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
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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
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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
[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