-oxidation and electro-coagulation.

These methods are effective since decolorization of dyes

with the concomitant reduction of the chemical oxygen

demand (COD). COD is commonly used to indirectly

measure the amount of organic compounds in water

determining the amount of organic pollutants found

in surface water (e.g. lakes and rivers) or wastewater,

making COD a useful measure of water quality. However,

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the rate of dye removal depends on the material of the

anode as well as its working potential, which represent the

main drawbacks due to high electricity costs (Gupta and

Suhas 2009).

2. Photocatalysis

It is a series of advanced oxidation steps where light energy

excites an electron from the catalyst (zinc oxide, zirconium

oxide, etc), which will initiate a chain of reactions resulting

in the formation of hydroxyl radicals. These have high

oxidizing potential and can therefore attack most organic

structures. The degradation of dyes depends, among other

factors, on the presence of electron acceptors such as

hydrogen peroxide and ammonium persulfate (Gupta and

Suhas, 2009).

Although most of these treatment methods are effective,

their disadvantages include high costs, the formation of

hazardous by-products and elevated energy requirements

(Ramsay and Nguyen 2002; Ramya et al., 2007). In

addition, some of them have short operational lifetimes

(Kaushik and Malik 2009), which will determine the color

remaining in the wastewater (Dos Santos et al., 2004).

They can also generate a significant amount of polluted or

non-polluted sludge (Ramsay and Nguyen 2002; Ramya

et al., 2007).

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Therefore, it has been suggested that enhanced microbial

decolorization may provide a less expensive and more

environmentally acceptable alternative to chemical

treatment (Selvam et al., 2003).

C. Biological methods for treatment of dyes

1. Bacteria

Bacterial treatment of azo dyes can be performed under

anaerobic and aerobic conditions (Gopinath et al., 2009).

Anaerobically, the decolorization of azo dyes is achieved

by reductive cleavage of the azo bond to produce amines

(Martins et al., 2001), which can be toxic and/or

carcinogenic. The carcinogenic effect is related to the

presence of azoreductase enzyme in the bacteria (Feng et

al., 2010; Dawkar et al., 2010). However, studies with

Bacillus spp. revealed that other enzymes (laccase and

lignin peroxidase) could also participate in the reductive

cleavage of the azo-dye (Dawkar et al, 2010). The role of

bacterial laccase in decolorization of azo dyes was

described by the application of Bacillus. spp. (Pereira et

al., 2009). In this enzymatic pathway, azo dyes are

oxidized non-specifically via a free radical mechanism,

forming phenolic type compounds, able to undergo

polymerization.

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Among the disadvantages of bacterial dye treatment is the

production of amines represents the most important

limitation; however, bacteria remain the most applied

organism for treatment of azo dyes (Saratale et al., 2011)

since high yields of decolorization can be achieved. In

addition, bacterial treatments can be coupled to other

methods to degrade the produced aromatic amines. So far,

aerobic approaches (Lourenço et al., 2006),

electrochemical techniques (Carvhalo et al., 2007) and

cultivating a mixture of bacterial strains (Saratale et al.,

2011) have been shown to be effective in degrading

aromatic amines.

2. Fungi

Fungi are applied for treatment of dyes in two ways. The

first treatment involves the mycelial adsorption of dyes by

application of living or dead cells (Fu and Viraraghavan,

2001; Kaushik and Malik, 2009), while the second

includes the application of living cells and their

extracellular enzymes (Kapdan et al., 2000; Wesenberg et

al., 2003). The efficiency of the treatment will depend on

the fungal source as well as on the type of dye to be treated.

Extracellular fungal enzymes demonstrate advantages over

the living whole cells since they provide lower mass

transfer limitations (Kaushik and Malik, 2009).

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3. Lignolytic enzymes

One of the most important and essential applications of

enzymes is the detoxification of industrial effluents. The

enzymes are capable of oxidizing a wide array of phenolic

and non-phenolic compounds based on their ability to

oxidize lignin which is a major component in wood

structure. Lignin is the second most abundant polymer on

earth, second only to cellulose, and is the most abundant

aromatic material (Leonowicz et al., 1999), it may also be

considered among the most recalcitrant natural products

produced in the biosphere, and thus bioconversion of lignin

is among the most important processes occurring in the

carbon cycle .

The extremely complex nature of lignin shown in Figure

(2) requires an array of oxidative enzymes to be involved in

its complete degradation. Although a wide range of

enzymes have been reported to be involved in the tedious

process of lignin degradation, only a few enzymes like

lignin peroxidase (LiP), manganese peroxidase (MnP) and

laccase play a major role in the process. These enzymes are

mainly produced by fungi and bacteria to achieve the task

of degrading the highly complex aromatic polymer, all

coming under the general category of lignin degrading

enzymes or lignolytic enzymes. Fungal attack is an

26

oxidative and non-specific process, which decreases

methoxy, phenolic, and aliphatic content of lignin, cleaves

aromatic rings, and creates new carbonyl groups (Kirk and

Farrell, 1987; Hatakka, 2001). These changes in the

lignin molecule result in depolymerization and carbon

dioxide production (Kirk and Farrell, 1987).

Fig 2: Lignin structure

3.1. Manganese peroxidase

It is a common peroxidase among white rot fungi (Eggert

et al., 1996; Hatakka, 1994); it requires the co-substrates

hydrogen peroxide and Mn2+ for enzyme activity. It has

been proposed that this enzyme uses a similar method for

the degradation of lignin as laccase, but instead of using a

phenolic substrate to oxidize lignin, it oxidizes Mn2+ to

Mn3+, which in turn oxidizes lignin (Wariishi et al., 1989).

The presence of similar peroxidases that do not require

Mn2+ as a co-substrate has been reported (Keharia and

27

Madamwar, 2002; Lobarzewski, 1981). These enzymes

have been termed manganese-independent peroxidases

(MIP).

3.2 Lignin peroxidase (LiP)

It is characterized by its ability to oxidize high redox

potential substrates using hydrogen peroxide as its co-

substrate (Martínez, 2002). LiP is a heme-containing

glycoprotein differentiated from the other ligninolytic

enzymes by its ability to oxidize veratryl alcohol as a

substrate (Linko, 1992), which includes polyphenols

(Bourbonnais and Paice, 1996), methoxy-substituted

monophenols and aromatic amines (Archibald et al., 1997;

Bourbonnais et al., 1995).

3.3. Laccase

Laccase (E.C. 1.10.3.2) (p-diphenol: dioxygen

oxidoreductases; benzenediol dioxygen oxidoreductases) an

important member of the lignolytic enzymes family is an

oxidative enzyme that uses molecular oxygen as the

electron acceptor, to oxidize the phenolic units of lignin

(Claus 2004; Riva 2006). Laccase enzyme is found in

higher plants, bacteria, fungi, insects and lichens (Arakane

et al., 2005; Riva, 2006). The first report dates from 1883

when Yoshida detected laccase-like activity in Rhus

vernicifera plant in Japan (O´Malley et al., 1993).

28

However, it was not until 1962 that laccase was designated

a p-diphenol oxidase (Benfield et al., 1964), and was

accepted as part of the lignification process in plants

(O´Malley et al., 1993).

Most monomeric laccase molecules contain four copper

atoms in their structure that can be classified in three

groups using UV/visible and electron paramagnetic

resonance (EPR) spectroscopy as shown in Figure (3)

(Leontievsky et al., 1997).

The type I copper (T1) is responsible for the intense blue

colour of the enzymes at 600nm The T2 and T3 copper

sites are close together and form a trinuclear centre

(Leontievsky et al., 1997) that are involved in the catalytic

mechanism of the enzyme (Palmieri et al., 1998).

Fig 3: Laccase 3D structure

29

3.3.1 Mechanism of action of laccase

The reaction mechanism may be described by the fact that

laccase is able to reduce one molecule of dioxygen to two

molecules of water without the step of hydrogen peroxide

formation (Yaropolov et al., 1994), while performing one-

electron oxidation of a wide range of aromatic compounds

(Thurston, 1994). This oxidation results in an oxygen-

centred free radical, which can then be converted in a

second enzyme-catalysed reaction to quinone as shown in

Figure (4). The quinone and the free radicals can then

undergo polymerization (Thurston, 1994).

Fig 4: Mechanism of action of laccase

30

The reaction is formed of three major steps Figure (5)

(Gianfreda et al., 1999):

(i) Type-1 copper reduction by the reducing substrate,

(ii) Internal electron transfer from type 1 copper to type 2

and type 3 copper trinuclear cluster,

(iii) Molecular oxygen reduction to water at type-2 and

type-3 copper atoms

Fig 5: Reaction locations in copper molecules

The characteristics of this enzyme differ widely according

to its microbial sources with great variations between

different strains.

3.3. 2 Microbiological sources of laccase:

3.3.2.1. Bacteria

In bacteria, the first reported laccase was found in

Azospirrullum lipoferum, where laccase was associated

with the melanin production for cell pigmentation (Faure

et al., 1994). In other bacterial species, it was related with

Type 1 Copper

Type 2 & 3 Copper

atoms

31

morphogenesis (Endo et al., 2002) or the resistance of

spores against hydrogen peroxide and UV (Held et al.,

2005; Sharma et al., 2007). Characterization of bacterial

laccases has revealed that they have a low redox potential

(0.45-0.54 V) (Durão et al., 2006) but they are active and

stable at high temperatures (66 hour at 60°C), pH (7-9) and

in high salt concentrations (Held et al., 2005; Dwivedi et

al., 2011).

These characteristics represent advantages for industrial

applications, since many processes are carried out under

similar conditions where other types of laccases might

easily be inactivated.

3.3.2.2 Fungi

Fungal laccase is involved in delignification of

lignocellulosic material (Robene-Soustrade & Lung-

Escarmant, 1997). However, in some fungi; the reactions

of laccase are unrelated to ligninolysis (Thurston, 1994).

Laccase plays a role in the formation of fruiting body,

fungal morphogenesis, protection against toxic compounds,

sporulation (Dwivedi et al., 2011) and synthesis of

molecules with virulent activity (i.e., melanin) to cause

fungal diseases (Riva, 2006).

The number of fungal laccase producers is immense and

they belong to the basidiomycetes and ascomycetes but

32

laccase activity has never been reported in the lower fungi

belonging to the zygomycete (glomeromycete) and

chytridiomycete (Rodríguez Couto and Toca-Herrera

2006; Morozova et al., 2007). Figure (6) shows (a.

Pleurotus ostreatus, b. Lentinula edodes and c.Trametes

veriscolor) that belong to basidiomycetes.

a. Pleurotus ostreatus

b. Lentinula edodes c.Trametes veriscolor

Fig 6: Different sources of fungal laccase

In the white rot basidiomycetes Trametes versicolor,

Lentinus edodes and Pleurotus ostreatus, laccase is

involved in lignin degradation. Laccase from Pycnoporus

33

cinnabarinus functions in both lignin degradation and the

biosynthesis of cinnabarinic acid, an antimicrobial with

activity against a variety of bacterial species. Other

functions, such as oxidation of humic acids and oxidation

of Mn+2 to Mn+3 have also been proposed for fungal

laccases (Octavio et al., 2006).

3.3.3 Industrial production of laccase

The industrial importance of laccase has led to the need to

enhance the laccase-producing ability of fungi (Lorenzo et

al., 2002). Screening of laccase producing microrganisms is

important for selecting suitable laccase producing strains

(Herpoël et al., 2000).

Since the microbial production of ligninolytic enzymes is

highly regulated by nutrients (Ben Hamman et al., 1999;

Miura et al., 1997), culture media optimization and

investigation into alternative nutrient sources are useful

tools for improving production of ligninolytic enzymes

from microbes especially fungi. The lignolytic enzymes

production pattern of fungi has been shown to be species

dependent, and even strain dependent (Pickard et al.,

1999; Rogalski et al., 1991), consequently medium

optimization provides a useful tool for an improvement in

lignolytic enzyme production.

34

3.3.4 Strain improvement

A natural approach to increasing the ligninolytic enzyme

production in basidiomycetes is through the genetic

crossing of monokaryotic strains derived from spores, and

screening of the resultant dikaryotic strains for improved

production of enzymes. Using this methodology

(Eichlerová and Homolka, 1999) increased the production

of laccase ten-fold in Pleurotus ostreatus.

Increased production of laccase may be achieved through

the mutation of wild-type fungal strains, followed by

selection for the improved character. The use of a mutation-

selection strategy was demonstrated successfully by

(Dhawan et al., 2003) and achieved a 6-fold increase in the

production of laccase from cyathus bulleri after mutation

with ethidium bromide. The major disadvantage of

employing this strain improvement technique is the

development of undesirable side effects, such as

pleiotropism where a change in a single gene affects a

number of phenotypic traits in the same organism

(Eichlerová and Homolka, 1999).

35

3.3.5 Medium optimization

3.3.5.1 Technique of fermentation

The concept of using solid substrates is probably the oldest

method used by man to make microorganisms work. By

using solid-state fermentation (SSF), which is defined as

the fermentation process in which microorganisms grow on

solid materials without the presence of free liquid.

SSF was known from ancient times in Asian countries and

was used in the production of koji and sake that are among

their traditional drinks. However, in western countries SSF

was nearly ignored after 1940. This was due to fact that

submerged fermentation (SMF) technique had become a

model technology for production of any compound by

fermentation as a result of the development of penicillin

Table (2). Pandey (1992) gives a brief summary of the

historical evolution of SSF.

Table 2: A brief summary of the historical evolution of SSF

Period Development

2,600 BC* Bread making by Egyptians

1,000 BC in Asia Cheese making by Penicillium roqueforti

500 BC Fish fermentation/preservation with

sugar, starch, salts, etc

36

7th Century by

Buddhist priests Koji process from China to Japan

18th Century Vinegar from pomace

Gallic acid used in tanning, printing, etc

1860-1900

Sewage treatment

1900-1920 Fungal enzymes (mainly amylases), kojic

acid

1920-1940 Fungal enzymes, gluconic acid, rotary

drum fermenter, citric acid

1940-1950

Huge development in fermentation

industry

Penicillin production by SSF and SmF

1950-1960 Steroid transformation by fungal cultures

1960-1980 Production of mycotoxins, protein

enriched feed

1980-present Various other products like alcohol,

gibberellic acid

*B.C, before Christ

Most enzyme manufacturers produce enzymes by (SMF)

techniques. However, in the last decades there has been an

increasing trend towards the use of the (SSF) technique for

the development of several bioprocesses, products and

production of several enzymes.

37

Solid-state offers greatest possibilities when fungi are used.

Unlike other microorganisms, fungi typically grow in

nature on solid substrates such as pieces of wood, seeds,

stems, roots and dried parts of animals such as skin, bones

and fecal matter.

Listed above the relative advantages and disadvantages of

SSF over SMF.

Advantages of SSF over traditionally employed SMF

(Pérez-Guerra et al., 2003):

• Similar or higher yields than those obtained in the

corresponding submerged cultures.

• The low availability of water reduces the possibilities

of contamination by bacteria and yeast. This allows

working in aseptic conditions in some cases.

• Similar environment conditions to those of the

natural habitats for fungi which constitute the main group

of microorganisms used in SSF.

• The inoculation with spores (in those processes that

involve fungi) facilitates their uniform dispersion through

the medium.

• Culture media are often quite simple. The substrate

usually provides all the nutrients necessary for growth.

• Simple design reactors with few spatial requirements

can be used due to the concentrated nature of the substrates

38

• Low energetic requirements (in some cases

autoclaving or vapour treatment, mechanical agitation and

aeration are not necessary).

• The low moisture availability may favour the

production of specific compounds that may not be

produced or may be poorly produced in SMF.

• Due to the concentrated nature of the substrate,

smaller reactors in SSF with respect to SMF can be used to

hold the same amounts of substrate.

Disadvantages:

• Only microorganisms that can grow at low moisture

levels can be used.

• Usually the substrates require pre-treatment (size

reduction by grinding, rasping or chopping,

homogenization, physical, chemical or enzymatic

hydrolysis and cooking.

• Biomass determination is very difficult.

• The solid nature of the substrate causes problems in

the monitoring of the process parameters (pH, moisture

content, and substrate, oxygen and biomass concentration).

• Agitation may be very difficult. For this reason static

conditions are preferred.

• Many important basic scientific and engineering

aspects are yet poor characterized.

39

• Information about the design and operation of

reactors on a large scale is scarce.

• There is a possibility of contamination by

undesirable fungi.

• In some SSF, aeration can be difficult due to the high

solid concentration.

• Cultivation times are longer than in SMF.

3.3.5.2 Influence of carbon source on laccase production

Increasing the production of lignolytic enzymes may be

achieved by modifying the source of carbon and nitrogen in

the medium (Gayazov and Rodakiewicz-Nowak, 1996).

Alterations in the source of carbon and nitrogen have

provided significant improvements in the extracellular

ligninolytic enzyme production experienced by several

researchers.

The carbon sources in the medium play an important role in

ligninolytic enzyme production.

a. Chemical carbon sources

Mansur et al. (1997) showed that fructose induced 100-

fold increase in laccase production of Basidiomycete spp.

Trametes versicolor is an excellent producer of laccase in

fermentation of mandarin peels. Glucose and cellobiose

40

were efficiently and rapidly utilized by Trametes pubescens

increasing laccase activity.

b. Agricultural and industrial wastes

Investigation into the use of agricultural wastes such as

cotton stalk extract (Ardon et al., 1996; Castillo et al.,

1997) and corn straw extracts. Crestini et al., (1996) have

yielded significant quantities of laccase, in T. versicolor

lignocellulosic material (barly bran) increased laccase

activity almost 50-fold compared to the control culture with

glucose.

Industrial effluents such as sugar cane baggase (Perumal

and Kalaichelvan, 1996) and olive oil mill wastewater

(Sanjust et al., 1991) have also been investigated as

potential nutrient sources for the production laccase. This

strategy of using industrial wastes provides not only a less-

expensive growth medium, but also has the added benefit

of solving an environmental problem.

c. Food wastes

Another potential solution to the relative expense of the

enzyme for industrial application is to decrease their

production costs by investigating less expensive substrates

as food wastes, opposed to the use of a chemically defined

medium (Berka et al., 1997; Yaver et al., 1999; Papinutti

41

et al., 2003). Diverse food wastes, such as apple, orange

and potato, which were screened for laccase production,

under solid-state fermentation conditions, by the white-rot

fungus Trametes hirsuta. Potato peelings gave the highest

activity, reaching about 5000 U/L within 8 days.

In a medium with the following carbon sources (mandarine

peelings and grapevine sawdust), both Pleurotus eryngii

and Pleurotus ostreatus strain No. 493, showed the highest

laccase activity (Stajic et al. 2006).

3.3.5.3 Influence of nitrogen source on laccase

production

White rot fungi ligninolytic systems are mainly activated

during the secondary metabolic phase of the fungus and are

often triggered by nitrogen depletion. Buswell et al. 1995

found that laccase was produced at high nitrogen

concentrations. Laccase was also produced earlier when the

fungus was cultivated in a substrate with a high nitrogen

concentration.

In a study by Elisashvili et al. served highest laccase

activity in C. unicolor IBB 62 in a medium with

ammonium sulphate as the nitrogen source (Elisashvili et

al., 2001). D’Souza- Ticlo et al. showed that well defined

organic nitrogen sources such as glutamic acid and glycine

42

were better than beef extract and corn steep liquor for

laccase production (D’Souza- Ticlo et al., 2006).

The use of malt extract as the carbon and nitrogen source in

Cyathus bulleri resulted in a much higher yield of laccase

than in mineral medium (Vasdev and Kuhad, 1994). Bran

flakes have successfully been used as a carbon source, and

provided a 794 fold increase in laccase production in

Trametes versicolor UAMH 827 and a 3190 fold

improvement in laccase production in Coriolopsis gallica

UAMH 8260 (Pickard et al., 1999).

3.3.5.4 Inducers

Inducers are compounds that elicit a positive response on

laccase production. The regulation of laccase genes differs

from organism to organism as the expression of some genes

is probably constitutive whereas in other cases it is

sensitive to culture conditions (Klonowska et al., 2001).

Laccase is usually constitutive or non-inducible as it does

not react readily to dissolved compounds that exhibit

properties similar to their substrates (Gianfreda et al.,

1999 and Yaver et al., 1996White rot fungi constitutively

produce low concentrations of laccase when they are

cultivated in submerged culture or on wood (Robene-

Soustrade and Lung-Escarmant, 1997). But higher

43

concentrations can be obtained by the addition of various

inducers.

The inducible laccase in other microorganisms is not

constitutive but the addition of certain inducers can

increase the concentration of a specific laccase or can

induce the production of new isoforms.

Some inducers interact variably with different fungal

strains found and their secretion of laccase (Eggert et al.,

1996; Lu et al., 1996). Single inducers may not elicit the

desired response of laccase production but higher

concentrations were obtainable with the addition of various

supplements to media (Lee et al., 1999). The exact action

of inducers is however unknown. It has been demonstrated

that fungi may possess several isozymes of laccase encoded

by several laccase genes and these may be differentially

regulated (Collins and Dobson, 1997). It has further been

suggested that the action of certain inducers may be as a

direct result of their toxicity to the fungus and the

capability of laccase to polymerize and detoxify them

(Collins and Dobson, 1997; Fenice et al., 2003;

Thurston, 1994). The use of inducers does however suffer

from several disadvantages including their toxicity and the

extra expense associated with the addition of an inducer.

44

Inducers include:

a. Metal ions

The addition of low concentrations of copper to the

cultivation media of laccase producing fungi (Assavanig et

al., 1992) or cadmium (Baldrian and Gabriel, 2002)

stimulates laccase production.

Addition of 150 μM copper sulphate to the cultivation

media resulted in a fifty-fold increase in laccase activity

compared to a basal medium (Palmieri et al. 2000).

Palmieri et al. found that the copper ions may increase

laccase production. As Cu+2 forms an integral prosthetic

group within the enzyme (Baldrian and Gabriel, 2002;

Dittmer et al., 1997), or by increasing laccase mRNA

translation (Collins and Dobson, 1997).

b. Phenolic or low molecular weight aromatic compounds

(lignin derivatives):

Many of these phenolic compounds resemble lignin

molecules or other phenolic chemicals (Marbach et al.,

1985; Farnet et al., 1999) examples like veratryl alcohol

and 3, 5-xylidine (Fig 7 .a) (Xavier et al., 2001). There is

evidence, in Trametes versicolor, that these compounds

cause an increase in mRNA levels and also it has been

suggested that the addition of veratryl alcohol in Figure

(7.b) may not elicit an inductive effect, rather it may act as

45

a protective agent against inactivation by hydrogen

peroxide produced endogenously by the fungus, thereby

indirectly eliciting a higher enzyme production (Dosoretz

et al., 1990).

3, 5-xylidine Veratryl alcohol

(a) (b)

Fig 7: Phenolic inducers

c. Organic acids

Gallic acid and ferulic acid Figure (8) are other examples of

inducers which enhance production of laccase from

Botrytis cinerea and Pleurotus sajor-caju, respectively

(Octavio et al., 2006). Ferulic acid (Figure 8. b) forms an

integral prosthetic group within the enzyme (Leonowicz

and Trojanowski, 1975).

The promoter regions of the genes encoding for laccase

contain various recognition sites that are specific for

xenobiotics and heavy metals (Sannia et al., 2001). These

inducers can bind to the recognition sites when present in

the substrate and induce laccase production.

46

Gallic acid b. Ferulic acid

(a) (b)

Fig 8: Organic acids

d. Alcohols

Some of these alcohols affect the metabolism or growth

rate (Froehner & Eriksson, 1974) while others, such as

ethanol, indirectly trigger laccase production (Lee et al.,

1999). Lee et al. investigated the induction effect of

alcohols on the laccase production in Trametes versicolor.

The enhanced laccase activity was comparable to those

obtained using 3, 5-xylidine and veratryl alcohol (Mansur

et al., 1997).

It was postulated that the addition of ethanol to the

cultivation medium caused a reduction in melanin

formation. The monomers, when not polymerised to

melanin, then acted as inducers for laccase production (Lee

et al., 1999). The addition of ethanol as an indirect inducer

47

of laccase activity offers a very economical way to enhance

laccase production.

3.3.5.5 Surfactants

The addition of detergents, e.g. Tween 20 or 80 has

resulted in higher yields of ligninolytic enzymes in certain

fungi. There is evidence that these detergents result in

higher permeability of oxygen and extracellular enzyme

transport through the cell membranes of fungi (Leštan et

al., 1994; Rothschild et al., 1995). Effective induction of

laccase from Pleurotus floridae with anionic and cationic

surfactants has been demonstrated (Dombrovskaya and

Kostyshin, 1996).

3.3.6 Influence of temperature on laccase production

It has been found that the optimal temperature for fruiting

body formation and laccase production is 25 °C in the

presence of light, but 30 °C for laccase production when

the cultures are incubated in the dark (Thurston, 1994). In

general the fungi are cultivated at temperatures between

25 °C and 30 °C for optimal laccase production (Arora

and Gill, 2000; Fåhreus and Reinhammar, 1967).

3.3.7 Immobilization of laccase

It is a proven method of enhancing the stability of enzymes

in unfavorable conditions (Ahn et al., 2002). The use of a

natural support medium such as clay or soil is desirable

48

since it poses no environmental risk, and is therefore

beneficial for terrestrial bioremediation. The benefits of

immobilization may however be offset by the increased

cost and loss of enzyme activity during immobilization

(Ahn et al., 2002).

Factorial design

The rapid advances in the field of computational

techniques inspired by biological phenomenon have the

potential to provide solutions to many complex

optimization problems. The use of statistical techniques is a

powerful tool for searching the key factors rapidly from a

multivariable system for the optimization of fermentation

medium composition (Montgomery, 2004). In the

optimization by (OFAT) one factor at time experiments, we

vary only one factor or variable at a time while keeping

others fixed. The process is laborious, time-consuming and

often overlooks the interaction effects of the parameters.

This obvious limitation of OFAT approach necessitates the

use of a more powerful technique by which multiple

variables can be optimized in relatively few experiments.

Factorial design is a statistically designed experimental

system that has been frequently used to vary several factors

simultaneously and efficiently (Box and Behnken, 1960).

49

It is important to describe the advantages of factorial design

over OFAT experimentation

Advantages of factorial over (OFAT) experiments

1. A designed experiment is a more effective way to

determine the impact of two or more factors on a

response than an OFAT experiment, as: It requires

fewer resources (experiments, time, material, etc.) for

the amount of information obtained. This can be of

major importance in industry, where experiments can

be very expensive and time consuming.

2. The estimates of the effects of each factor are more

precise because more observations are used to estimate

an effect results in higher precision.

Gamma radiation

Gamma radiation is a phenomenon of natural radioactivity.

When an atom emits either an alpha or beta particle, it often

emits a gamma ray at the same time. Gamma rays are a

type of electromagnetic radiation similar to X-rays, but of

shorter wavelengths. Treatment of materials with gamma

radiation, commonly used in medical and pharmaceutical

applications, involves exposing those materials to a

radioactive Cobalt 60 source under tightly controlled

conditions. The high doses of gamma rays from this source

causes the ionization of water and the formation of highly

50

reactive and short-lived hydroxy radicals, which inactivate

most microorganisms present by damaging or destroying

their nucleic acids. However, low doses of gamma radiation

can cause a state of “Hormesis” defined as the stimulation

of any system by low doses of any agent whether

environmental, biotic or abiotic stress factors as pathogens,

physical and chemical agents (Luckey, 1980). Radiation

hormesis is the stimulation, often considered to be

beneficial, from low doses of ionizing radiation where the

productivity of different metabolites e.g proteins,

glycoproteins, polysaccharides, pharmaceuticals, amino

acids and vitamins, by microorganisms is increased. That

depends, on the response of the organism to physical stress

factors depending on the cell type and stage of growth.

Most physiologic reactions in living cells are stimulated by

low doses of ionizing radiation. This evidence of

radiogenic metabolism (metabolism promoted by ionizing

radiation) includes enzyme induction, photosynthesis,

respiration and growth. Low level irradiation increased the

growth (replication) rate in protozoa (Luckey, 1991).

51

Laccase applications

Laccase exhibits a broad natural substrate range, which is a

major reason for the attractiveness of laccase to

biotechnological applications (Nyanhongo et al., 2002)

The broad substrate range of laccase that is derived from

their non-specific formation of a free radical from a

suitable substrate, and the use of air oxygen as a second

substrate, make laccases attractive candidates for industrial

application. However, (Gianfreda et al., 1999) noted that

the use of laccase in industrial operations is hampered by

several drawbacks as the high expenses and unavailability

of a suitable source (Gianfreda et al., 1999).

1. Pulp and paper industry

In the industrial preparation of paper, the separation and

degradation of lignin in wood pulp are conventionally

obtained using chlorine- or oxygen-based chemical

oxidants. Non-chlorine bleaching of pulp with laccase was

first patented in 1994 using an enzyme treatment to obtain a

brighter pulp with low lignin content (Luisa et al., 1996).

Oxygen delignification process has been industrially

introduced in the last years to replace conventional and

polluting chlorine-based methods. In spite of this new

method, the pretreatments of wood pulp with laccase can

provide milder and cleaner strategies of delignification that

52

also respect the integrity of cellulose (Barreca et al., 2003;

Gamelas et al., 2005).

2. Pharmaceutical sector

Due to their specificity and bio-based nature, potential

applications of laccase in the field are attracting active

research efforts.

Laccase can be used in the synthesis of complex medical

compounds as anesthetics, anti-inflammatory, antibiotics,

sedatives, etc., including triazolo(benzo)cycloalkyl

thiadiazines, vinblastine, mitomycin, penicillin X dimer,

cephalosporins, and dimerized vindoline (Xu, 1999).

One potential application is laccase-based in situ generation

of iodine, a reagent widely used as disinfectant (Xu, 1999).

Also, laccase has been reported to possess significant HIV-

1 reverse transcriptase inhibitor activity (Wang et al.,

2004).

Also a new enzymatic method based on laccase was

developed to distinguish simultaneously morphine and

codeine in drug samples injected into a flow detection

system (Bauer et al., 1999).

3. Cosmetics

Laccase is known to catalyze the oxidation, transformation

and cross-linking of various precursors (phenols and

53

anilines), which are used in hair dyeing processes (Xu,

2005).

In personal hygiene, laccase can oxidize thiol, sulfide,

ammonia and amine compounds that cause bad breath (Xu,

2005).

A novel application field for laccase is in cosmetics and

deodorants for personal hygiene products, including

toothpaste, mouthwash, detergent, soap, and diapers

( Roriz et al., 2009).

4. Food industry

Many laccase substrates, such as carbohydrates,

unsaturated fatty acids, phenols, and thiol-containing

proteins, are important components of various foods and

beverages. Their modification by laccase may lead to new

functionality, quality improvement, or cost reduction

(Minussi et al., 2002);

Sometimes, oxygen is detrimental to the quality or storage

of food/beverage because of unwanted oxidation. Laccase

may be used as oxygen scavengers for better food packing.

The flavor quality of vegetable oils can be improved with

laccase by eliminating dissolved oxygen (Farneth, 2005).

54

Various enzymatic treatments have been proposed for fruit

juice stabilization, among which it can be found the use of

laccase (Piacquadio et al., 1998).

Laccase is added to the dough used for producing baked

products, to exert an oxidizing effect on the dough

constituents and to improve the strength of gluten structures

in dough and/or baked products (Marbach-Ringhandt et

al., 2002).

5. Removal and degradation of other organic pollutants

Laccase can be used for removal/degradation of a number

of environmental pollutants besides dyes. The enzyme

alone has been able to catalyze the removal of phenolic

compounds (Dec & Bollag 1990; Ullah et al., 2000),

endocrine disrupting chemicals (Cabana et al., 2007) and

estrogens from wastewater samples (Auriol et al., 2008).

Nanotechnology

At the modern technological time, there is a strong demand

to develop new techniques to fabricate nanoparticles.

Significant advancement have been done over the last

decades in that direction. Nanosize particle (particles sized

less than 100 nm) (Ahmad et al. 2003), results in specific

physicochemical characteristics such as high surface area to

volume ratio, resulting in unique physical and chemical

properties compared to their bulk counterparts and these

55

properties are highly promising for a variety of

technological applications.

One of the most fascinating and useful aspects of nano

materials is their optical properties. Applications based on

such physical properties include optical detectors, laser,

sensor, imaging, display, solar cell, photocatalysis, and

biomedicine.

In the chemistry field, gold nano particles was used as soft

Lewis acid and also as a catalyst in the field of organic

synthesis.The ability of gold to produce heat after

absorbing light provides a medicinal usage named as photo

thermal therapy which can be added to their well-known

role in treatment of cancer.

Application of gold nano particles in gene therapy and drug

delivery systems increased studies on development of

methods for gold nano particles production (Shih et al.,

2009 and Kalishwaralal et al., 2009). As in targeted drug

delivery techniques adopting nanoparticles in the

pharmacotherapy of neurodegenerative disorders like

Alzheimers’ disease where this extremely successful

strategy aids therapeutic moieties to penetrate the CNS very

effectively (Sahni et al., 2011).

Beyond nanomedicine, the applications of nanoparticles

extend to water purification technology, where noble metal

56

particles in the nano scale range are employed successfully

for removal of heavy metal particles, microorganisms and

pesticides including halogenated organics (Pradeep et al.,

2009; Lee et al., 2009).

Metal oxide nanoparticles like those of molybdenum have

found use as renewable source of energy (Lee et al., 2009).

Some of the period four metal nanoparticles also have

antifouling potential (Chapman et al., 2010).

In cosmetic industry nano titanium and zinc oxides are

being used in its lightening and sun screen products

(Sadrieh et al., 2010).

Gold nanoparticles synthesis

Due to the unique applications of gold nanoparticles and

wide usages in different fields, the number of publications

on the preparation and characterization of gold nano

particles has extensively increased during recent decade.

Although preparation of by physical procedures (such as

laser ablation) provides narrow range of particle size, it

needs expensive equipment and has low yield (Alanazi et

al., 2010). Hazardous effects of organic solvents, reducing

agents and toxic reagents applied for synthesis of gold nano

particles on environment, encouraged researchers to

develop eco-friendly methods for preparation of gold

nanoparticles (Ahmad et al. 2003).

57

Green synthesis of gold nano particles

Preparation of nanometals using physical methods such as

attrition and pyrolysis supply nanoparticles with narrow

and controlled size ranges, however, these methods require

very expensive equipment and the final yield is low

(Safaepour et al.,2009)

Although chemical reduction using compounds like sodium

borohydride (NaBH4) and stannous chloride (SnCl2) is the

most common technique for synthesis of nanoparticles,

hazardous effects of byproducts and solvents persuaded

investigators to apply biological systems and their

biodegradable products, e.g. protein/ enzyme,

carbohydrates and chitosan, for nanoparticles preparation.

Green synthesis using microorganisms or enzymes

provides advancement over chemical and physical method

as it is cost effective, environment friendly, easily scaled up

for large scale synthesis and in this method there is no need

to use high pressure, energy, temperature and toxic

chemicals.

Green synthesis of gold nanoparticles (GNPs) either intra-

or extra-cellularly using bacteria (Du et al., 2007,

Husseiny et al., 2007 and Kalishwaralal et al. 2008),

fungi (Ahmad et al., 2003 and Castro-Longoria et al.,

58

2011) and microalgal strains (Shakibaie et al. 2010) as

well as herbal extracts (Dubey et al., 2009; Sathyavathi et

al., 2010) has been recently reported.

(Kalishwaralal et al., 2010 and Dubey et al., 2009)

demonstrated that the secreted proteins/ enzymes and

reducing agents such as amino acids, peptides and organic

acids in biological entities were found to be responsible for

nanoparticle production.

In the study of (Wang et al., 2007), the synthesis of GNPs

using the peptide sequence of MS14

(MHGKTQATSGTIQS) was determined. Also

(Kalishwaralal et al. 2010) revealed that the amylase

produced by Bacillus licheniformis is the reductive agent

responsible for biosynthesis of GNPs. (Rangnekar et al.,

2007) also showed that amylase is responsible for synthesis

of GNPs.

Due to the above mentioned important properties,

characteristics and applications of laccase enzyme.

The present study focuses on the optimization of

production of fungal laccase using factorial design to study

the effect of several variables upon the enzyme activity.

Partial purification and characterization of the obtained

ezyme was done. Some of the potential applications of the

enzyme were studied.

59

III. Materials and Methods

1. Materials:

1.1 Fungal strains

Seven locally isolated fungal strains namely (Gliocladuim

virens, Sclerotiam rolfsii, Penicilluim chrysogenum,

Pleurotus ostreatus, Gliocladuim deliquescence,

Rhizoctania solani and Penicilluim citrinum) were used in

the study from the culture collection in the Pharmaceutical

Microbiology laboratory Drug Radiation Research

Department (NCRRT, Egypt). All strains are

microscopically identified and kept on potato dextrose agar

(PDA) at 4°C and periodically subcultured to attain

viability. All strains were screened for production of

laccase enzyme.

1.2. Media for preservation of fungi

Potato dextrose agar (PDA): Potato extract (200 gm

potatoes boiled in one litre distilled water and filtered), agar

20 gm , glucose 20 gm were mixed and autoclaved for 20

minutes at 121 ̊ C with pH 4-5.5 at 25 ̊ C.

60

1.3. Substrates

Banana peelings (dried in oven at 55°C for 36 hours), spent

coffee ground (brought from local coffee factory), rice

straw and wheat bran flakes (both bought from local

market). Banana peelings and rice straw were both ground

to 30 mesh powders screen using electric grinder.

1.4. Chemicals

D-L methionine and syringaldazine (for laccase assay)

were bought from Sigma Aldrich, Germany. The five used

dyes (reactive ramazol brilliant blue (RBB-R), reactive

ramazol brilliant red (RBR-R), reactive ramazol brilliant

yellow (RBY-R), methyl orange and trypan blue) were

provided from Dye Star company, Germany and

tetrachloroauric acid from Alpha Chemica, India.

Ammonium sulphate, ammonium chloride, resorcinol,

tannic acid, Tween-80, malt extract, tryptone, urea and

yeast extract were all from ADWIC, Egypt.

61

2. Methods

2.1. Inoculum preparation

Fungi were cultivated on PDA slants at 30 ̊ C for 7 days.

The resulted spores were suspended in solution containing

0.01% Tween-80 by scratching with sterile loop. The

fermentation medium was inoculated with 2 ml of spore

suspension (~ 8-9×10 6 spores ⁄ ml).

2.2. Fermentation medium

Fermentation was done in 250 ml Erlenmeyer flasks, 8 ml

of distilled water were added to 5 gm carbon source (66%)

moisture content to which chosen concentrations of the

tested substances were added (according to the experiment

design) and autoclaved at 121°C for 20 minutes. The

fungus was added to the medium as a 2 ml spore

suspension (~ 8-9 ×106 spores/ml) and incubated at 29°C

statically in complete darkness.

2.3. Extraction of enzyme

After seven days, the whole contents of the flask were

soaked in 100 ml citrate phosphate buffer (pH 5) for 2

hours and put in a shaker at 200 rpm (LAB -Line R Orbit

Environ, U.S.A) and then extracted in tincture press,

centrifuged in cooling centrifuge (Hettich Universal 16 R,

62

Germany) for 10 min at 6° C and 2415 RCF to remove

particulate and then frozen for further use.

2.4. Enzyme assay

Laccase activity was performed spectrophotometrically

(JASCO V/560 UV/Vis, Japan) at λ 525 nm in a reaction

medium containing 0.2 ml of 1 mM syringaldazine (Ɛ=65

mM-1cm-1) and 0.6 ml of 50 mM phosphate buffer pH 5 and

1.2 ml of the culture filtrate. Oxidation of syringaldazine

was monitored by measuring the increase in absorbance for

4 minutes. Enzyme activity was expressed in units (U); 1U

was defined as 1 micro mole of syringaldazine oxidized per

minute (Leonowicz and Grzywnowicz, 1981).

2.5. Screening of carbon source

Four agricultural wastes were screened as carbon sources

for their ability to produce laccase. Banana peelings, spent

coffee ground, rice straw and wheat bran flakes were used.

2.6. Screening of nitrogen source

Six nitrogen sources of natural and synthetic origin were

screened. They are yeast extract, tryptone, malt extract,

urea, ammonium chloride and ammonium sulphate.

63

2.7. Experimental factorial design

The statistical software package Minitab 16, by (Minitab

Inc. U.S.A) was used for designing the experiment; the

chosen concentrations of the six variables in 2 level forms

are shown in Table (3). Sixty four experiments were done

to fully investigate the effect of these variables on the

production of laccase enzyme and the results were

recorded. The statistical software chooses 32 experiments

to completely analyze them.

Table 3: The six variables and their corresponding high and low

concentrations

Levels

Variables

Malt extract Tween-

80 CuSO₄ Resorcinol Methionine

Tannic

acid

High

Level

(+1)

1 gm (2%N

content)

0.02 %

(v/v)

1.25

mM

20 mg 10 mg 5 mg

Low

Level

(-1)

0.5 gm(1%N

content)

0.01 %

(v/v)

0.625

mM

10 mg 5 mg 2.5 mg

*N content = Nitrogen content

The main effects of parameters on laccase production were

estimated by subtracting the mean responses of variables at

their lower levels from their corresponding higher levels

and divided by the total number of experimental runs.

64

Regression analysis of the experimental data and plotting

the relation between variables are done.

The adequacy of the model was tested and the parameters

with statistically significant effects were identified using

Fisher’s test for the analysis of variance (ANOVA). The

possible interactions between them were investigated.

2.8. Gamma irradiation of fungus

The process of irradiation was carried out using Co-60

Gamma Chamber (4000-A-India) at a dose rate 10.28

kGy/hr at the time of experiment. Seven days old slant

about (~8-9 ×106 spores/ml) was irradiated at different

doses (0.1, 0.25, 0.5, 0.75, 1, 1.5 and 2 kGy) then cultivated

at optimized conditions for laccase production. Non-

irradiated culture was used as control.

2.9. Laccase enzyme partial purification and

characterization

2.9.1. Ammonium sulphate precipitation

Ammonium sulphate was added to 1500 ml of the cell free

filtrate obtained to attain 80% saturation. The flask was

kept at 4°C for 48 hours. Content was centrifuged at 5,000

rpm for 15 min at 4°C and the supernatant was discarded.

65

The pellet was dissolved in 50 ml citrate phosphate buffer

pH 5.0 to be dialyzed.

2.9.2. Desalting by dialysis

The precipitate was desalted by dialysis bag (Dialysis

membrane-150, Himedia laboratories Ltd; Mumbai, India)

according to (Karthik et al., (2011) to remove low

molecular weight substances and other ions that interfere

with the enzyme activity.

2.9.3. Protein measurement

Protein concentration was quantified using the Bradford

assay (Bradford, 1976) with Bradford reagent and Bovine

serum albumin as standard at wavelength 595 nm.

2.9.4 Effect of pH/ temperature on laccase activity

The effects of pH on the activity of partially purified

enzyme were studied by incubating it with the following

buffers for 7 minutes: citrate phosphate buffer for pH 3, pH

4 and pH 5 and sodium phosphate buffer for pH 6, pH 7

and pH 8. The effect of temperature on the partially

purified enzyme activity was determined by incubating it in

water bath in the range from 30°C to 90°C with 10°C

increments for 15 minutes. Results were expressed as

percentage of the control (non-treated).

66

2.9.5. Effect of activators and inhibitors on laccase

activity

The effects of several activators and inhibitors such as

Cu+2, Zn+2 and Mg+2 used as sulphate salts and Ca+2,

Cd+2, Co+2 and Ba+2 used as chloride salts and EDTA with

the concentration of 1 mM on laccase activity were

monitored under standard assaying conditions. The reaction

assay mixture of laccase was incubated with activators or

inhibitors, optimized buffer and syringaldazine and at

respective optimum temperature; the change in absorbance

was measured spectrophotometrically to evaluate the

influence of these activators and inhibitors on enzyme

activity. Results were expressed as percentage of the

control (non-treated laccase).

2.9.6. Effect of gamma radiation on laccase activity

The effects of five doses of gamma radiation (2, 3, 4, 5 and

6 kGy) on the activity of laccase were studied. Results were

expressed as percentage of the control (non-irradiated).

2.10. Decolorization of dyes

Five dyes [methyl orange, trypan blue, (RBR-R), (RB-B)

and (RBY-R)] were chosen to test the enzyme’s ability to

remove their color, a volume of 0.1 ml of the stock solution

67

( 20 ppm ) was added to 2 ml distilled water and 2 ml of

the partially purified enzyme extract with activity 417 U/ml

respectively, the percentage reduction of color was

monitored for 3 hours and was determined

spectrophotometrically ( JASCO V/560 UV/Vis, Japan) by

monitoring the absorbance at the characteristic wavelength

of each dye. The percentage of decolorization (R %) was

calculated by the following equation :

R % = [(Initial absorbance - final absorbance) / (initial

absorbance)] x 100

Where the initial absorbance indicated absorbance of the

untreated dye at the characteristic peak and the final

absorbance indicated absorbance of dye after treatment

with laccase at the same peak after 3 hours.

2.11. Preparation & characterization of GNPs:

GNPs were prepared according to the method described by

(Song and Kim, 2009 and Noruzi et al., 2011). Briefly, to

3 ml laccase enzyme 0.1 ml tetrachloroauric acid with

concentration of (10 mg /ml and purity 49% gold metal)

was added. The reaction mixture was stirred properly with

magnetic stirrer; at room temperature violet color was

detected visually indicating formation of GNPs and

68

measured spectrophotometrically at 550 nm due to surface

plasmon resonance of GNPs (Yonezawa et al., 2006).

The effects of temperature (40 to 100 ̊ C with 10 ̊ C

increments), radiation (2, 2.5, 3, 3.5, 4, 5 and 6 kGy) and

different volumes of tetrachloroauric acid (0.1, 0.2, 0.3, 0.4

and 0.5 ml) were assessed to study their effect on the

formation of GNPs.

2.11.1. UV/Vis spectroscopy:

UV/Visible Spectra of GNPs were recorded using (JASCO

V-560UV/Vis, Japan) spectrophotometer operated at a

resolution of 1nm from range of 200 to 700 nm.

2.11.2. Dynamic Light Scattering (DLS):

Average particle size and size distribution were determined

by (PSS-NICOMP 380-ZLS) particle sizing system St.

Barbara, California, USA.

2.11.3. Fourier Transform Infra-Red Spectroscopy (FT-

IR):

FT-IR measurements were carried out to obtain information

about chemical groups present around GNPs for their

stabilization and to understand the transformation of

functional groups due to reduction process. Measurements

were carried out using (JASCO FT/IR-6300 infra–red

69

spectrophotometer, Japan) by employing potassium

bromide (KBr) pellet technique.

2.11.4. Transmission Electron Microscopy (TEM):

The size and morphology of synthesized GNPs were

recorded using TEM electron microscope (JEM-

100CX.TEM, JEOL, Japan) studies were carried by drop

coating of GNPs onto carbon–coated TEM grids. The film

on the TEM grids was allowed to dry and the extra solution

was removed using blotting paper.

70

IV. Results

1. Screening for laccase producing fungi

After screening of seven fungal strains, Pleurotus ostreatus

showed relatively high laccase activity (4610 U/gfs) i.e.

number of units of enzyme produced from 5 gram

fermented substrate compared to other laccase producing

fungi as shown in Table( 4).

Table 4: Screening of laccase producing fungi results

Fungal strain Laccase activity

(U /gfs)

Pleurotus ostreatus 4610

Gliocladuim virens 1647

Sclerotiam rolfsii 1200

Penicilluim chrysogenum 720

Penicilluim citrinum 267

Gliocladuim deliquescence 200

Rhizoctania solani 20

71

2. Screening of carbon source

The high cost of the enzyme is a major limitation in the

using of laccase on industrial scale; using agricultural

wastes not only decrease the cost but also solve an

environmental problem. The screening of the four

agricultural wastes showed that wheat bran which is an

abundant byproduct formed during wheat flour preparation

was the highest in laccase activity as shown in Table (5).

Table 5: Screening of carbon source results

Carbon source Laccase activity

(U /gfs)

Wheat bran 4610

Banana peelings 1280

Rice straw 569

Spent coffee ground 135

Wheat bran had been selected as carbon source to perform

all further experiments throughout the study.

3. Screening of nitrogen source

The screening of the six nitrogen sources showed that malt

extract gave higher laccase activity (8460 U/gfs) compared

to other nitrogen sources as shown in Table (6).

72

Table 6: Screening of nitrogen source results

Nitrogen source Laccase activity

(U /gfs)

Malt extract 8460

Yeast extract 7205

Tryptone 6500

Amm. sulphate 5420

Urea 1789

Amm.chloride 819

Malt extract was selected as a nitrogen source for the

production of laccase.

4. Experimental factorial design results

The effects of the six variables and the possible interactions

between them were investigated using Minitab version 16.

The different combinations of variables mentioned in Table

(3) for all experiments and the corresponding enzyme

activity are shown in (Table 7).

Maximum enzyme activity (32450 U/gfs) was obtained in

the 28th run as shown in Table (7) at malt extract (1gm),

Tween-80 (0.02% (v/v), CuSO₄ (1.25 mM), Resorcinol (20

mg), DL-Methionine (10 mg) and Tannic acid (2.5 mg),

73

where all variables were in their higher level except for

tannic acid which was in the lower level

Table 7: The factorial experiments done and the results obtained

Run N2

T-

80 Cu+2 M R T

Enzyme Activity

(U/gfs)

1 1 1 1 -1 -1 -1 28044

2 -1 -1 -1 1 -1 -1 2435.8

3 1 -1 1 1 1 -1 22834

4 1 -1 -1 -1 -1 -1 13858

5 -1 1 -1 -1 1 1 1217.5

6 1 1 1 -1 -1 1 14679

7 -1 1 1 1 -1 -1 1507

8 -1 1 -1 -1 -1 -1 410

9 1 1 -1 1 -1 1 15789

10 -1 -1 -1 1 1 -1 2447

11 1 -1 1 1 -1 -1 15846

12 1 1 1 -1 1 -1 14474

13 1 -1 -1 -1 1 1 8843.8

14 -1 1 1 1 1 1 1427

15 1 -1 -1 -1 1 -1 14564.8

16 1 -1 1 1 -1 1 12225

17 1 1 -1 1 -1 -1 18294

18 -1 -1 -1 1 1 1 904.5

19 1 1 1 -1 1 1 12788

20 -1 -1 1 -1 -1 -1 840

21 1 -1 1 -1 1 -1 14420

74

22 -1 -1 1 1 1 1 2610

23 -1 1 1 -1 -1 1 1564

24 -1 1 -1 1 1 -1 5076.7

25 1 1 1 1 -1 1 12820

26 1 -1 1 -1 1 1 10015

27 -1 1 -1 1 1 1 1057.7

28 1 1 1 1 1 -1 32450

29 -1 -1 -1 -1 1 -1 1314.5

30 -1 1 1 -1 1 1 923

31 1 1 1 1 1 1 30191

32 1 -1 -1 1 1 -1 10230

*1=High level -1=Low level

** N2=Nitrogen source T -80=Tween -80

Cu+2= Copper sulphate M=Malt extract R=Resorcinol

T=Tannic acid

The main effects of the six variables on laccase production

were estimated by subtracting the mean responses of

parameters at their lower levels from their corresponding

higher levels and dividing by the total number of

experimental runs as shown in Figure (9). Main effects

plotting showed the positive effect of nitrogen source,

Tween-80, CuSO4 and methionine. In case of nitrogen

source a major difference between means was noticed.

75

However, tannic acid had negative effect in its higher

concentration as shown in Figure (9).

1-1

16000

12000

8000

4000

0

1-1 1-1

1-1

16000

12000

8000

4000

0

1-1 1-1

Nitrogen Source

Me

an

Tween 80 CuSO4

Methionine Resorcinol Tannic Acid

Main Effects Plot for Enz.ConData Means

Fig 9: Main effects of variables

The adequacy of the model was tested and the parameters

with statistically significant effects were identified using

Fisher’s test for the analysis of variance (ANOVA). The

analysis of variance for the selected factorial model showed

that the model was significant with a model F-value of

17.75 Table (8). The model determination coefficient

(R=0.81) suggested that the fitted model could explain 81%

of the total variation. This implies a satisfactory

representation of the process by the model. The coeffecient

76

of determination R value always lies between 0 and 1. The

closer the value of R is to 1, the stronger the model and

the better it predicts the response.

Table 8: Analysis of Variance (ANOVA) results for experiments

Source F** P***

Regression 17.75 0.000

R2 = 81.0% R2 (adjusted) = 76.4%

*DF= Degree of freedom **F=F- value ***P=Probability

Both the T-value and P-value statistical parameters were

used to confirm the significance of the effect of the six

variables factors studied as shown in Table (9), the

significance was tested using one way (ANOVA).

Table 9: Test of significance of variables

Term T** P***

Constant 10.23 0.000*

Nitrogen

Source 8.94 0.000*

Tween -80 2.61 0.015*

CuSO₄ 1.63 0.115

Methionine 1.99 0.057

Resorcinol 1.26 0.218

Tannic Acid -2.59 0.016*

*Significance of variable where P = < 0.05

77

** T= T-value **P=Probability

Results shown in (Table 10) expressed significance of

nitrogen source ,Tween -80 and tannic acid.

The T-value measures how large the coefficient is in

relationship to its standard error. The P-value is the chance

of getting a larger T-value (in absolute value) by chance

alone. The larger the magnitude of the-t-value and smaller

the P-value, the more significant is the corresponding

coefficient (Myers&Montgomery, 2002)

The mathematical expression for relationship between the

six variables and for the laccase production is given in the

equation shown:

Enzyme activity predicted = 8511 + 7460 Nitrogen

Source + 2207 Tween-80 + 1397 CuSO₄ + 1590

Methionine + 1054 Resorcinol -2197 Tannic Acid

The predicted enzyme would be determined using this

equation

Figure (10) shows the actual enzyme activity and the

predicted activity with an equation describing the line.

78

35000300002500020000150001000050000

30000

25000

20000

15000

10000

5000

0

Enz.activity

pre

dic

ted

S 3680.77

R-Sq 81.0%

R-Sq(adj) 80.4%

Fitted Line Plotpredicted = 1937 + 0.8099 Enz.activity

Fig 10: Actual enzyme activities versus predicted

5. Effect of gamma irradiation on growth of Pleurotus

ostreatus:

The effect of gamma radiation on growth of Pleurotus

ostreatus was done. The results revealed that as the dose

increased, the growth of Pleurotus ostreatus decreased

gradually as seen in Figure (11).

79

Fig 11: Pleurotus ostreatus growth after different doses of gamma

irradiation

6. Effect of Gamma irradiation of Pleurotus ostreatus

on laccase production:

The produced laccase was highly affected by irradiating the

fungus as it decreased to almost half 17,200(U/gfs) Figure

(12) compared to the enzyme obtained from non-irradiated

fungi (32,450 U/gfs). This decrease was directly

proportional to the increase in the dose until complete loss

in enzyme activity at 1.5 and 2 kGy.

0.1kGy 0.25kGy 0.5kGy 0.75kGy

1kGy

1.5kGy

80

02000400060008000

100001200014000160001800020000

0.1 0.2 0.5 0.7 1 1.5 2 .0

En

zym

e a

ctiv

ity

(U

/gfs

)

Gamma radiation dose (kGy)

Fig 12: Laccase activity after gamma irradiation (kGy) of Pleurotus

ostreatus

7. Laccase enzyme partial purification

After precipitation of the enzyme using ammonium

sulphate total activity decreased from 675000 to 622000

U/1500 ml of enzyme but the specific activity increased

from 112.5 to 204 U/mg Table (10).

81

Table 10: Partial purification of laccase produced from Pleurotus

ostreatus

Purification

step

Total

activity

(U) /1500

ml

Total

protein

(mg)

Specific

activity

(U/mg)

Purification

Yield

(%)

Crude

extract 675000 6000 112.5 1 100

(NH4)SO4 622000 305 204 1.8 55

8. Effect of pH/ temperature on laccase activity

Figure (13) shows that laccase from Pleurotus ostreatus

exhibited a bell shaped pattern with optimum activity at pH

5 and keeping 90% of activity at pH6 with abrupt decrease

before and beyond them.

82

0%

20%

40%

60%

80%

100%

120%

3 4 5 6 7 8

lacca

se a

cti

vit

y

(Rela

tiv

e %

)

pH

Fig 13: Effect of pH on laccase activity

Study of the thermal stability of purified laccase is shown

in Figure (14).

At temperature 40 °C laccase exhibited the highest activity,

above 60 °C laccase activity decreased sharply and at 80 °C

only 10 % of initial activity remained after 15 min

incubation.

83

0%

20%

40%

60%

80%

100%

120%

30 40 50 60 70 80

La

cca

se a

ctiv

ity

(Rel

ati

ve%

)

Temperature (⁰C)

Fig 14: Effect of Temperature (̊C) on laccase activity

9. Effect of activators and inhibitors

Several activators/inhibitors such as Cu+2, Zn+2, Mg+2, Ca+2,

Cd+2, Co+2 and Ba+2 with a concentration of up to 1 mM

were evaluated for the effect on enzyme activity. Activators

included Cu+2, Zn+2, Mg+2 and Ca+2

had an enhancing effect

on activity of laccase with different extents Figure (15).

Whereas inhibitors as Cd+2, Co+2 and Ba+2 caused inhibition

of activity as shown in Figure (16). However, EDTA did

not inhibit its activity at the concentration used.

84

0%

2%

4%

6%

8%

10%

12%

14%

16%

Cu+2 Zn+2 Mg+2 Ca+2

% L

acca

se e

nzy

me

acti

va

tio

n

Activator ions

Fig 15: Showing the effect of different activators

0%

10%

20%

30%

40%

50%

60%

70%

80%

Cd+2 Co+2 Ba+2 EDTA% L

acca

se e

nzy

me

inh

ibit

ion

Inhibitors

Fig 16: Showing the effect of different inhibitors

85

10. Effect of gamma radiation on on laccase activity

Gamma irradiation of the enzyme decreased its activity to

half, up to 5 and 6 kGy where the activity remained

constant Figure (17).

0%

10%

20%

30%

40%

50%

60%

2 3 4 5 6

La

cca

se a

cti

vit

y

(Rela

tive %

)

Gamma radiation dose (kGy)

Fig 17: The effect of gamma radiation dose (kGy) on laccase activity

11. Decolorization of dyes

Visual decolorization in one of the treated dyes (trypan

blue) Figure (18) and the decrease in absorbance in the

characteristic wavelength of every dye (Table 11) were

monitored for 3 hours.

86

Fig 18: Visual decolorization of trypan blue

Figure (19) shows the characteristic peak of trypan blue

before and after laccase treatment.

87

Fig 19: Characteristic peak of trypan blue at 607 nm before and after

treatment by laccase

88

Not all dyes were decolorized to the same extent; the

decolorization percentage was shown in Table (12) .The

highest decolorization percentage was obtained in case of

methyl orange and trypan blue.

Table 11: The used dyes characteristic wavelength

and % of reduction in colour

Dye Wavelength

(nm)

Reduction

%

Methyl Orange 480 86

Trypan Blue 607 83

Ramazol Red 525 66

Ramazol Blue 595 53

Ramazol Yellow 465 0.4

89

12. Preparation & characterization of GNPs:

Violet color was formed after 90 minutes at room

temperature indicating formation of GNPs shown in Figure

(20).

Fig 20: Violet color indicating formation of GNPs

12.1. UV/Vis spectroscopy:

GNPs gave a characteristic peak at 550 nm indicating

formation of GNPs due to surface plasmon resonance of

GNPs Figure (21).

.

Fig 21: Characteristic peak of GNPs at 550 nm

90

12.1.1. Effect of temperature on synthesis of GNPs

Incubation of laccase in the presence of chloroaurate ions at

different temperatures gave different absorbance at

different time at the characteristic wavelength of GNPs

(550 nm). Absorbance increased with temperature

increase which indicated higher concentration of GNPs.It

was noticed that as temperature increased the time of

formation of GNPs significantly decreased Table (12).

Table 12: Effect of temperature on GNPs synthesis

Temperature(°C) Abs at

550nm

Time

(min)

30(room temp.) 2 90

40 2.3 77

50 2.5 75

60 2.6 70

70 1.8 60

80 2.8 40

90 3 20

100 3.2 10

12.1.2. Effect of Gamma Radiation on GNPs synthesis

Different doses of gamma radiation were tested for

assessing their effect on GNPs formation. Increasing the

91

dose of radiation increased the production of GNPs; with

absorbance increasing to 4 at 5 kGy after that decrease in

the absorbance was found as shown in Table (13).

Table 13: Effect of gamma radiation on GNPs synthesis

Dose (kGy) Abs

at 550nm

2 1.1

2.5 2.2

3 2.7

3.5 2.9

4 3

5 4

6 3.1

12.1.3. Effect of different HAuCl4 volumes on GNPs

synthesis

Testing the effect of increasing the volume of HAuCl4

showed that the best volume of HAuCl4 was 0.3 ml as it

gave the highest production of GNPs; further increase

caused decrease in GNPs production as shown in Table

(14).

92

Table 14: Effect of different HAuCl4 volumes on GNPs synthesis

12.2. Dynamic Light Scattering (DLS):

DLS representing size distribution of GNPs with 100 % of

particles in sample with mean diameter 22.5 as shown in

Figure (21).

Fig 21: Showing DLS images of GNPs formed by laccase from Pleurotus

ostreatus.

HAuCl4

(ml) Abs at 550nm

0.1 4

0.2 4

0.3 4.6

0.4 3.6

0.5 3.32

0 20 40 60 80 100

100

80

60

40

20

0

Mean Diameter = 22.5 nm Standard Deviation (5.4%)

93

12.3 Fourier Transform Infra-Red Spectroscopy (FT-

IR):

The FTIR spectrum of GNPs in Figure (22) showed the

change in the corresponding peaks of functional groups

before and after formation of GNPs, expressing change in

major peak at 3016 cm−1 peak corresponding to OH and/or

NH functional groups and the peak of 1631 cm−1 indicated

presence of carbonyl group, both could be ascribed to

secondary amide structure.

94

Fig 22: FTIR spectrum of laccase by Pleurotus ostreatus before GNPs

synthesis

Fig 23: FTIR spectrum of laccase by Pleurotus ostreatus after GNPs

synthesis

95

12.4. Transmission Electron Microscopy (TEM):

TEM image of gold nanoparticles are illustrated in Figure

(24), showing highly mono dispersed GNPs with size range

of 22-39 nm.

Fig 24: TEM images of synthesized GNPs by laccase from Pleurotus

ostreatus

96

V. Discussion

Laccase (E.C. 1.10.3.2) (p-diphenol: dioxygen

oxidoreductases; benzenediol dioxygen oxidoreductases) is

an important member of the lignolytic enzymes family that

is an oxidative enzyme, which uses molecular oxygen as

the electron acceptor, to oxidize the phenolic units of lignin

(Claus 2004; Riva 2006). Most of the laccases studied are

of fungal origin especially from the classes of white-rot

fungi. Fungal laccases play an important role in plant

pathogenesis, pigment production, and degradation of

lignocellulosic materials (Gianfreda et al., 1999 and

Thurston, 1994).

The most efficient lignolytic fungi are basidiomycetes.

They could be either white or brown-rot fungi, both of

which are taxonomically so close to each other that they

sometimes appear in the same genus (Hatakka, 2001).

Almost all species of white rot fungi were reported to

produce laccase to varying degrees (Hatakka, 2001). After

screening of the seven fungal strains, Pleurotus ostreatus (a

well-known white rot fungus) was chosen due to its

relatively high laccase activity compared to other laccase

producing fungi as shown in Table (4). Pleurotus ostreatus

is a common edible mushroom also known as Oyster

mushroom was first cultivated in Germany as a subsistence

97

measure during the World War I (Eger et al., 1976). It is

now grown commercially around the world for food.

Oyster mushrooms can also be used industrially for

mycoremediation; which is a form of bioremediation where

fungi are used to degrade or sequester contaminants in the

environment, reducing toxins in-situ (Thomas, 2000). The

Oyster mushroom also may be considered a medicinal

mushroom since it contains statins such as lovastatin which

is used to reduce cholesterol (Gunde-Cimerman and

Cimerman, 1995).

Increasing the production of lignolytic enzymes may be

achieved by modifying the source of carbon and nitrogen in

the medium. Since the high cost of the enzyme is a major

limitation in the using of laccase on industrial scale; using

agricultural wastes not only decreases the cost but also

solves an environmental problem (Gomez et al, 2005).

Wheat bran is an abundant byproduct formed during wheat

flour preparation, has been selected to perform the present

study for its high yield of laccase as shown in Table (5)

Wheat bran’s physical integrity serves as a supporting

material and it provides the fungus with an environment

similar to its natural habitat therefore encouraging growth

of the fungus, also it is an abundant source for

hydroxycinnamic acids, particularly ferulic and p-coumaric

98

acids, which are known to stimulate laccase production

(Neifar et al, 2009) and it was successfully used in laccase

production by Trametes versicolor UAMH 8272 providing

a several fold increase in laccase activity (Osma et al,

2006).

For the nitrogen source to be used, malt extract was

selected as it gave significantly high yield of laccase

compared to other synthetic nitrogen sources Table (6).

Malt extract served as nitrogen and also as carbon source,

in Cyathus bulleri where it resulted in a much higher yield

of laccase than in mineral medium (Vasev and Kuhad,

1994).

For laccase application in industrial processes, large

amounts of enzyme are required. The major aim of the

study was to find optimized conditions for maximum

laccase production, to reach that aim using the conventional

one factor at a time technique would be quite laborious and

time consuming. Statistical experimental designs are one of

the recently applied techniques in optimization experiments

acting as powerful tools for searching the key factors

rapidly from a multivariable system and to define the

optimum settings of these factor levels (Phan-tan-luu and

Cela, 2009 and Baati et al, 2006). One of the currently

available statistical designs to predict the behavior of a

99

reaction is the factorial design. Such design of experiments

completely explains the reaction and brings out the finer

details by carrying out selected experiments.

The variables chosen to assess their effect on laccase

production were either nutrients or surface active agents or

possible inducers for enzyme production or activity. Their

choice depended on previous studies done; based on the

nature of the enzyme and its chemical structure Table (3).

Nitrogen source had always been an important nutrient for

the growth of fungi and the production of enzymes.

However; several fungi require the concentration of

nitrogen to be in excess to produce laccase, while other

fungi produce laccase only when induced by nitrogen

starvation. Lentinula edodes (Buswell et al., 1995) and

Phanerochaete chrysosporium (Dittmer et al., 1997)

provide examples of improved laccase production in

nitrogen sufficient media. A nitrogen deficient medium was

however required for high production of laccase in

Pycnoporus sanguineus (cinnabarinus) (Eggert et al.,

1996), the results of this study supported the first finding

showing that laccase production was in excess with the

higher concentration of malt extract (2% Nitrogen content)

and actually was a significant variable (p=0.000 ) Table

(9) which is probably due to the fact that, fungi require

100

nitrogen for their growth and general metabolic processes

and so providing nitrogen in excess subsequently increases

enzyme production .

For the surfactant Tween-80, it was a significant variable

(p=0.015) in Table (9), as high concentration of the

enzyme was usually accompanied by high concentration of

Tween-80. The addition of the surfactant Tween-80 has

resulted in higher yields of ligninolytic enzymes in certain

fungi because there is evidence that these surface acting

agents result in higher permeability of oxygen and

extracellular enzyme transport through the cell membranes

of fungi (Leštan et al., 1994 and Rothschild et al., 1995).

Most common inducers used for laccase production mainly

included phenolic compounds, which are structurally

related to lignin or lignin derivatives which is the natural

substrate for laccase and that was the main reason for

choosing both resorcinol and tannic acid, the effect of

resorcinol was not actually significant but the optimized

laccase production was observed at high concentration of

resorcinol. Although, attempts were made to increase

laccase production by the addition of the reported laccase

inducer tannic acid to enhance the expression of laccase

gene at the transcription level in the growth medium

(Bourbonnais et al, 1997), the optimized production

101

condition required low concentration of tannic acid with

significance of (p=0.016) Table (8), which might be due to

the reaction between the produced laccase and tannic acid,

consequently, making laccase in an undetectable state by

syringaldazine since tannic acid is one of the traditional

screening reagents for laccase (Harkin and Obst, 1973).

The effect of copper on laccase synthesis was studied in

Trametes versicolor (Collins and Dobson, 1997) and

Pleurotus ostreatus (Palmieri et al., 2000) among several

other white rot fungi. As laccase is a multi-copper oxidase

containing copper in its structure, the availability of copper

in the medium might allow the synthesis of the enzyme in

addition, the presence of copper in Pleurotus ostreatus

cultures decreases the activity of extracellular proteases

which might degrade laccase (Palmieri et al., 2000).

However, copper present in high concentration was

extremely toxic to microbial cells (Labbé and Thiele

1997). In the present study, copper was not a significant

variable indicting that copper was not a critical variable in

both concentrations which was quite unexpected.

The use of amino acid methionine was to provide a

principle amino acid that is required for protein production

and so inducing general enzyme production, but it did not

give the induction required.

102

Gamma radiation was used in many cases to induce general

metabolic processes and consequently increases enzymes

production due to the well-known phenomena of Hormesis;

where stimulation of any system by low doses of

environmental, biotic and abiotic stress factors including

pathogens, physical and chemical agents) (Luckey, 1998).

However; the reduction of growth and decrease of enzymes

production by gamma radiation had also been recorded by

other studies. The results obtained showed that at the doses

used, as the radiation dose increased Pleurotus ostreatus

growth decreased, which was in agreement with the

findings of (Abo-State et al, 2011) in case of her strain

Pleurotus sajor-caju.

That decrease in growth accompanying the increase in dose

(up to 1.5 kGy) and subsequent decrease in laccase

production shown in Figure (12), might be due to reduction

in the viable count of fungi as a result of the over

accumulation of free radicals that usually accompanies the

gamma irradiation process, when these rays interact with

water molecules in an organism, they generate transient

free radicals that can cause additional indirect damage to

DNA and so causes injury in the microbial cells resulting

in incomplete inhibition (Aubrey, 2002). Complete

103

inhibition of fungal growth and subsequent loss of enzyme

activity were detected with 2 kGy, which might be due to

break down of DNA structure of cells by that dose of

gamma irradiation resulting in complete death (Smith and

Pillai, 2004).

For a variety of industrial applications characterization of

produced enzyme would be a crucial step in order to know

the conditions (pH, temperature, gamma radiation and

activating and inhibiting ions) that the enzyme could

function.

Optimum pH for laccase exhibited variation may be due to

changes in the reaction caused by the substrate

(syringaldazine), oxygen or the enzyme itself. The highest

activity of the produced laccase was at pH 5 with

syringaldazine as a substrate in agreement with (Manole et

al., 2008).

Relatively high thermostability is an attractive and

desirable characteristic of an enzyme. In general, the

optimum temperature for laccase activities can differ from

one strain to another, with a range for most fungal laccases

being 50 - 70°C (Luisa et al., 1996) but laccase had

optimum temperature at 30-50°C and rapidly lose activity

at temperatures above 60°C (Galhaup et al., 2002;

Palonen et al., 2003), which might be due to breaking

104

down the integrity of laccase protein structure and so losing

much of its activity.

In general, laccase responds similarly to several inhibitors

of enzyme activity. Many ions such as azide, halides and

fluoride and can bind to the type 2 and type 3 copper

atoms, resulting in the interruption of internal electron

transfer with the subsequent inhibition of activity

(Kunamneni et al., 2007). EDTA did not inhibit laccase

activity at the concentration used as was observed with the

laccase obtained from an unidentified basidiomycete

(Jordaan et al., 2004).

Some of the most toxic dyes are amino-substituted azo

dyes, which are often mutagenic and carcinogenic (Selvam

et al., 2003). Current methods for dye-decolorization are

chemically derived and include adsorption, chemical

transformation, and incineration (Zilly et al., 2002). It has

been suggested that enhanced microbial decolorization of

dyes may provide a less expensive and more

environmentally acceptable alternative to chemical

treatment. An advantage of using fungal oxidative

mechanisms to degrade azo dyes over other

microorganisms is that it is possible to avoid the formation

of hazardous breakdown products such as anilines formed

by the reductive cleavage of azo dyes (Martins et al.,

105

2003). The laccase oxidative transformation of dyes

depends on their chemical structure. The presence of ortho-

hydroxy groups with respect to the azo link was found to

enhance the decolorization rates of azo-dyes with laccase

whereas nitro groups stabilized the dye molecules against

laccase action (Kandelbauer et al., 2004).

Preparation of nanometals using physical methods such as

attrition and pyrolysis supply nanoparticles with narrow

and controlled size ranges, however, these methods require

very expensive equipment and the final yield is low

(Safaepour et al., 2009)

Although chemical reduction using compounds like sodium

borohydride (NaBH4) and stannous chloride (SnCl2) is the

most common technique for synthesis of nanoparticles,

hazardous effects of byproducts and solvents persuaded

investigators to apply biological systems and their

biodegradable products, e.g. protein, enzyme,

carbohydrates and chitosan, for nanoparticles preparation.

Green synthesis using microorganisms or enzymes

provides advancement over chemical and physical method

as it is cost effective, environment friendly, easily scaled up

for large scale synthesis and in this method there is no need

to use high pressure, energy, temperature and toxic

chemicals.

106

Green synthesis of gold nanoparticles (GNPs) either intra-

or extra-cellularly using bacteria (Du et al., 2007,

Husseiny et al., 2007 and Kalishwaralal et al., 2008),

fungi (Ahmad et al., 2003 and Castro-Longoria et

al.,2011) and microalgal strains (Shakibaie et al., 2010) as

well as herbal extracts (Dubey et al., 2009; Sathyavathi et

al., 2010) has been recently reported.

A lot of researchers have focused on the preparation of

GNPs by peptide sequences and proteins. (Farmarzi and

Frootenfar, 2011). Kalishwaralal et al., 2010 and Dubey

et al., 2009 demonstrated that the secreted proteins/

enzymes and reducing agents such as amino acids, peptides

and organic acids in biological entities were found to be

responsible for nanoparticle production.

In this study, laccase from Pleurotus ostreatus served as a

rich source of the proteins and free amino groups reducing

gold into GNPs. These compounds contains functional

groups that play a role in the reduction and hence the

synthesis as well as the stabilization of nanoparticles (Gole

et al., 2001, Tortora et al., 2004). These proteins create

electrostatic attraction of negatively charged carboxylic

groups (Rastogi and Arunachalam 2011), therefore

107

stabilizing these nanoparticles by "capping" preventing

their aggregation through the creation of repulsive forces.

Laccase was performing the reduction process as a peptide

and not as an active enzyme as laccase in its active form

was actually catalyzing oxidation and upon exposure to

increasing temperature or gamma radiation. Laccase lost its

activity as breaking down the integrity of its protein

structure and exposing of various amino acids began.

FTIR measurements Figure (23 and 24) are carried out to

identify the possible interactions between gold ions and

enzyme protein which acted as reducing agent to synthesize

and stabilize gold nanoparticles. Enzyme protein contains

three main functional groups, including the amino,

carboxylic, and thiol group, which are easily used as active

sites to modify the other molecules or nanomaterials. FTIR

spectrum confirmed the presence of the functional groups,

3016 cm−1 peak corresponded to OH and/or NH functional

groups and presence of carbonyl group could be ascribed to

the peak of 1631 cm−1 (Noruzi et al. 2011).

Our finding was in agreement with the study of (Shakibaie

et al., 2010) characterized the GNPs produced by marine

microalgal strain of Tetraselmis suesica. According to

(Shakibaie et al., 2010) these functional groups could be

108

used in bioconjugation and/or immobilization of various

compounds.

The broad band contour which appears in the range of

3000–3400 cm−1 is the summation of associated

intermolecular hydrogen bonds arising from -NH2 and -OH

groups in protein molecules, which becomes much broader

and more intense after the reaction with gold ions,

indicating that the N-H vibration is affected due to the gold

attachment and revealing that nitrogen atoms are the

binding sites for gold on protein (Sanghi and Verma,

2010). The peaks at 1637 cm-1 and 1151 cm−1 arise from a

carbonyl stretching vibration and phenolic groups, which

shows the carbonyl stretching vibration from the

carboxylate ions and the hydroxyl stretching vibration from

the phenolic ions in the protein (Mandal et al., 2005).

This spectrum indicates that the secondary structure of the

protein of laccase is affected as a consequence of reaction

with the gold ions or binding with the GNPs.

Based on the study of (Kalishwaralal et al., 2010), the key

role of exposing thiol groups of α-amylase for GNPs

formation is high temperature (70°C) that destructs the

appropriate folding of α-amylase and exposes hydrophobic

and hidden groups with reductive ability and make it

possible to form nanometallic structures.

109

The effect of temperature was determined by carrying out

the reaction using (0.3 ml) of HAuCl4 at different

temperatures. As shown in Table (12), it was found that as

temperature increases, the GNPs synthesis rate increases

and the time taken for color conversion was much reduced.

At room temperature, there was an initial lag period for the

formation of GNPs and the synthesis time was longer

reaching 90 minutes. At 100°C only 10 minutes were

required to get the highest absorbance (3.2) indicating the

highest productivity of GNPs as shown in Table (12), this

result was in agree with (Farmarzi and Frootenfar, 2011).

The radiation-induced synthesis is one of the most

promising strategies because it is simple, clean and has

harmless feature (Mostafavi et al., 1993). During radiation

when aqueous solution is exposed to gamma radiation; it

creates solvated electrons which are able to reduce metal

ions forming nano metals.

Exposure of the extract to different doses of radiation was

performed after addition of HAuCl4 surface plasmon

resonance (SPR) band was noted for all doses Figure (21),

maximum absorbance (4) was found at a dose of 5 kGy as

shown in Table (13), after which further increase in

radiation dose resulted in decrease in absorbance at 550

110

nm, while no peak was recorded in blank sample (radiation

before mixing with HAuCl4).

The formation of GNPs can be attributed to the radiolytic

reduction, which generally involves radiolysis of aqueous

solutions that provides an efficient method to reduce metal

ions. In the radiolytic method, when aqueous solutions are

exposed to gamma rays, they create solvated electrons,

which reduce the metal ions and the metal atoms eventually

coalesce to form aggregates (Marignier et al,. 1985).

Exposure of water or aqueous solutions to ionizing

radiation leads to formation of primary species H3O+, H•,

OH•, H2O2. These free radicals have major importance in

radiolytic chemical reactions. The combined effect of both

radiolytic reduction and presence of peptide resulted in

formation of GNPs by radiolytic reactions and stabilization

by prevention of aggregates formation by "capping".

The higher concentration of GNPs indicated by in

absorbance 4 with radiation compared to 3.2 with

temperature, gave an obvious advantage for radiation over

temperature in the production of GNPs.

The volume of HAuCl4 added strongly affects the reaction.

Absorbance increased with increase in volume HAuCl4, the

best tested volume of HAuCl4 was 0.3 ml Table (14), which

indicates increased rate of reaction by increasing the

111

volume of HAuCl4 used, further increase as reported causes

decrease in formation of GNPs used due to aggregation as

reported earlier by (Noruzi et al., 2011).

112

VI. Summary

After screening of seven locally isolated fungal

strains, Pleurotus ostreatus was chosen as it showed

relatively high laccase activity compared to other

laccase producing fungi.

Four agricultural wastes were screened as carbon

sources for their ability to produce laccase. Wheat

bran had been selected as carbon source to perform

all further experiments throughout the study.

The screening of the six nitrogen sources of natural

and synthetic origin showed that malt extract gave

higher laccase activity compared to other nitrogen

sources.

The statistical software package Minitab 16, U.S.A

was used for designing the experiment to study the

effect of the chosen concentrations of six variables in

2 level forms namely (malt extract (0.5 gm - 1 gm),

Tween-80 (0.01% (v/v) - 0.02% (v/v), CuSO₄ (0.625

mM - 1.25 mM), Resorcinol (10 mg-20 mg), DL-

Methionine (5 mg-10 mg) and Tannic acid (2.5 mg-5

mg)).

Sixty four experiments were done to fully investigate

the effect of these variables and to get the optimized

conditions for the production of laccase enzyme; the

statistical software chose 32 experiments to

completely analyze them.

The optimization of production conditions gave

maximum enzyme activity (32450 U/gfs) in the 28th

113

run at (2% N content, 0.02% v/v Tween -80, 1.25

mM CuSO4, 20 mg methionine, 10 mg resorcinol

and 2.5 mg tannic acid) with incubation at 29°C for 7

days.

The analysis of variance (ANOVA) for the selected

factorial model showed that the model was

significant with a model F-value of 17.75 and p=000

where P = < 0.05.

The model determination coefficient (R=0.81)

suggested that the fitted model could explain 81% of

the total variation.

The analysis of variance (ANOVA) for the selected

variables expressed significance of nitrogen source,

Tween-80 and tannic acid where P = < 0.05, where

other variables were not statistically significant.

The effect of gamma radiation on growth of

Pleurotus ostreatus was done by irradiating seven

days old slant at seven doses. The results revealed

that as the dose increased, the growth of Pleurotus

ostreatus decreased gradually

The produced laccase from the irradiated fungus was

highly affected as it decreased to almost half 17, 200

(U/gfs) at 0.1 kGy compared to the enzyme obtained

from non-irradiated fungi (32, 450 U/gfs). Complete

loss in enzyme activity was obtained at 1.5 and 2

kGy.

Laccase enzyme was partially purified using

ammonium sulphate precipitation and the precipitate

114

was desalted by dialysis bag. Protein concentration

was quantified using the Bradford assay.

Total activity decreased from 675000 to 622000

U/1500 ml but the specific activity increased from

112.5 to 204 U/mg.

Characterization of the partially purified enzyme was

done; the effects of pH and temperature on the

activity of partially purified enzyme were studied,

the optimum activity for laccase was at pH5 and

keeping 90% of activity at pH6 and 40 °C.

The effects of several activators and inhibitors were

investigated. Activators included Cu+2, Zn+2, Mg+2

and Ca+2 giving an enhancing effect on the activity of

laccase with different extents. Whereas inhibitors as

Cd+2, Co+2 and Ba+2 caused inhibition of activity.

EDTA did not inhibit its activity at the concentration

used.

Gamma irradiation of the enzyme (5 doses of gamma

radiation), decreased its activity to half, up to 5 and

6 kGy where the activity remained constant

Five dyes [methyl orange, trypan blue, (RBR-R),

(RB-B) and (RBY-R)] were chosen to test the

enzyme’s ability to remove their color, showing

highest decolorization in case of methyl orange and

trypan blue with 86 and 83 % respectively.

Laccase was used to prepare GNPs, characterization

of the produced GNPs using DLS, TEM and FTIR

was done.

115

The effects of temperature, gamma radiation and

different volumes of tetrachloroauric acid were

assessed to study their effect on the formation of

GNPs. Highest amount of GNPs was at 100°C, 5

kGy and 0.3 ml of tetrachloroauric acid.

116

Conclusion

From the above mentioned results we can conclude that

laccase production has been shown to depend markedly on

the fungal strain, carbon, nitrogen content and inducer

compounds, and we were able to reach the conditions that

helped in multiplying the enzyme concentration to almost

10 folds (compared to fermentation of wheat bran alone)

indicating that factorial design can be a practical useful tool

for enhancing the activity of laccase.

Using the partially purified enzyme in the decolorization of

5 different reactive and azo dyes was able to get relatively

high percentage of decolorization in the used dyes which

facilitates further insight into dye decolorization using

fungal enzymes, the decolorization of type model dyes is a

simple method to assess the aromatic degrading capability

of laccase. The enzyme was able to produce GNPs, using

temperature and gamma radiation was able to increase the

production of GNPs.

The applications investigated reflect the possible

implication of laccase in further biotechnological and

industrial processes.

117

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