TABLE
OF CONTENT
CHAPTER
ONE
INTRODUCTION AND
LITERATURE REVIEW
1.1
Enzyme
1.1.1 Types of enzymes
1.1.2
Characteristics of enzymes
1.2
industrial applications of enzymes
1.3Fungi
as a micro-organism.
1.3.1
How fungi reproduce
1.3.2
Growth and physiology of fungi
1.4. Production of
enzyme by fermentation technologies
1.4.1. Solid-State Fermentation (SSF)
1.4.2. Submerged Fermentation (SmF)/Liquid
Fermentation (LF)
1.4.3. Substrates used for fermentation
1.5. INVERTASE
1.5.1. Benefits of Invertase
1.5.2.Measurement
of invertase activity:
1.6. Objective
CHAPTER TWO
MATERIALS AND METHODS
2.1. Materials
2.1.1. Apparatus
2.1.2. Culture Media
2.1.3. Reagents
2.2. Methods
2.2.1. Sample Processing
2.2.2. Isolation
of fungal species
2.2.3. Inoculation of basal medium for enzyme
production
2.2.4 Screening for invertase
CHAPTER THREE
RESULTS
3.1 characterization of the fungal isolates
CHAPTER FOUR
DISCUSSION
CONCLUSION
REFERENCES
CHAPTER
ONE
INTRODUCTION AND
LITERATURE REVIEW
1.1 Enzyme
Enzymes are large
biological molecules responsible for thousands of chemical inter-conversions
that sustain life (Smith, 1997). All known enzymes are proteins. They are high
molecular weight compounds made up principally of chains of amino acids linked
together by peptide bonds, they are denatured at high temperature and
precipitated with salts, solvents and other reagents. They have molecular
weights ranging from 10,000 to 2,000,000 units. Enzymes do not cause reactions
to take place, but rather they enhance the rate of reactions that would have been slower without their presence and
still remains unused and unchanged.
Many enzymes
require the presence of other compounds - cofactors - before their catalytic
activity can be exerted. This entire active complex is referred to as the
holoenzyme; i.e. apoenzyme (protein portion) plus the cofactor (coenzyme,
prosthetic group or metal-ionactivator) is called the holoenzyme (Alexopoulos et al., 1996)
The living cell is
the site of tremendous biochemical activity called metabolism. It is the
process of chemical and physical change which goes on continually in the living
organism involving the build-up of new tissues, replacement of old tissue,
conversion of food to energy, disposal of waste materials, reproduction - all
the activities that we characterize as "life."Thephenomenon of enzyme
catalysis makes possible biochemical reactions necessary for all life
processes. Catalysis is defined as the acceleration of a chemical reaction by
some substance which itself undergoes no permanent chemical change. Synthetic
molecules called artificial enzymes also display enzyme like catalysis
(Grovesm, 1997).
The catalysts of biochemical
reactions are enzymes and are responsible for bringing about almost all of the
chemical reactions in living organisms. Without enzymes, these reactions take
place at a rate far too slow for the pace of metabolism(Bairoch, 2000).
Enzymes actually
work by lowering the activation energy of a reaction. This is achieved when it
creates an alternative pathway which is faster for the reaction hence speeding
it up such that products are formed faster. Enzyme catalysed reactions are
million times faster than uncatalysed reactions, they alter the rates but not
the equilibrium constant of the reaction being catalysed (Ashokkumar et al., 2001). A few RNA molecules
called ribozymes also catalyse reactions, with an important example being some
parts of ribosome (Lilley, 2005).
1.1.1
Types of enzymes
Metabolic
enzymes: These have been called the spark of
life, the energy of life and the vitality of life. These descriptions are not
without merit. Metabolic enzymes catalyse and regulate every biochemical
reaction that occurs within the human body, making them essential to cellular
function and health (Sangeethaet al.,2005).
Digestive enzymes turn the food we eat into energy and unlock this energy for
use in the body. Our bodies naturally produce both digestive and metabolic
enzymes as they are needed. They either speed up or slow down the chemical
reactions within the cells for detoxification and energy production. The enable
us to see, hear, and move and think. Every organ, every tissue and all 100
trillion cells in our body depend upon the reaction of metabolicenzymes and
enjoy their energy factor. Without these metabolic enzymes, cellular life would
beimpossible.
Food
enzymes:These are introduced to the body through
the raw foods we eat and throughconsumption of supplemental enzyme products.
Raw foods naturally contain enzymes providing asource of digestive enzymes when
ingested(Hossainet al., 1984).
However, raw food manifests only enough enzymesto digest that particular food,
not enough to be stored in the body for later use (the exceptionsbeing
pineapple and papaya, the sources of the enzymes bromelain and papain). The
cooking andprocessing of food destroys all of its enzymes. Since most of the
foods we eat are cooked orprocessed in some way and since the raw foods we do
eat contain only enough enzymes toprocess that particular food (Persike et al., 2002) our bodies must produce
the majority of the digestive enzymes werequire, unless we use supplemental
enzymes to aid in the digestive process. A variety ofsupplemental enzymes are
available through different sources. It is important to understand
thedifferences between the enzyme types and ensure that one is using an enzyme
product which willmeet one’s particular needs.
Plant
based enzymes:These are the most popular choice of
enzymes. They are grown in a laboratorysetting and extracted from Aspergillus species. The enzymes
harvested from Aspergillusspecies are
called plantbased, microbial and fungal. Of all the choices, plant based
enzymes are the most active. Thismeans they can break down more fat, protein
and carbohydrates in the broadest pH range than any other sources (Ashokkumar et al., 2001).
1.1.3
Characteristics
of enzymes
Protein
nature:Enzyme is a protein. The
main components of an enzyme is protein.
Temperature:Enzymes
are sensitive to temperature. Many work best at temperatures close to body
temperatures and most lose their ability to catalyse if they are heated above
60 or 70o C. (Ashokkumar et
al., 2001).
Acidity and alkalinity:Many
enzymes work best at a particular pH and stop
working if the pH becomes too acidic or alkaline.
Catalytic
effect:It acts as catalyst, enzyme
functions in accelerating chemical reaction, but the enzyme itself does not
change after the reaction ends.
Specificity:It functions specifically. The enzyme only
catalyzes one kind of substrate and cannot function for many substrates. The
term is called one enzyme one substrate.
Reversibility: It means the enzyme does not determine the direction
of reaction, but it only functions in accelerating reaction rate until it
reaches equilibrium. The enzyme also functions in substance synthesis and
substance breaking down reaction.
Small
quantity:It is required, in small
amount. A small amount of enzyme is able to catalyze a chemical reaction (Nason, 1968).
Table
1: Classification of enzyme by nature and the reaction they catalyse
Classification Type
of Reaction Catalysed
Oxidoreductases Oxidation-reduction
reactions
Transferases Transfer
of functional groups
Hydrolases Hydrolysis
reactions
Lyases Group
elimination to form double bonds
Isomerases Isomerization
Ligases Bond
formation coupled with ATP
hydrolysis
1.1.3 Classes of enzymes
Oxidoreductase:
These enzymes catalyze redox reactions, i.e., reactions involving the transfer
of electrons from one molecule to another. In biological systems we often see
the removal of hydrogen atoms from a substrate. Typical enzymes catalyzing such
reactions are called dehydrogenases. For example, alcohol dehydrogenase
catalyzes reactions of the type R-CH2OH + A → R-CHO + AH2,
where A is a hydrogen acceptor molecule. Other examples of oxidoreductases are
oxidases and laccases, both catalyzing the oxidation of various substrates by
dioxygen, and peroxidases, catalyzing oxidations by hydrogen peroxide.
Catalases are a special type, catalyzing the disproportionation reaction 2H2O2
→ O2 + 2H2O, whereby hydrogen peroxide is both oxidized
and reduced at the same time. (Alexopoulus et
al., 1996)
Transferases:
Enzymes in this class catalyze the transfer of groups of atoms from one
molecule to another or from one position in a molecule to other positions in
the same molecule. Common types are acyltransferases and glycosyltransferases.
CGTase (cyclodextrin glycosyltransferase) is one such enzyme type, which moves
glucose residues within polysaccharide chains in a reaction that forms cyclic
glucose oligomers (cyclodextrins).
Hydrolases:
Hydrolases catalyze hydrolysis, the cleavage of substrates by water. The
reactions include the cleavage of peptide bonds in proteins by proteases,
glycosidic bonds in carbohydrates by a variety of carbohydrases, and ester
bonds in lipids by lipases. In general, larger molecules are broken down to smaller
fragments by hydrolases (Alexopoulus et
al., 1996)
Lyases:Lyases
catalyze the addition of groups to double bonds or the formation of double
bonds though the removal of groups. Thus bonds are cleaved by a mechanism
different from hydrolysis. Pectate lyases, for example, split the glycosidic
linkages in pectin in an elimination reaction leaving a glucuronic acid residue
with a double bond.
Isomerases:
Isomerases catalyze rearrangements of atoms within the same molecule; e.g.,
glucose isomerase will convert glucose to fructose.
Ligases:
Ligases join molecules together with covalent bonds in biosynthetic reactions.
Such reactions require the input of energy by the concurrent hydrolysis of a
diphosphate bond in ATP, a fact which makes this kind of enzyme difficult to
apply commercially.
1.2 industrial
applications of enzymes
The
catalytic ability of enzymes has made it of utmost importance in various
industry such as food industries, pharmaceutical industries, Health sector,
agricultural sectors, clothing and textile etc. Enzymes found in nature have
been used since ancient times in the production of food products, such as
cheese, sourdough, beer, wine and vinegar, and in the manufacture of
commodities such as leather, indigo and linen and processes relied on either
enzymes produced by spontaneously growing microorganisms or enzymes present in
added preparations such as calves’ rumen or papaya fruit although the enzymes
were, accordingly, not used in any pure or well-characterized form.
In the last century, several developments has made it
possible to manufacture enzymes in purified and well-characterized preparations
and even on a large scale. This developments allowed the introduction of
enzymes into true industrial products and processes, for example, within the
detergent, textile and starch industries. The use of recombinant gene
technology also has further improved manufacturing processes and enabled the
commercialization of enzymes that could previously not be produced.
Latest developments within modern biotechnology
i.e. the introduction of protein engineering and directed evolution, have
further revolutionized the development of industrial enzymes. These advances
have made it possible to provide tailor-made enzymes displaying new activities
and adapted to new process conditions, enabling a further expansion of their
industrial use.
Wine manufacturing:
Much of the early interest in enzymology was developed by scientists like
Pasteur, Payen and Persoz, who were associated with food, wine, and beer
industries. Pasteur was perhaps best known to the French nation as the “saviour
of the wine industry” because his pasteurization process salvaged an ailing
industry beset with problems of microbial contamination. Papain is used in
brewing industry as a stabilizer for chill-proof beer, because it removes small
amounts of protein that cause turbidity in chilled beer (Shafiq et al., 2002).
Cheese making:
Since long the animal rennin (or rennet) is employed in making cheese. The
enzyme rennet is obtained on a commercial scale from the fourth or true stomach
of the unweaned calves which are specifically slaughtered for this purpose. One
calf produces only 5 to 10 gm. of rennet. The enzyme helps in coagulating the
casein of milk. Certain preservatives (boric acid, benzoic acid or sodium
chloride) are, sometimes, added to prevent decomposition of the enzyme
preparations by bacteria. An enzyme lipase is added to cheese for imparting
flavor to it. Many vegetarians are unaware that the cheese made in India
contains animal rennet. However, an international charitable trust concerned
with the welfare of animals, the Beauty Without Cruelty (BWC) has, with the
help of Aurey Dairy, Mumbai, undertaken successful experimental trials in
cheese making using non animal rennet .(Stanieret al.,
1970).
Candy making:
An enzyme, invertase helps preventing granulation of sugars in soft-centred
candies. Another enzyme, lactase prevents formation of lactose crystals in ice
cream which would otherwise not allow the product seem sandy in texture.
Bread whitening:
Lipoxygenase is used for whitening the bread.
Clarifying fruit-juices:
The enzymes are being used in processing of fruit juices such as apple juice
and grape juice. The juices are clarified by adding a mixture of pectic enzymes
which hydrolyze the pectic substances causing turbidity.
Tenderizing meat:
Because hydroxyprolyl residues create bends in collagen helices, which
contribute to the tough and rubbery texture often associated with cooked meat,
treating the meat with a protease (bromelain or papain) prior to its cooking
hydrolyzes peptide bonds, and thus tenderizes it. (Schell et al., 2002)
Desizing fabrics:
The woven fabrics are sized by applying starch to the warp (lengthwise) threads
to strengthen the yarn before weaving. But when these fabrics are printed or
dyed, the sizing should be removed. Desizing may be done by acids, alkalis’ or
enzymes. Enzymatic desizing is, however, preferred as it does not weaken the
fabrics. Enzymes for this purpose are obtained from a variety of sources including
bacteria, fungi and malt.
Destaining fabrics:
In dry-cleaning, the stains due to glue, gelatin or starch are removed by
employing certain enzymes, such as alcalase.
Dehairing hide:
In the manufacture of leather, the hide is made free from hair. This is done by
employing pancreatic enzymes which hydrolyze the proteins of the hair
follicles, thus freeing the hair so that it may be easily scraped off from the
hide.
Recovering silver:
Pepsin is used to digest gelatin in the process of recovering silver from
photographic films (Wang, 1998).
.Correcting digestion: When the enzymes
are present insufficiently in the body, certain digestive disorders come up.
These may be cured by supplying the lacking enzymes. Pepsin, papain and
amylases aid digestion in the stomach while pancreatic enzymes act in the
duodenum.
Wound healing:
Proteolytic enzymes from pig pancreas are used to alleviate skin diseases, bed
sores and sloughing wounds. These enzymes act by destroying proteolytic enzymes
of man, that prevent the healing of such wounds. The enzymes commonly used for
wound debridement are the proteases such as streptodornase, ficin and trypsin.
Analyzing biochemical: Certain
enzymes are used in clinical analysis. For example, uricase and urease are
employed in the determination of uric acid and urea respectively in
blood.Besides, sucrose and raffinose contents in sugar mixtures are determined
by polarimetrybefore and after treating the solutions with the enzymes, sucrase and melibiase.
Dissolving blood clot: The
enzyme urokinase, which is
manufactured from urine, is being used effectively in Japan in the treatment of
blood clot in brain, artery and other circulatory diseases. A team of Soviet
scientists led by Yevgeni Chazov, Director of the National Cardiological
Research Centre, Moscow, have, in 1982, developed an effective enzyme streptodekase, which can dissolve blood
clots in vessels. The new enzyme is particularly useful in preventing heart
attacks as clots are responsible for 9 out of 10 fatal cases of cardiac arrests
(Hansen, 1990).
Changing the blood type: Scientists
have successfully employed several types of specific enzymes in an epoch-making
experiment to freely change human blood types. They found that the composition
of polysaccharide on the surface of blood corpuscles determines each person’s
type of blood. Different kinds of sugar characteristics of each blood type form
on the surface of RBCs due to the function of a synthetic enzyme. If the sugar
is separated from the surface of RBCs by using a specific decomposition enzyme,
type A blood and type B blood can be reverted to type O, the prototype of the
two blood types. If this breakthrough can be put to practical use, it will
fulfil a long-cherished dream of doctors to administer blood transfusions
irrespective of the type of blood a patient has by merely changing the
patient’s blood type to match the blood available (Koshland, 1984).
Diagnosing hypertension: A
new method called radio immunoassay procedure for diagnosing cases of
hypertension has been developed by Bhabha Atomic Research Centre (BARC). In it,
the activity of renin, a proteolytic
enzyme secreted by the kidneys, is calculated indirectly by measuring
angiotensin-I which is formed by the action of renin. Renin acts as part of a
complex feedback mechanism for regulating blood volume and pressure (Ashokkumar
et al., 2001).
Augmenting surgery: A
technique using the enzyme trypsin as
an adjunct to cataract surgery has been developed. With older techniques, it
required an incision about 2.5 cm long in the white of the eye to remove the
clouded lens. Modern microsurgery has, however, reduced this cut to only 0.3
cm. But the new method involves a still smaller cut wide enough for a needle
0.025 cm wide. The hollow needle is used to inject a microscopic amount of
trypsin, a digestive enzyme secreted by the pancreas. Trypsin digests and
liquefies the semisolid interior of the lens without harming other parts of the
eye. Once the enzyme liquefies the lens—which takes from a few hours to
overnight—the lens is removed by suction through the same hollow needle. This
eliminates the necessity of intervention in the eye, the constant passing in
and out of the instruments and suturing. The lesser the tissue is wounded, the
quicker it recovers. This enzyme surgery for cataracts could be done as an
outpatient operation. The patients would come in one day to have the enzyme
injected and return the next day to have the cataract removed (Dahot and
Noomrio, 1996).
Breaking down chemicals: Recently,
in 1993, a group of scientists from the Netherlands led by Han G. Brunner have
found a tiny genetic defect that appears to predispose some men toward
aggression, impulsiveness and violence. The afflicted persons often react to
the mostmildly stressful occasions with aggressive outbursts, cursing or
assaulting the persons they deem a threat. The researchers have linked the
abnormal behaviours to mutations in the gene responsible for the body’s
production of monamine oxidase-a, an
enzyme critical for breaking down chemicals that allow brain cells to
communicate. It is proposed that lacking the enzyme, the brains of afflicted
men end up with excess deposits of potential signalling molecules like
serotonin, dopamine and noradrenaline. Those surplus neurotransmitters, in
turn, stimulate often hostile conduct. The erratic behaviour is due to point
mutation. The gene is on the X chromosome, which explains why only males, with
their single copy of the X chromosome, can suffer from the enzyme deficiency.
Women can serve as carriers of the genetic defect, but are themselves protected
from its symptoms by their possession of a second, good copy of the gene,
sitting on their second X chromosome. Although the number of persons afflicted
with this disease is not known but based on other types of hereditary
disorders, the researchers estimate that the illness is likely to be quite rare
in the general population, i.e., no
more than one in 100,000 people (Benkebliaet
al., 2007)
Destroying acids: Sprouts
are the natural health boosters. They are basically the young new plants and
are, in fact, the organic answer to simple natural health. Almost any edible
seed can be sprouted. Far from being the invention of food faddists, sprouting
dates back to 2939 B.C. in China. Sprouts are found in all shapes and colours
and it is best to choose these from legume plants. Sprouting greatly improves
the safety and nutritional quality of all pulses, seeds and grains. The enzymes
which go into action during sprouting not only neutralize trypsin-inhibiting
factors but also destroy harmful acids like phytic acid. Phytic acid, an
integral constituent of grains, tends to bind minerals, making them unavailable
to the body.
Syrup manufacturing:
An immobilized enzyme is one that is
physically entrapped or covalently-bonded by chemical means to an insoluble
matrix, e.g., glass beads,
polyacrylamide or cellulose. Immobilization of an enzyme often greatly enhances
its stability, which makes its prolonged catalytic life a valuable industrial
trait (Kotwal and Shankar, 2009). These days, immobilized glucoseisomerase is being successfully used in the
production of high-fructose corn syrup, esp.,
in the United States.
1.3Fungi as a
micro-organism.
Fungi are heterotrophic organisms belonging to the
eukaryotic kingdom. Occurring worldwide, there are some 70,000 species of fungi
including mushrooms, moulds, rusts, smuts, truffles, puffballs, morels and
yeasts (Alexopoulos et al., 1996).
About 80 000 to 120 000 species of fungi have been
described to date, although the total number of species is estimated at around
1.5 million (Papagianni, 2004). This would render fungi one of the
least-explored biodiversity resources of our planet. It is notoriously
difficult to delimit fungi as a group against other eukaryotes, and debates
over the inclusion or exclusion of certain groups have been going on for well
over a century. In recent years, the main arguments have been between
taxonomists striving towards a phylogenetic definition based especially on the
similarity of relevant DNA sequences, and others who take a biological approach
to the subject and regard fungi as organisms sharing all or many key ecological
or physiological characteristics-the ‘union of fungi’ (Moore-Landecker, 1996). Being
interested mainly in the way fungi function in nature and in the laboratory, we
take the latter approach and include several groups in this book which are now
known to have arisen independently of the monophyletic ‘true fungi’ (Eumycota)
and have been placed outside them in recent classification schemes
Based on their lifestyle, fungi may be circumscribed
by the following set of characteristics(Moore-Landecker,
1996):
Nutrition: Heterotrophic
(lacking photosynthesis), feeding by absorption rather than ingestion.
Vegetative state: On or in the
substratum, typically as a non-motile mycelium of hyphae showing internal
protoplasmic streaming. Motile reproductive states may occur (St-Germain and
Summerbell ,1996).
Cell wall: Typically present,
usually based on glucans and chitin, rarely on glucans and cellulose
(Oomycota).
Nuclear status: Eukaryotic, uni-
or multinucleate, the thallus being homo- or heterokaryotic, haploid,
dikaryotic or diploid, the latter usually of short duration (but exceptions are
known from several taxonomic groups).
Life cycle: Simple or, more
usually, complex.
Reproduction: The following
reproductive events may occur: sexual (i.e. nuclear fusion and meiosis) and/or
parasexual (i.e. involving nuclear fusion followed by gradual de-diploidization)
and/or asexual (i.e. purely mitotic nuclear division).
Propagules: These are
typically microscopically small spores produced in high numbers.
Motile spores are confined to certain groups.
Sporocarps: Microscopic or
macroscopic and showing characteristic shapes but only limited tissue
differentiation.
Habitat: Ubiquitous in
terrestrial and freshwater habitats, less so in the marine environment.
Ecology: Important
ecological roles as saprotrophs, mutualistic symbionts, parasites, or
hyperparasites.
Distribution: Cosmopolitan
1.3.1 How fungi reproduce
Fungi are capable of both sexual and asexual
reproduction.When a fungus reproduces sexually it forms adiploid zygote, as do
animals and plants. Unlike animalsand plants, all fungal nuclei except for the
zygote are haploid,and there are many haploid nuclei in the commoncytoplasm of
a fungal mycelium (Nguyenet al.,
2005). When fungi reproducesexually, hyphae of two genetically different mating
typescome together and fuse (Nakano et
al., 2000). In two of the three phyla offungi, the genetically different
nuclei that are associatedin a common cytoplasm after fusion do not combine
immediately.Instead, the two types of nuclei coexist formost of the life of the
fungus. A fungal hypha containingnuclei derived from two genetically distinct
individuals iscalled a heterokaryotic hypha. If all of the nuclei are
geneticallysimilar to one another, the hypha is said to behomokaryotic. If
there are two distinct nuclei withineach compartment of the hyphae, they are
dikaryotic. Ifeach compartment has only a single nucleus, it ismonokaryotic.
Dikaryotic hyphae have some of the geneticproperties of diploids, because both
genomes aretranscribed. These distinctions are important in understandingthe
life cycles of the individual groups (Kumar and Jam, 2003).
Cytoplasm in fungal hyphae normally flows
throughperforated septa or moves freely in their absence.
Reproductivestructures are an important exception to this generalpattern. When
reproductive structures form, they arecut off by complete septa that lack
perforations or haveperforations that soon become blocked. Three kinds of
reproductivestructures occur in fungi:
Sporangia:
whichare involved in the formation of spores
Gametangia:structures
within which gametes form; and
Conidiophores:structures
that produce conidia, multinucleateasexual spores.Spores are a common means of
reproduction amongfungi. They may form as a result of either asexual or
sexualprocesses and are always non-motile, being dispersed bywind. When spores
land in a suitable place, they germinate,giving rise to a new fungal hypha.
Because the spores arevery small, they can remain suspended in the air for
longperiods of time. Because of this, fungal spores may beblown great distances
from their place of origin, a factor inthe extremely wide distributions of many
kinds of fungi.Unfortunately, many of the fungi that cause diseases inplants
and animals are spread rapidly and widely by suchmeans. The spores of other
fungi are routinely dispersed byinsects and other small animals. (Alexopoulos et al., 1996).
1.3.2 Growth and
physiology of fungi
The growth of fungi as hyphae on or
in solid substrates or as single cells in aquatic environments is adapted for
the efficient extraction of nutrients, because these growth forms have high surface area
to volume ratios (Hayden and Maude
1994). Hyphae are specifically adapted for growth on solid surfaces, and
to invade substrates
and tissues.They can exert large penetrative mechanical forces; for example,
the plant
pathogenMagnaporthe grisea
forms a structure called an appressorium
that evolved to puncture plant tissues. The pressure generated by the
appressorium, directed against the plant epidermis,
can exceed 8 megapascals
(1,200 psi) (Mammaet
al.,2008). The filamentous fungus Paecilomyces
lilacinus uses a similar structure to penetrate the eggs
of nematodes.
The mechanical pressure exerted by
the appressorium is generated from physiological processes that increase
intracellular turgor by
producing osmolytes
such as glycerol
(Tafintaet al., 2013).Adaptations
such as these are complemented by hydrolytic enzymes
secreted into the environment to digest large organic molecules—such as polysaccharides,
proteins,
and lipids—into
smaller molecules that may then be absorbed as nutrients (Pereiraet al.,2007; Schalleret al.,2007). The vast majority
of filamentous fungi grow in a polar fashion—i.e., by extension into one
direction—by elongation at the tip (apex) of the hypha. Other forms of fungal
growth include intercalary extension (longitudinal expansion of hyphal
compartments that are below the apex) as in the case of some endophytic
fungi(Hayden and Maude,
1994)or growth by volume expansion during the development of mushroom stipes
and other large organs. Growth of fungi as multicellular structures
consisting of somatic
and reproductive cells—a feature independently evolved in animals and
plants—has several functions, including the development of fruit bodies for
dissemination of sexual spores (see above) and biofilms for
substrate colonization and intercellular
communication (Collier et al.,
1998).
The fungi are traditionally considered heterotrophs,
organisms that rely solely on carbon fixed
by other organisms for metabolism(Vaija and
Linko, 1986).
Fungi have evolved
a high degree of metabolic versatility that allows them to use a diverse range
of organic substrates for growth, including simple compounds such as nitrate,
ammonia,
acetate,
or ethanol(Marzlufet al., 1981;
Heyneset al., 1994). In some
species the pigment melanin may play
a role in extracting energy from ionizing radiation,
such as gamma
radiation. This form of "radiotrophic"
growth has been described for only a few species, the effects on growth rates
are small, and the underlying biophysical
and biochemical processes are not well known. This process might bear
similarity to CO2 fixation
via visible light,
but instead using ionizing radiation as a source of energy (Hayden and Maude, 1994).
1.4. Production of
enzyme by fermentation technologies
Fermentation technology is a field which involves the
use of microorganisms and enzymes for production of compounds which have
application in the energy, material, pharmaceutical, chemical and the food
industry. Though fermentation processes are used for generations for the
requirement for sustainable production of materials and energy is demanding
creation and advancement of novel fermentation processes. Efforts are directed
both to the advancement of cell factories and enzymes as well as of design of
new processes, concepts and technologies for fermentation process. Through
fermentation, we can produce enzymes for industrial purposes. Process of
Fermentation includes the use of microorganisms, like yeast and bacteria for
the production of enzymes. Mainly, there are two methods of fermentation which
are used to produce enzymes. First is submerged fermentation and second is
solid-state fermentation.
1.4.1.
Solid-State Fermentation (SSF)
SSF utilizes solid
substrates, like bran, bagasse, and paper pulp(Arandaet al., 2006). The main advantage of using these substrates is that
nutrient-rich waste materials can be easily recycled as
substrates(Gutierrez-Correaet al., 1999).
In this fermentation technique, the substrates are utilized very slowly and
steadily, so the same substrate can be used for long fermentation periods.
Hence, this technique supports controlled release of nutrients. SSF is best
suited for fermentation techniques involving fungi and microorganisms that
require less moisture content (Kubicek and Röhr, 1989). However, it cannot be
used in fermentation processes involving organisms that require high Aw (water
activity), such as bacteria. (Ashokkumar et
al., 2001)
1.4.2.
Submerged Fermentation (SmF)/Liquid Fermentation (LF)
SmF utilizes free flowing
liquid substrates, such as molasses and broths. The bioactive compounds are
secreted into the fermentation broth. The substrates are utilized quite
rapidly; hence need to be constantly replaced/supplemented with nutrients. This
fermentation technique is best suited for microorganisms such as bacteria that
require high moisture content(Leangon
and Maddox, 2000). An additional advantage of this technique is that
purification of products is easier. SmF is primarily used in the extraction of
secondary metabolites that need to be used in liquid form (Ashokkumar et al., 2001).
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