1

CHAPTER 1

INTRODUCTION

Enzymes can be defined as soluble colloidal organic catalysts which are

produced by living cells and are capable of acting independently of the cells

[1].

1.1 Microbial Enzyme

Microbial enzymes can be roughly classified into three major fields of

application: 1) those that can be used to synthesize useful compounds; 2) that

can stereo specifically carry out important bioconversion reactions; and 3) that

are able to hydrolyze polymers into interesting monomers [1].

1.2 Laccase Enzyme

Laccase [E.C. 1.10.3.2, p-benzenedial: oxidoreductases] is an oxido-

reductase able to catalyze the oxidation of various aromatic compounds

[particularly phenol] with the concomitant reduction of oxygen to water [2].

Laccase belongs to the small group of enzymes called the blue copper

proteins or the blue copper oxidases along with the plant ascorbate oxidase

and the mammalian plasma protein ceruloplasmin among others[1,3]. These

proteins are characterized by containing 2-4 copper atoms per molecule. One

copper is placed at the T1 site, where reducing substrate binds, and it is

responsible in the characteristic blue-greenish colour in the oxidizing resting

2

state Cu2+ [1,4].The other three coppers are clustered in the T2/T3 site in

which molecular oxygen binds. Comparative studies of fungal laccases have

shown that these enzymes are similar in their catalytic activity with phenolic

compounds, regardless of their origin, but differ markedly in their inducibility,

number of enzyme forms, molecular mass and optimum pH [5,6]. In the

presence of an appropriate redox mediator, such as 2,2'-azino-bis(3-

ethylbenzothiazoline-6-sulfonate) (ABTS) or 1-hydroxybenzotrizole (HBT),

laccase also catalyzes the oxidation of non-phenolic lignin model compounds

[7] and degrades polycyclic aromatic hydrocarbons [8] and various dye

pollutants.

1.2.1 Occurrence and Distribution

Laccase is widely distributed in higher plants and fungi [9] and has

been found also in insects and bacteria. Recently a novel polyphenol oxidase

with laccase like activity was mined from a metagenome expression library

from bovine rumen microflora [10].

Yoshida [2]first described laccase in 1883 when he extracted it from

the exudates of the Japanese lacquer tree, Rhus vernicifera. In 1896 laccase

was demonstrated to be present in fungi for the first time by both Bertrand and

Laborde [1,11]. Since then, laccases have been found in Ascomycetes,

Deuteromycetes and Basidiomycetes; being particularly abundant in many

white-rot fungi that are involved in lignin metabolism [12,13]. Fungal laccases

have higher redox potential than bacterial or plant laccases (up to +800 mV),

and their action seems to be relevant in nature finding also important

applications in biotechnology. Thus, fungal laccases are involved in the

degradation of lignin or in the removal of potentially toxic phenols arising

during lignin degradation [2].

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The enzyme is widely distributed in fungi; however its biological

function is still not totally clarified [14,15]. Fungal laccases have been

implicated in the physiological functions such as sporulation, rhizomorph

formation, pathogenesis, formation of fruity bodies, cell detoxification,

pigment synthesis and lignin degradation [13,16]. In addition, fungal laccases

are hypothesized to take part in the synthesis of dihydroxynaphthalene

melanins, darkly pigmented polymers that organisms produce against

environmental stress [17] or in fungal morphogenesis by catalyzing the

formation of extracellular pigments [18].

The white-rot fungi belonging to Basidiomycetes are the most efficient

degraders of lignin and also the most widely studied. The enzymes implicated

in lignin degradation are: (1) lignin peroxidase, which catalyses the oxidation

of both phenolic and non-phenolic units, (2) manganese-dependent peroxidase,

(3) laccase, which oxidises phenolic compounds to give phenoxy radicals and

quinones; (4) glucose oxidase and glyoxal oxidase for H2O2 production, and

(5) cellobiose-quinone oxidoreductase for quinone reduction [19].

Laccase oxidizes phenolic units in lignin to phenoxy radicals, which is

the same process as that brought about by the chelated Mn (III) produced by

MnP. However, in the presence of appropriate “primary’’ substrates (such as

ABTS), the effect of laccase apparently can be enhanced; laccase/primary

substrate systems have recently been reported to degrade lignin in Kraft pulp

[11] and to oxidize non-phenolic compounds that otherwise are unattached by

laccase.

The laccase producing fungi can be isolated from various habitats like

decaying woods, composting yards, forest soil, paper mill effluents etc., [9].

Many wood decaying fungi produces laccase enzyme and this also suggests

their role in lignin biodegradation [10].

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1.2.2 Laccase Producing Fungi

Fungi constitute a kingdowm including eukaryotic organisms such as

mushrooms, yeasts and molds. The principal characteristics include the

presence of cells walls, basically containing chitin and glucans, their

heterotrophic behavior and the ability to produce extracellular hydrolytic

enzymes in order to obtain the nutrients [20], which can be absorbed through

the cell wall and cell membrane. From an evolution point of view, fungi are

believed to be closer to animals than plants, since they have chitin rather than

cellulose structures, store nutrients such as glycogen and produce spores

(similar to gametes) [20]. Their life cycle includes theformation of spores,

germination, the development of hyphae and mycelia and the growing of fruit

body [21].

Fungi have a significant role in nature by breaking down organic

material, and have importance for humans as producers of important

antibiotics, enzymes, foods, etc., on one hand, and as agents of food spoilage,

plant and animal diseases on the other. They are the only organisms that are

able to completely mineralize lignocelluloses, the most abundant recalcitrant

renewable material available in nature. They do so by producing several sets

of enzymes for breaking down polysaccharides, celluloses and hemicelluloses

as well as lignin, a natural aromatic polymer. Ligninases or ligninolytic

enzymes constitute a group of oxidoreductases that are highly specialized in

polymerization as well as in the degradation of lignin. These enzymes are

mostly produced by the so called white-rot fungi and litter-decomposing fungi.

1.2.2.1. White Rot Fungi

White-rot fungi are a physiological group of fungi capable of

biodegrading lignin. The name white-rot derives from the white appearance of

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the wood when is attacked by these fungi, where the wood gets this bleached

appearance due to lignin removal [22]. Though some white-rot fungi are

Ascomycete, taxonomically talking, most of them are Basidiomycetes [23].

They can grow in a wide range of temperatures and withstand a wide range of

pH [24], but no growth has been observed below 10 ºC.

Based on the enzyme production patterns of white-rot fungi, Hatakka,

1994, [25] suggested three categories of fungi: 1) lignin peroxidase-

manganese peroxidase group, 2) manganese peroxidase-laccase group and 3)

lignin peroxidase-laccase group.

The most efficient lignin degraders are able to mineralize lignin to CO2

and belong to the first category of fungi. Only moderate and very poor

mineralization of lignin occurs in the second and third category of fungi

respectively.

The genus Trametes belonging to the kingdom of fungi, phylum

Basiomycota, class Basidiomycetes, subclass Agaricomycetidae, order

Polyporales and family Polyporaceae, is assumed to be one of the main

laccase producers [26]. Trametes is probably one of the most widely

investigated Basiodiomycota for ligninolytic enzyme production and

application [27]. Like other white-rot fungi, Trametes genus is attractive due

to the extracellular secretion of non-specific ligninolytic enzymes and the

possibility of growing on cheap media such as lignocellulosic wastes [27].

1.2.3 Laccase Expression

In several organisms, laccases are constitutively produced in small

amounts. However, their production can be considerably enhanced by a wide

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variety of substances, including aromatic or phenolic compounds, metal ions,

alcohol, and detergents [28].

1.3 Production Methods for Laccase Enzyme

Fermentation techniques used for the production of laccase enzyme can

be divided into two main groups: solid-state fermentation (SSF) and

submerged fermentation (SmF). The difference between these two techniques

consists in the quantity of free flowing liquid present in the system. SSF

involves the growth of microorganisms on solid materials in the absence or

near-absence of free flowing water, whereas in SmF the microorganisms grow

on a continuous liquid phase [29].

Solid state and submerged fermentation techniques are common and

conventional biotechnology processes in view of production of value-added

products such as enzyme, biopharmaceuticals, organic acid, biosurfactant,

vitamin, flavoring compounds, biofuel, biopesticides etc., [1].

1.3.1 Submerged Fermentation

Submerged fermentation (SmF), more strongly developed from the

1940’s onwards because of the necessity to produce antibiotics on a large scale

has been characterized as fermentation in the presence of excess water. Almost

all the large-scale enzyme producing facilities have been used the established

approach of SmF owing to better monitoring and ease of handling [3].

1.3.2 Solid State Fermentation

Cultivating fungus using an inert substrate without any free-flowing

water is known as solid state fermentation (SSF). SSF utilizes agro-industrial

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wastes as the substrates in the enzyme production. Substrates (agro-industrial

wastes) act as either inert or non-inert material, supporting the fermentation

process. Inert substrates only act as an attachment place for the fungal growth,

while non-inert substrate also supply nutrients for the fungal growth [30].

Solid state fermentation is a suitable process for enzyme production

using filamentous fungi since, SSF is well adapted to the metabolism of the

fungus and it gives the natural habitat for fungal growth [31]. Smaller

substrate particle would provide large surface area for microbial metabolism,

which is a desirable factor in enzyme production. However, too small

substrate particle will lead to substrate agglomeration, which may interfere

with microbial respiration/aeration, and result in poor cellular growth [32].

In SSF, the moisture necessary for microbial growth exists in an

absorbed state or complex within the solid matrix. This solid matrix can be

either a natural support (e.g.lignocellulosic materials) or an inert support (e.g.

plastic foams) [32].Although most researchers consider solid-state and solid-

substrate fermentation essentially one and the same, Pandey et al.,[33] have

distinguished these two as separate processes. According to them, solid-

substrate fermentation includes those processes in which the substrate itself

acts as the carbon source and occurring in the absence or near-absence of free

flowing water, whereas SSF is defined as any fermentation process occurring

in the absence or near-absence of free flowing water, using a natural substrate

or an inert substrate as solid support.

Due to the small quantity of water present in the SSF, the formation of

specific products, which are not produced under SmF, can take place. For the

products that can be obtained using both techniques, SSF presents higher

volumetric productivity and better performance than SmF [34].

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1.3.3. Assay and Detection of Laccase Enzyme

For detecting laccase, some assay methods including HPLC method,

manometry, order spectrum method and spectrophotometry are involved [35].

Compared with other methods, spectrophotometry is widely used owing to it’s

simply and sensitive characteristics. Usually, there are several compounds that

have been used as substrates by spectrophotometry methods such as 2, 2'-

azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) [36], syringaldazine

[37], o-dianisidine [38] and Guaiacol [39]. Assay sensitivity for enzyme is

largely depended upon the efficiency of substrates. Thus, sensitivity of

substrates is vital to evaluate enzyme activity.

1.3.4 Optimization of Laccase Enzyme Production

Selection of nutrients such as carbon, nitrogen and other nutrients is

one of the most critical stages in an efficient and economic process

development. Yield of any microbial product can be improved by optimization

of medium components that are required in fermentation processes. The

methodologies used for screening the nutrients fall into two categories;

1.Classical Method, and 2.StatisticalMethod.

Optimization by classical Method, also called one-factor-at a time

method, involves keeping one variable fixed and varying other variables. It is

time consuming and laborious and does not include interactive effects among

the variables.

The application of statistical methodologies in fermentation process

development has numerous advantages in terms of rapid and reliable short

listing of nutrients. Understanding the interactions among nutrients at varying

concentrations and tremendous reduction in total number of experiments

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resulting in less time consumption,glassware, chemicals and man power [40].

Application of statistical methodologies in fermentation process development

can result in improved yield of the product, reduced process variability, closer

confirmation of the output response (product yield/ productivity) to normal

and target requirements, reduced development time with overall costs.

1.3.5 Immobilization of Laccase Enzyme

The main drawbacks of many important enzymes for their use in

industrial applications are their low stability and productivity and high

production costs [41]. The most frequently used stabilization method is

immobilization, which in addition provides many other process benefits such

as reduction of enzyme replacement, facilitation of separation and reuse of the

catalyst and assistance of reaction control [42, 43]. Moreover, it is well known

[44, 43, 42] that immobilization shifts the enzyme properties like: optimum

values of pH and temperature, kinetics parameters and strengthens protein

structure. Especially higher thermo-stability of the enzyme allows conducting

the processes at higher temperatures and so it reduces reaction time. The

advantages and disadvantages of enzyme immobilization are shown in Table

1.1[45, 46].

Different methods based on physical and chemical mechanisms are

used for enzyme immobilization on solid materials and gels [14, 47]. The

chemical methods include covalent bonds between the enzyme and the

support, cross-linking between the enzyme and the support and enzymatic

cross-linking by multifunctional agents. The physical methods involve

adsorption, entrapment of enzymes in insoluble polymeric gels (polymeric

entrapment) or in micelles (encapsulation) [14].

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The immobilization of laccase renders possible the recycling of the

enzyme and protection against deactivation. Thus, the immobilization

procedure determines how effective and stable the enzyme will become.

Chitosan is a polymer of amino-D-glucose groups produced from deactylation

of chitin. Chitosan is a non-toxic and biodegradable polymer, soluble in water

under acidic conditions and insoluble in neutral and alkaline environments

[48]. It has been applied for the treatment of dyes due to its high capacity of

adsorption and its reusability [49]. Its mechanical property and low cost render

it suitable to be applied as a matrix for the immobilization of proteins [50].

Laccase immobilized on chitosan demonstrated higher yields of decolorization

of an azo- and tri-phenyl methane dye as well as a faster decolorization of the

azo-dye when compared to the free enzyme.

Table 1.1: Advantages and disadvantages of enzyme immobilization

� Advantageso Easier separation and recuperation of the enzyme

and productso Reusabilityo Increase of thermal stability and resistance against

denaturalizing agentso Reaction can be stopped easiero Continuous operations can be easier to achieveo Higher flexibility in the design of bioreactorso Prevents the contamination caused by the protein in

the final producto Microbial contamination is easier to control

� Disadvantageso Lower enzymatic activity caused by the

immobilization processo Increase of the Michaelis-Menten constant

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1.4 Agro Waste

Efficient utilization of agro-industrial waste for the production of value

added products such as enzymes and organic acids is one of the most

economical and ecological recycling process using fungi and other microbes

[51]. The nutrient composition of the fermentation media is one of the major

limiting factors for better conversion of lignocellulosics into enzymes. This

needs optimization of fermentation media to ensure balanced proportion of

nutrients to get optimum microbial growth and enzyme production [52]. The

cakes or whole vegetable meals like sorghum, wheat bran, cotton seed meal

and soybean meal are the most commonly used fermentation additives [53]

ground nut cake, neem seed cake [54] and cotton seed cake [55] for

submerged fermentation.

1.4.1 Sapota Seed

The Sapotaceae family consists of large evergreen trees and shrubs,

distributed throughout the tropic Asia, Africa and America. It consists of about

40 genuses, one among them being Minusops manikara also known as Achras

sapota, which is an important edible fruit bearing tree in India and Pakistan

[56].

The flowers of the tree are glabrous, long pedicelled. The fruits are

glabose, fleshy berry, 5-10 cm in diameter. Seeds 2-3 shining black, obovate 2

cm long and are nuts. The wood of the plant is reddish brown, hard with radial

groups, fine medullary rays and irregular narrow wavy transverse lines.

Leaves are crowded near the end of the thick branchlets, shining in

appearance, lanceolate in shape; usually they are broad and large. The plant

bark and leaves are used in folk medicine [56].

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The seeds are reported to have saponins, fatty acid esters of

triterpenoids, polyphenols and tannins. The nut kernel saponins were reported

to have basic acids like aglycone and arabinose, rhamnose and glucose as

sugars [57].

1.5 Enhancers for Laccase Induction

Although most laccases are constitutively produced in a small amount,

the production of laccases could be significantly enhanced by a wide variety of

substances [58-61]. There enhancing effect of various inducers(phenolics,

alcohols, heavy metals, vitamins, amino acids, antibiotics) for laccase

production were also reported [62].

Nearly 104 times enhancement of laccase activity (759.8 U/l per day)

was obtained using complex-inducer-supplemented (copper, xylidine, and

phenolic mixture) medium due to the cooperative effect between the inducers

on laccase production [63]. However, most potent laccase inducers, such as

aromatic compounds, are volatile, toxic, and expensive precluding their use

from industrial application. Furthermore, laccase production generally requires

long fermentation time, which is still not appreciated for industrial

applications. In recent times there has been a great degree of attempt to use the

agro based wastes for the production of laccase enzyme, in order to reduce the

cost.

1.5.1 Vermi Wash

Vermi wash is the coelomic fluid of earth worms, which is obtained

after the death of earth worms. It contains many nutrients like soluble

nitrogen, phosphorus and potash. Hormones such as cytokinins, oxyn, etc.,

amino acid, vitamin, enzymes etc., are present in the vermi wash. The vermi

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wash is rich in dissolved nutrients and the amino acids present in it are easily

available to the plants on application to soil. Vermi wash has nutritional,

pesticidal and plant disease control potential. The composition of vermi wash

is given in the table 1.2. [64].

Table 1.2: Composition of Vermi Wash

Component Quantity

pH 6.09

Dissolved oxygen 1.14 ppm

Dissolved nitrogen 2.0 ppm

Total phosphorus 60 ppm

Total potassium 69 ppm

Sodium 122 ppm

Salt 70 ppm

Chlorides 110 ppm

Sulphates 177 ppm

Calcium 175 ppm

Magnesium 200 ppm

1.6 Lignin

Lignin may be defined as amorphous; polyphenolic material arising

from an enzyme mediated dehydrogenative polymerization of three phenyl

propanoid monomers namely: coniferyl, sinapyl and p-coumaryl alcohols.

Lignin is a hard material embedded in the cellulose matrix of vascular

plant cell walls that functions as an important adaptation for support in

terrestrial species [65]. It is a highly polymeric substance, with a complex,

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cross-linked, highly aromatic structure of molecular weight about 10,000

derived principally from coniferyl alcohol (C10 H12 O3) by extensive

condensation polymerization.

It is found mostly between cells and also within the cells. Lignin holds

cellulose and fibers together. The high concentration of this recalcitrant

polymer is found in the middle lamella, where it acts as a cement between

wood fibers, but it is also present in the layers of the cell wall (especially the

secondary cell wall), forming, together with hemicelluloses, an amorphous

matrix in which the cellulose fibrils are embedded and protected against

biodegradation. It’s function is to regulate the transport of liquid in the living

plant partly by reinforcing cell walls and keeping them from collapsing, partly

by regulating the flow of liquid and it enables trees to grow taller and compete

for sunshine [66].

Lignin is the third largest biomass after cellulose and hemi cellulose in

plants accounting for about 25-30%. The primary wall of green plant is made

of cellulose; the secondary wall contains cellulose with variable amount of

lignin and hemi cellulose.

In softwoods, the lignin is usually referred to as guaiacyl lignin, which

is derived from coniferyl alcohol and accounts for 95% of total lignin while

the remaining 5% are p-coumaryl and sinapyl alcohols. Where as in the hard

wood, guaiacyl and syringyl lignins will be there in majority and these are

derived from coniferyl and sinapyl alcohols in varying ratios. Grass lignin’s

are also like hard wood lignin but in addition they contain a small amount of

coumaryl derivative [18].

The lignocelluloses composition of various plants, common

agricultures waste and residues are given in the table 1.3 [67].

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Table 1.3:Lignocellulose contents of common agricultures waste and residues

Lignocellulosic materials Cellulose(%)

Hemi cellulose(%)

Lignin(%)

Hardwood stems 40-55 24-40 18-25Softwood stems 45-50 25-35 25-35Corn cobs 45 35 15Nutshells 25-30 25-30 30-40Paper 85-99 0 0-15Wheat straw 30 50 15Rice straw 32.1 24 18Sorted refuse 60 20 20Leaves 15-20 80-85 0Cotton seeds hair 80-95 5-20 0Newspaper 40-55 25-40 18-30Waste paper from chemicals pulp

60-70 10-20 5-10

Primary wastewater solids 8-15 NA 24-29Fresh bagasse 33.4 30 18.9Swine waste 6 28 NASolid cattle manure 1.6-4.7 1.4-3.3 2.7-5.7Coastal Bermuda grass 25 35.7 6.4Switch grass 45 31.4 12.0

NA= Not Available

Lignin is the well-known complex substance covalently bound to side

chains of xylans of cell-walls. It represents an obstacle to microbial digestion

of structural carbohydrates both because it is a physical barrier and because of

the depressing effect on microbial activity, due to the phenolic compounds it

contains [68].

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The aromatic polymer lignin is well-known for resistance to microbial

degradation because of its high molecular weight and presence of various

biologically stable carbon-to-carbon and ether linkages. Microorganisms that

degrade plant lignin via an oxidative process, are fungi [69], Actinomycetes

[70] and to a lesser extent, bacteria [71].

Only white-rot Basidiomycetes are responsible for the complete

mineralization of this polymer. Phanerochaete chrysosporium, the best studied

white-rot fungus, secretes two heme peroxidases, lignin peroxidase(LiP) and

manganese peroxidase (MnP) under ligninolytic conditions (M).

Biological lignin degradation is interesting for two reasons: it is an

essential component of global carbon cycling and it is biochemically unusual.

Almost all terrestrial fixed carbon consists of lignified polysaccharides or of

lignin itself and would be recalcitrant to biodegradation were it not for

ligninolytic microorganisms. These organisms are, in large part, higher

(basidiomycetous) fungi that inhabit decaying wood and soil litter [72, 73].

Many different organisms causes damage to wood but fungi remains

the primary one. Wood decaying fungi can also bring about the lignin removal

or degradation. Wood decay fungi may be divided into three categories: white,

brownand soft-rot fungi, where white and brown-rot fungi are the two most

important wood-destroying organisms. Brown and white-rot fungi decay wood

by distinctly different mechanisms. The white- rot fungi are able to fragment

the major structural polymers of wood and other lignocellulosics- lignin,

cellulose, and hemicellulose and further metabolize the fragments. The hyphae

of fungi rapidly invade wood cells and lie along the lumen walls where they

secrete the enzyme to depolymerize the hemi cellulose, cellulose and

fragmentation of lignin. A few white-rot fungi, termed selective white-rot

fungi, remove lignin and hemicellulose preferentially during early stages of

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decay. In contrast, most brown-rot fungi only utilize cell wall hemicelluloses

and cellulose leaving the lignin essentially undigested, but modified [74-76].

The only microorganisms which to any extent can degrade lignins are the

white-rot fungi.

Three lignolytic enzymes namely, lignin peroxidase (LiP), manganese

peroxidase (MnP), and laccase are responsible for the initial fragmentation of

the lignin polymer and production of low molecular mass breakdown products

in white-rot fungi [77]. Several white-rot fungi are known to secrete the above

enzymes [62]. Various enzymes that are involved in the lignin breakdown are

given in the Table1.4[25].

In paper and pulp industries, lignin is the coloured material that must be

removed in pulp bleaching because it is what makes the mechanical pulp

fibers stiff and makes newsprint turn yellow [78]. Lignin is what makes the

cellulose and hemi cellulose in wood indigestible. Pulp and paper industry use

wood and non-wood materials in the production of chemical pulp, wherein

lignin is degraded and dissolved almost completely (90-95%) in black liquor.

If not removed from the treated wastewater, the lignin presents a serious

pollution and toxicity problem in aquatic ecosystems, owing to its low

biodegradability and high range of colour [79]. A number of physio-chemical

methods have been developed for the treatment of lignin from paper mill

wastewater. However, these processes are not very effective and are costly

[80]. In biological treatment systems a wide variety of microorganisms

including fungi, Actinomycetes and bacteria have been implicated in lignin

biodegradation and decolourization of pulping effluent [81-83]. Among them,

white-rot fungi have received extensive attention due to their powerful lignin-

degrading enzymatic systems [25].

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Table 1.4: Enzymes involved in the degradation of lignin

Enzyme Activity,Abbreviation

CofactororSubstrate,“Mediator”

Main Effect or Reaction

Ligninperoxidase,Lip

H2O2,veratry alcohol

Aromatic ring oxidized to cation radical

Manganeseperoxidase,MnP

H2O2, Mn, organic acid aschelator, thiols, unsaturatedlipids

Mn(II) oxidized to Mn(III); chelated Mn(III) oxidizes phenolic compounds to phenoxyl radicals; other reactions inthe presence of additional compounds

Laccase, Lacc O2; mediators, e.g.,Hydroxyl benzatriazole or ABTS

Phenol are oxidized to phenoxyl radicals; other reactions in thepresence of mediators.

Glyoxal oxidase,GLOX

glyoxal, methyl gloxal

Glyoxal oxidized to glyoxylic acid; H2O2

production

Aryl alcohol oxidase,AAO

Aromatic alcohols (anisyl,veratryl alcohol)

Aromatic alcohol oxidized to aldehydes; H2O2production

OtherH2O2producing

Many organiccompounds

O2 reduced to H2O2

1.6.1 Eucalyptus Sp.

Eucalyptus sp is one of the most widely used plant species as a source

of wood and fiber in the pulp and paper making industry. Eucalyptusis the

most valuable and widely planted hardwood in the world (18 million ha in 90

countries). Eucalyptus are grown extensively as exotic plantation species in

tropical and subtropical regions throughout Africa, South America, Asia, and

19

Australia and in more temperate regions of Europe, South America, North

America, and Australia. A few eucalypt species and hybrids constitute the

majority of these plantations. Most domesticated eucalypts are from the

subgenus Symphyomyrtus, the largest of the 10 subgenera currently

recognized within Eucalyptus, containing over 75% of the species. Four

species and their hybrids from this subgenus, Eucalyptus grandis(EG), E.

urophylla(EU), E. camaldulensis, and E. globulus, account for about 80% of

the eucalypt plantations worldwide. EG is the most widely used species in

plantation forestry worldwide in tropical and subtropical areas not only as a

pure species, but also as a parental species in hybrid breeding. It has the fastest

growth and widest adaptability of all Eucalyptus species [84].

The greatest area of plantations of EG (Eucalyptus grandis)and its

hybrids with other species is in Brazil and several other Central and South

American countries. It has been planted extensively in India, South Africa,

Zambia, Zimbabwe, Tanzania, Uganda, and Sri Lanka and is grown in

California, Florida, and Hawaii in the United States. EU (E. urophylla)has

been widely planted in the tropics for many years. E. globulusis the premier

species for temperate zone plantations in Portugal, Spain, Chile, and Australia.

For pulp production and increasingly for solid wood, EG, EU, and the EG x

EU hybrid are the most favored in tropical and subtropical regions [84].

Eucalypts are utilized worldwide for a wide array of products including

pulp for high quality paper[85], lumber, plywood, veneer, solid and

engineered flooring, fiberboard [65, 66], wood cement composites [86, 87],

mine props, poles, firewood, charcoal, essential oils [88-91], honey, tannin,

and landscape mulch [92] as well as for shade, windbreaks, and

phytoremediation [93-95]. The expansion of eucalypt plantations throughout

the world is largely attributable to eucalypts superior fiber and pulping

properties and the increased global demand for short-fiber pulp.

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1.7 Azo Dyes

Dyes are widely used within the food, pharmaceutical, cosmetic, textile

and leather industries. During industrial processing, up to 40% of the used dye

stuffs are released into the process water, producing highly colored

wastewaters that affect aesthetics, water transparency, and gas solubility in

water bodies. Moreover and most importantly, there is a general concern

regarding toxicity of some of these dyes. Because of both the high discharged

volumes and the effluent composition, wastewaters from the textile industry

can be considered as the most polluting among all industrial sectors, thus

greatly requiring appropriate treatment technologies [96]. Biotechnological

approaches were proven to be potentially effective in treatment of this

pollution source in an eco-efficient manner (97-101). The white-rot fungi

(WRF) are, so far, the microorganisms most efficient in degrading synthetic

dyes, with basidiomycetous fungi that are able to depolymerize and mineralize

lignin. This WRF’s property is due to the production of extracellular lignin-

modifying enzymes (LME’s) and because of their low substrate specificity and

also able to degrade a wide range of xenobiotic compounds [20, 22, and 102]

including dyes [103-106]. The main LME’s are manganese peroxidases

(MnP), E.C. 1.11.1.13, [107], lignin peroxidases (LiP), E.C. 1.11.1.14 and

laccases (Lac), E.C. 1.10.3.2, [108]. LiP, MnP, and laccase play significant

roles in dye metabolism by White-rot Fungi [109], due to the structural

similarity of the most commercially relevant dyes to lignin (sub) structures

amenable to be transformed by LME’s.

Large quantities of Azo dyes (about 50–70% of the dyes available on

the market today) are manufactured worldwide and used in a variety of

applications. It is estimated that about 15% of the total world production is lost

during synthesis and processing. Therefore azo dyes are extensively contained

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in wastewater generated from the textile and dyestuff industries. Synthetic azo

dyes are heavily used in the textile, paper, cosmetics, pharmaceutical and food

industries. Such colored dye effluents pose a major threat to the surrounding

ecosystems owning to their non biodegradability, toxicity and potential

carcinogenic nature. Azo dyes are considered non degradable under aerobic

conditions with bacteria. This makes it very difficult to remove azo dyes by

conventional wastewater systems.

1.7.1 Acid Orange-7

Acid orange-7 {IUPAC name sodium 4-[(2E)-2-(2-oxonaphthalen-1-

ylidene) hydrazinyl] benzenesulfonate,C16H11N2NaO4S (sodium salt)} is a

reddish yellow color turns dark brown and gives various colours with H2SO4

(Megenta Red) and NaOH (Brownish Yellow). It is soluble in water, ethyl

alcohol and HCL solubility in water being high 116 g/L (30 ºC). Its melting

point is 164°C and molecular weight is 350.32 g/mol. It is irritative to

eyes(R36), respiratory system (R37) and skin (R38). It is widely used for

dyeing and printing of wool, nylon, silk, paper dyeing, leather (combine

dyeing & printing points under one head) and also as biological stain and

indicator, paper coating, transparent pigments in tin printing and as molding

powders.

1.7.2 Biological Removal of Azo Dyes

Colored industrial wastewater is usually treated by physico-chemical

processes. These processes include flocculation, flotation, electro-flotation,

membrane-filtration, ion exchange, irradiation, precipitation, ozonation and

adsorption using activated carbon or biological adsorption using bacteria,

fungi, algae or plant biomass [98,110 and 111]. Both living and dead cells

have been used for bio-adsorption [112].

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Biological treatment, either aerobic (in the presence of oxygen) or

anaerobic (in the absence of oxygen), is generally considered to be the most

effective means of removing the bulk of pollutants from wastewater. Different

species have been used for the treatment of various dye effluents. The major

advantage of this technique is the low running costs. Bacteria and fungi are the

two groups of microorganisms that have been widely studied in the treatment

of dye wastewater. The enzymes secreted by aerobic bacteria can break down

the organic compounds. The isolation of aerobic bacterial strains capable of

degrading different dyes has been carried out for more than two decades [113].

Also, fungal strains and their enzymes have been studied in detail by various

authors [114,112 and 115]. However, it is important to remark that different

factors like concentration of pollutants, dyestuff concentration, initial pH and

temperature of the effluent affect the discoloration process.

White-rot fungi have been also widely used for dye removal. This color

removal process can take place by adsorption of dyestuff on fungal mycelia,

by real degradation or by a combination of both. Nevertheless, there are some

important drawbacks in the use of white-rot fungi. The addition of reagents for

having appropriate growth conditions is necessary, since dye degradation is

attributed to secondary metabolic pathways. Also, the production of the

enzymes involved in the dye degradation is not constant with time and is

influenced by inhibitors that can be present in the effluents. For overcoming

these problems, the use of the enzymes involved in the dye degradation,

instead of the whole fungal cultures, is getting more attention.

Exhaustive reviews on decolorization of synthetic dyes [116,117] and

dye wastewaters using white-rot fungi and their lignin degrading enzymes

have appeared [112, 118]. A textile effluent besides containing dyes also has

extreme pH values and contains salts, often at very high ionic strength. The

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role of laccase in terrestrial fungi in decolorization of dyes and dye

wastewaters is undisputed [116, 112 and 118].

1.8 Cypermethrin

Cypermethrin is a composite pyrethroid; a broad spectrum, non-

cumulative insecticide and a fast-acting neurotoxin with good contact and

stomach action. Active against a wide range of insect pests, particularly leaf

and fruit eating Lepidoptera, Coleoptera and Hemiptera; cattle ectoparasites,

sheep scab and lice. Cypermethrin may be applied to a wide range of fruit,

vegetables, vines, tobacco, and several non-food crops. It is of moderately

high toxicity to mammals. In general pyrethroid poisoning is characterized by

hyperactivity and hypersensitivity (somatosensory). Cypermethrin in

particular is included among a group of compounds producing the CS-

syndrome in rodents, in sequence: pawing and burrowing behavior, salivation,

coarse tremors progressing to choreoathetosis and occasionally terminal clonic

seizures. Toxic for fish, aquatic arthropods, and honey-bees in laboratory tests

[119]. Most susceptible species is possibly the mouse. Cypermethrin is a

neurotoxic agent most probably acting through the central nervous system to

cause repetitive nerve activity. It is readily absorbed from the gastrointestinal

tract, by inhalation of dust and fine spray mist and only minimally through

intact skin. The toxic oral dose in mammals is greater than 100-1000 mg/kg,

and the potentially lethal acute oral dose is 10-100 g. However, it is non-

carcinogenic and teratogenic.

In vertebrates and invertebrates, cypermethrin acts mainly on the

nervous system. Cypermethrin is both a stomach poison and a contact

insecticide [120]. In the peripheral nervous system of the frog, its primary

action is to induce noticeably repetitive activity and produce trains of nerve

impulses as a result of altering ion permeability of nerve membranes

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[121,122]. These long-lasting trains of nerve impulses can cause hundreds to

thousands of repetitive nerve impulses in the sense organs. This repetitive

activity is induced by pyrethroid damage to the voltage-dependent sodium

channel, causing sodium channels to stay open much longer than normal

[123]. Cypermethrin has been shown to inhibit ATPase enzymes involved in

movement of ions against a concentration gradient which are regulated by

active transport. This action is especially critical to fish and aquatic insects

where ATPase enzymes provide the energy necessary to active transport, and

are very important at sites of oxygen exchange. ATPase inhibition and

disruption of active transport possibly affect ion movement and the ability to

maintain ion balance, and disrupt respiratory surfaces; indicating that

cypermethrin is inherently more toxic to aquatic organisms [124].

The biodegradation of cypermethrin by several bacteria and

Actinomycetes has been discussed by several authors [123, 124 and 125].

1.9 Applications of Laccase Enzyme

Laccases have become important industrially relevant enzymes because

of a number of diverse applications, e.g. for biocatalytic purposes such as

delignification of lignocellulosics and cross linking of polysaccharides, for

bioremediation applications such as waste detoxification and textile dye

transformation, for food technological uses, and for biosensor and analytical

applications [13]. Lignocellulolytic enzymes have significant potential

applications in various industries including chemicals, fuels, food, pulp and

paper, textile and laundry, animal feed and agriculture [17]. Being specific,

energy-saving and biodegradable, laccase-based biocatalysts fit well with the

development of highly efficient, sustainable and eco-friendly industries.

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The laccase enzyme has potential applications in environmental

biotechnology. The saw mil waste fungi were the least studied one and the

usage of sapota seed based media was a novel approach for laccase enzyme

production by solid state fermentation. The acid orange-7 decolourization and

delignification of Eucalyptus sp. were studied on a comparative basis with 5

laccase producing fungi for the first time and the degradation of cypermethrin

by fungi was not studied so far(based on literature survey). For these reasons

this work was chosen.