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Abstract:
Feathers are one of the epidermal growths that form the distinctive outer covering
on birds. Feathers aid in flight, thermal insulation, water proofing and coloration that helps in
communication and protection. Feathers are produced as waste of poultry processing plants in
large quantities, millions of tons per year worldwide. Poultry feathers constitutes the most
abundant keratinous material in nature. The main component of feathers is Keratin, a
mechanically durable and chemically unreactive and insoluble protein, which renders it difficult
to digest by most proteolytic enzymes. Due to the insoluble nature of keratin, it is resistant to
enzymatic digestion by plant, animals and many known microbial proteases. Therefore, the
keratinase producing microorganisms have been described having the ability to degrade insoluble
keratin in feathers. Recycling of feathers is a subject of interest because it is a potentially cheap
and alternative protein supplement to be used in animal feed.
In the present study, we isolated keratinase producing fungi from the poultry waste
(decaying feathers) collected from tirupati urban waste dumps. We have screened 5 types of fungi
which showed keratinase activity which were isolated by serial dillution method. Their keratinase
activity was observed spectrophotometrically at 450,595, and 280 nm by the modified methods
of Yamamura et al.,yu et al.and,gradisar et al respectively among which three types of fungi
showed more keratinase activity. By the staining of fungi with lactophenol plus cotton blue and
microscopical observation, the studied fungi were primarily identified as Aspergillus sp. rhizopus
sp,mucor sp,. The study suggests that keratin degrading microbes are ubiquitous and need to
isolate high keratinase activity producing fungi for their large scale production in order to exploit
their activity.
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1. INTRODUCTION
Insoluble and hard-to-degrade animal proteins are ubiquitously present throughout
animal bodies. Enormous numbers of these proteins are generated in the meat industry in a mixture
of bones, organs and hard tissues, finally being converted to industrial wastes, the disposal of which
is tremendously difficult. World-wide poultry processing plants are producing millions of tons of
feathers as waste products annually, which consist of approximately 90% of keratin. Feathers
represent 5-7% of the total weight of mature chickens. These feathers constitute a sizable waste
disposal problem. Around 24 billion chickens are being killed per year across the world which is
discarding four billion pound (18, 14,369 tonnes) of poultry feather, according to Times of India.
Feather is generated in bulk quantities as a by-product of poultry industry. It is estimated that 400
million chickens are processed every week. Typically as each bird has up to 125gms of feather, the
weekly worldwide production of feather waste is about 3000 tons. Piling up of these waste materials
results in the accumulation of dumps. Disposal of this bulk waste is a global environmental problem
accounting to pollution of land and underground water resources. Thus, feather in spite of being
made up of almost pure keratin protein is neither profitable nor environmentally friendly forming a
production of high volume with low profit margin (Mc Govern, 2000).
Several different approaches are being used for the disposal of feather waste,
including land filling, burning, natural gas production and treatment for animal feed. Feathers
hydrolyzed by mechanical or chemical treatment can be converted to feedstuffs, fertilizers, glues and
foils or used for the production of amino acids and peptides. Most animal proteins (feathers) are
currently disposed of by incineration. This method, however, has ecological disadvantages in terms
of an apparent energy loss and the production of a large amount of carbon dioxide. Thus, an
innovative solution to these problems is urgently needed (Suzuki et al., 2006). An alternative to
decrease this pollution is the utilization of feather constitutes that can be used as animal feed,
preventing accumulation in the environment and the development of some types of pathogens.
Traditional ways to degrade feathers such as alkali hydrolysis and steam pressure cooking may not
only destroy the amino acids (Methionine, Lysine, and Histidine) but also consume large amounts of
energy. Utilizing poultry feathers as a fermentation substrate in conjunction with keratin-degrading
microorganisms or enzymatic biodegradation may be a better alternative to improve nutritional value
of poultry feathers and reduce environmental waste.
Currently a minor quantity of waste feathers is used in other industrial applications such as
clothing, insulation and bedding, producing biodegradable polymers and enzymes and also as a
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medium for culturing microbes. A higher quantity of pretreated feather is utilized to produce a
digestible dietary protein feedstuff for poultry and livestock. However, to decrease the risk of disease
transmission via feed and food chain legislation on the recovery of organic materials for animal feed
is becoming tighter (Commission of the European Communities, 2000).
Pretreatment methods for hydrolysis of poultry feathers
Because of the complex, rigid and fibrous structure of keratin, poultry feather is a challenge to
anaerobic digestion. It’s poorly degradable under anaerobic conditions. However, application of
appropriate pretreatment methods hydrolyzes feather and breaks down its tough structure to
corresponding amino acids and small peptides. For more than half a century many studies have been
performed and various pretreatment methods have been applied to improve the digestibility of
feather meal, dietary animal protein feedstuff and feather biogas potential. Feather meal treatment
methods are usually categorized into two groups:
1. Hydrothermal pretreatment
2. Microbial keratinolysis.
Hydrothermal pretreatment
Hydrothermal pre-treatment includes thermo-chemical treatment methods (such as acidic
hydrolysis and alkali hydrolysis), and also steam pressure cooking. These methods usually need high
temperatures or high pressure with addition of diluted acids such as hydrochloric acid (HCl) or
alkalis such as sodium hydroxide (NaOH). Acidic solutions promote the loss of some amino acids
such as tryptophan etc. Alkaline reactions are slow and degradation of some amino acids with
hydroxide is less. Hence the use of bases is recommended. A stepwise diagram for the hydrolysis of
protein rich material under alkaline condition is indicated in the figure-1 below.
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Fig1: Protein hydrolysis during thermo-chemical treatment.
As a whole, hydrothermal hydrolysis usually consumes high amount of energy and employs
expensive equipment during its lengthy processes (8 to 12 hrs). Thus, optimization of the treatment
conditions is an important issue from technological and economical points of view when applying
this method.
Biological pretreatment
Biodegradation of feathers is another alternative method. Some fungal strains can produce
keratinase proteases which have keratinolytic activity and are capable to keratinolyse feather α-
keratin. These enzymes help the fungi to obtain carbon, sulfur and energy for their growth and
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PROTEIN
α – keratin (hair), β – keratin, animal tissue, plant matter.
HYDROLYSIS
Peptide bond is broken. Smaller peptides and free amino acids are generated.
DEAMIDATION
GLN and ASN residue in protein react and form GLU and ASP residues, with ammonia as a
product.
SMALLER PEPTIDES & FREE AMINO ACIDS
Smaller peptides with a higher digestibility (structure) and free amino acids are dissolved in the liquid phase.
DEGRADATION
Several amino acids are not stable under alkaline conditions and undergo reactions
that generate different products (e.g. other amino acids, ammonia)
maintenance from the degradation of α -keratin. Various keratinases from different microorganisms
such as Aspergillus spp, Fusarium spp, Alternaria spp., Curvularia spp., Rhizopus spp.,
Trichoderma spp., Penicillium spp and Mucor spp has been isolated and studied to date. Microbial
proteases are classified into acidic, neutral, or alkaline groups, depends on the conditions required
for their activity and on the characteristics of the active site group of the enzyme, i.e. metallo-,
aspartic- , cysteine- or sulphydryl- or serine-type. Alkaline proteases which are active in a neutral
to alkaline pH, especially serine-types are the most important group of enzymes used in protein
hydrolysis, waste treatment and many other industrial applications. Alkaline protease from Bacillus
subtilis was used for the keratinolysis of waste feathers. Subtilisins are extracellular alkaline serine
proteases, which catalyse the hydrolysis of proteins and peptide amides. Savinase is one of these
enzymes; Alcalase, Esperase and Maxatase are others. These enzymes are all produced using species
of Bacillus. Maxatase and Alcalase come from B. licheniformis, Esperase from an alkalophilic strain
of a B. licheniformis, and Savinase from an alkalophilic strain of B. amyloliquefaciens .
An important advantage of enzyme treatment method is fully biodegradability of enzymes by
themselves as proteins. Hence, unlike other remediation methods, there is no buildup of unrecovered
enzymes or chemicals that must be removed from the system at the end of degradation process.
Although enzymatic treatment is a promising technology; it has some limitations and disadvantages,
as well. Currently, the main disadvantage of using alkaline proteases is the high cost of the enzymes
production. Much of the cost of producing enzymes is related to high purification of enzymes
solutions to avoid the side effects and side activities of the crude enzyme solution which is cheaper.
Furthermore, in contrast with microbes which can reproduce themselves and increase their
population to be able to consume a large quantity of substrate and survive in harsh environments,
extracellular enzymes like alkaline protease do not have reproducibility. Namely, increasing the
enzyme population must be done through adding new enzymes from outside into the system. On the
other hand, these alkaline proteases lose some reactivity after they interact with pollutants and could
eventually become completely inactive. Hence they do not have the adaptability to the harsh
environment even though they can survive in a wide range of environmental conditions. This means
that the enzyme concentrations must be monitored and controlled during the process in order to
optimize enzyme kinetics for site-specific conditions.
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Chemical-Biological pretreatment
Keratins are insoluble macromolecule comprises super coiled long polypeptide chains with
high degree of cross linked disulphide bonds between contiguous chains. According to the literatures
disulfide bonds in keratin significantly decrease protein digestibility and for complete easy
degradation of feather all enzymatic keratinolysis from any organism essentially needs to be assisted
by a suitable redox. Therefore, it has been suggested that some reductants, such as thioglycollate,
copper sulphate, ammonia and sodium sulphite and others, might cleave the disulfide bonds in
keratin and allows the proteases to have access to their peptide bond substrates, and consequently
improve the degradability of feathers. For instance Ramnani et al., 2007 found that savinase is
capable of near complete feather degradation (up to 96%) in the presence of sodium sulfite.
Keratin refers to a family of fibrous structural proteins. Keratin is the key of structural
material making up the birds feather. It is also the key structural component of hair and nails.
Keratin monomers assemble into bundles to form intermediate filaments, which are tough and
insoluble and form strong unmineralized tissues found in reptiles, birds, amphibians, and mammals.
The only other biological matter known to approximate the toughness of keratinized tissue is chitin.
Keratin filaments are abundant in keratinocytes in the cornified layer of the epidermis; these are cells
which have undergone keratinization. There two types of keratin α-keratin and β-keratin. The α-
keratins are present in the hair (including wool), horns, nails, claws and hooves of mammals. The
harder β-keratins found in nails and in the scales and claws of reptiles, their shells (Testudines, such
as tortoise, turtle, terrapin), and in the feathers, beaks, claws of birds and quills of porcupines. The
usefulness of keratins depends on their supermolecular aggregation. These depend on the properties
of the individual polypeptide strands, which depend in turn on their amino acid composition and
sequence. The α-helix and β-sheet motifs, and disulfide bridges, are crucial to the conformations of
globular, functional proteins like enzymes, many of which operate semi-independently, but they take
on a completely dominant role in the architecture and aggregation of keratins. The alpha keratin
helix is not a true alpha helix, as it only has 3.5 residues/turn, where the normal alpha helix has
3.6residues/turn. This is important for the different helices to form tight disulfide bonds. Also,
roughly every seventh residue is a leucine, so they can line up and help the strands stick together
through hydrophobic interactions.
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Structural details
Fibrous keratin molecules super coil to form a very stable, left-handed super helical motif to
multimerise, forming filaments consisting of multiple copies of the keratin monomer. A
preponderance of amino acids with small, nonreactive side groups is characteristic for structural
proteins, for which H-bonded close packing is more important than chemical specificity. In addition
to intra and intermolecular hydrogen bonds, keratins have large amounts of the sulfur-containing
amino acid cysteine, required for the disulfide bridges that confer additional strength and rigidity by
permanent, thermally-stable cross linking—a role sulfur bridges also play in vulcanized rubber.
Chicken feathers are composed of over 90% of keratin protein, small amounts of lipids and water.
Feathers keratin consists of high quantities of small and essential amino acid residues such as glycyl,
alanyl and seryl as well as cysteinyl and valyl. Keratin is also the main protein components of hair,
wool, nails, horn, and hoofs. Animal hair, hoofs, horns and wool contain β -keratin, and bird’s
feather contains α-keratin. The polypeptides in α-keratin are closely associated pairs of β helices,
whereas α-keratin has high proportion of β pleated sheets. This conformation confers an axial
distance between adjacent residues of 0.35 nm in β -sheets, compared to 0.15 nm in α-helices. The β
sheets have a far more extended conformation than the α–helices. Keratins are insoluble
macromolecule comprises a tight packing of super coiled long polypeptide chains with a molecular
weight of approximately 10 kDa. High degree of cross linked cysteine disulphide bonds between
contiguous chains in keratinous material imparts high stability and resistance to degradation.
Fig 2: Structure of keratin protein
Hence, a keratinous material is a tough, fibrous matrix being mechanically firm, chemically
unreactive, water insoluble and protease-resistant. Such a molecular structure makes feathers poorly
degradable under anaerobic digestion condition. Human hair is approximately 14% cysteine. The
pungent smells of burning hair and rubber are due to the sulfur compounds formed. Extensive
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disulfide bonding contributes to the insolubility of keratins, except in dissociating or reducing
agents. The more flexible and elastic keratins of hair have fewer inter chain disulfide bridges than
the keratins in mammalian fingernails, hooves and claws (homologous structures), which are harder
and more like their analogs in other vertebrate classes. Hair and other α-keratins consist of α-
helically-coiled single protein strands (with regular intra-chain H-bonding), which are then further
twisted into super helical ropes that may be further coiled. The β-keratins of reptiles and birds have
β-pleated sheets twisted together, then stabilized and hardened by disulfide bridges. Keratin
filaments are intermediate filaments. Like all intermediate filaments, keratin proteins form
filamentous polymers in a series of assembly steps beginning with dimerization; dimers assemble
into tetramers and octamers and eventually, the current hypothesis holds, into unit-length-filaments
(ULF) capable of annealing end-to-end into long filaments.
Keratin is an insoluble protein and is resistant to degradation by common
peptidases, such as trypsin, pepsin, and papain due to the constituent amino acid composition and
configuration that provide structural rigidity. The mechanical stability of keratin and its resistance to
biochemical degradation depends on the tightly packed protein chains in α-helix (α-keratin) and β-
sheet (β-keratin) structures. In addition, these structures are cross-linked by disulfide bridges in
cysteine residues. Keratinases are produced only in the presence of keratin containing substrate. It
mainly attacks on the disulfide (-S-S-) bond of the keratin substrate (Bockel et al., 1995). It was
found that keratinase produce by fungi were produced in nearly at alkaline pH and almost
thermophilic temperatures.
Keratins proteolysis like the other proteins is effectively directed by proteases. Nevertheless,
keratinases are known to have an effect on their hydrolysis. Keratinases have already been purified
from several microorganisms such as fungal species. Keratinase belongs to a group of proteinase
enzymes that have high level of activity on insoluble keratin, playing a crucial role in hydrolyzing
feather, hair, wool, collagen and casein in removing barriers in waste water treatment systems. Not
only have these enzymes been applied in sewage systems but have also recently emerged in many
applications including food, textile, medicine, and cosmetics industries. In fact, using of keratinases
in skin medications to get rid of acne and psoriasis as well as removing of human callus in medical
applications is well known. It is also utilized for the erection of a vaccine for dermatophytosis
therapy. More interestingly, keratinases are well identified in leather industry to have been employed
in dehairing process of animal skins instead of treating them with sodium sulfide. The majority of
known keratinases are endopeptidases belonging to the serine protease family.
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Sources of microbial keratinases: Diversity among keratinase-producing microorganisms
Keratinases are very widespread in the microbial world and they can be identified from
microorganisms of the three domains: Eucarya, Bacteria, and Archaea. These microorganisms have
been isolated from the most distinct sites, from Antarctic soils to hot springs, including aerobic and
anaerobic environments. Therefore, microbial keratinases present a great diversity in their
biochemical and biophysical properties. The characteristics of some microbial keratinases are
summarized in Table 1.
Table 1: Diversity of keratinolytic microorganisms and some biochemical properties of their keratinases, Microorganism
Catalytic type Molecular Optimal Optimal Reference mass (kDa) pH T (°C)
In natural environments, keratinolytic fungi are involved in recycling the carbon, nitrogen,
and sulfur of the keratins. Their presence and distribution seem to depend on keratin availability,
especially where humans and animals exert strong selective pressure on the environment. A number
of studies focused on the keratinolytic potential of dermathophytic fungi such as Trichophyton and
Microsporum, mainly due to their medical and veterinary implications. Although some studies on
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the biotechnological potential of such genera are available, little commercial interest attracted this
group because of their potential pathogenicity. Among nondermatophytic fungi, keratinases showing
attractive biochemical properties were reported to be produced by Aspergillus, Trichoderma,
Doratomyces, Myrothecium, Paecilomyces, Scopulariopsis, and also Acremonium, Alternaria,
Beauveria, Curvularia, and Penicillium. Besides the biotechnological interest, these investigations
may help in understanding the role of fungi in the degradation of complex keratinous substrates in
the nature. Several keratinases have been isolated from a diversity of bacteria. Bacillus spp. appears
as the prominent keratinase producer. Diverse strains of Bacillus licheniformis and Bacillus subtilis
are described as keratinolytic but other species such as Bacillus pumilus and Bacillus cereus also
produce keratinases. Furthermore, B. licheniformis is the source of Versazyme™, the first thermo-
resistant commercial keratinase developed by Shih and coworkers at Bioresource International, Inc.
Some thermophilic and alkaliphilic strains of Bacillus have also been described to show keratin-
degrading activity, such as Bacillus halodurans AH-101, Bacillis pseudofirmus AL-89, and B.
pseudofirmus FA30-01. Besides, microorganisms belonging to the same genus (Bacillus) can
produce different keratinases (Table 2.4). Keratinase producers have been also described among
actinomycetes, mainly from the Streptomyces genus. These microorganisms, isolated from several
different soil sites, are associated with the hydrolysis of a wide range of keratinous substrates like
hair, wool, and feathers. For example, two highly keratinolytic actinomycetes strains, Streptomyces
flavis 2BG (mesophilic) and Microbispora aerata IMBAS-11A (thermophilic), were isolated from
Antarctic soil. The thermophilic species Streptomyces gulbarguensis , Streptomyces
thermoviolaceus, and Streptomyces thermonitrificans have also been isolated from soils. Besides
these thermophilic strains, some mesophilic Streptomyces have also been characterized like
Streptomyces pactum DSM 40530, Streptomyces graminofaciens and Streptomyces albidoflavus . In
addition to these Bacillus sp. and actinomycetes, keratinase production has been associated to an
increasing number of bacteria. Since keratin degradation is facilitated at high temperatures and pH,
and thermostable hydrolases are employed in various industrial processes, the thermophilic and
alkaliphilic microorganisms are of great interest. Fervidobacterium pennavorans, Fervidobacterium
islandicum, Meiothermus ruber H328, Clostridium sporogenes, and strains of Thermoanaerobacter
spp. were isolated from extreme environments like hot springs, geothermal vents, solfataric muds,
and volcanic areas. Some alkaliphilic strains such as Nesternkonia spp. and Nocardiopsis spp. TOA-
1 have been also characterized, showing keratinase activity in strongly alkaline pH.
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Production of keratinases
The biotechnological application of keratinolytic proteases requires the production of these
enzymes in sufficient amounts for commercial purposes. Keratinase production is usually induced by
keratin and, thus, a keratinous substrate (chicken feathers, feather meal, and hair) is often added to
the cultivation medium. Such keratin-rich materials are produced in high amounts by agro-industrial
activities and are normally discarded as wastes. Therefore, this microbial technology connects the
production of valuable products (keratinases, microbial biomass, protein hydrolysates) from low-
cost substrates with an alternative and efficient way of waste management. However, the addition of
a keratinous substrate is not always required for keratinase production. Other non-keratinous
substrates, such as soy flour, soybean meal, skim milk, shrimp shell powder, gelatin, casein, and
cheese whey, have been reported to act as inducers of keratinase production. Furthermore, in some
cases, keratinase production appears to be constitutive. Recently, peptide limitation in culture media
induced the sequential production of collagenase, elastase, and keratinase by B. cereus. The
keratinolytic activities produced on keratinous substrates, in comparison to readily assimilable
substrates, may result from nitrogen limitation rather than keratin induction. In this case, keratinous
substrates would act only as indirect inducers. Supplementation of keratin-containing media with
different carbon and/or nitrogen sources might result in higher levels of keratinase production. For
instance, the addition of glucose, sucrose, starch, molasses, and bagasses and additional nitrogen
sources, such as urea, peptone, tryptone, yeast extract, ammonium chloride, and sodium nitrate are
reported to enhance enzyme yields. Conversely, the addition of supplementary substrates
carbohydrates, inorganic and/or organic nitrogen sources often decreases enzyme production by
some microorganisms, mainly due to catabolite repression mechanisms. Therefore, the effect of
different growth substrates on keratinase production is highly variable, depending on the
microorganism, the substrate, carbon and nitrogen concentration, implicating that the medium
composition should be determined on a case-by-case basis. Besides the composition of the culture
medium, incubation temperature, pH, and aeration are among the important variables investigated in
view to obtain high keratinase yields. Maximum keratinase activities are usually achieved in the late
exponential or stationary growth phases. In this sense, keratinase production was observed to be
growth-associated in B. licheniformis FK 14; similar results were observed with Chryseobacterium
spp. kr6 and Streptomyces gulbargensis DAS 131. Nevertheless, Serratia spp. HPC 1383 showed the
highest proteolytic activity in the initial phase of growth (24 hrs) on feather meal medium, whereas
maximum biomass was achieved after 96 hrs. Development of mutant and recombinant microbial
strains is also investigated, representing useful techniques to enhance keratinase production and
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keratin degradation. In the specific case of the opportunistic pathogen P. aeruginosa, cloning and
heterologous expression of its keratinase gene also represents a viable alternative to ensure safety.
The gene kerA, which encodes B. licheniformis keratinase, is expressed specifically for feather
hydrolysis; therefore, the presence of feather keratin as the sole carbon and nitrogen source in the
culture medium may result in preferential expression of the keratinolytic protease. This gene has
been cloned and expressed in heterologous microorganisms such as Escherichia coli and B. subtilis,
but the keratinase yields are lower than the wild strain. However, increased keratinase yield was
achieved by chromosomal integration of multiple copies of the kerA gene in B. licheniformis and B.
subtilis. The kerA gene was also cloned for extracellular expression in P. pastoris, resulting in a
recombinant enzyme that was glycosylated and even though active on azo-keratin. The vast majority
of investigations report keratinase production through submerged cultivations. Only recently the
production of keratinolytic enzymes through solid-state processes has been demonstrated. The
potential of keratinase production by immobilized microorganisms was also reported.
Emerging Applications for Keratinases
Although biotechnological processes related with the hydrolysis of waste keratin was an
early proposition for microbial keratinases, other promising applications have been associated with
keratinolytic enzymes. With the advance in the knowledge about keratinases and their action on
keratinolysis, a myriad of novel applications have been suggested for these enzymes (Fig. 3).
Enzymatic dehairing is increasingly seen as a reliable alternative to avoid the problem created by
sulfide in tanneries (Cantera 2001; Thanikaivelan et al. 2004). The advantages of enzymatic
dehairing are a reduction of sulfide content in the effluent, recovery of hair which is of good quality,
and elimination of the bate in the deliming. However, this potential benefit remains unfulfilled as
enzymes are more expensive than the conventional process chemicals and require careful control
(Schraeder et al. 1998). The potential for the commercial use of enzymes in leather production is
considerable because of their properties as highly efficient and selective catalysts. The resulting
savings in process time may increase the efficiency in leather production, which represents added
value to the tanner. A significant feature of the enzymatic dehairing process is the complete hair
removal and minimal usage of sulfide and the decomposition products formed from the tannery
wastewater with great improvement in wastewater quality as a result. Thus, the substitution of
chemical depilatory agents in the leather industry by proteolytic enzymes produced by
microorganisms has an important economical and environmental impact. Some microorganisms
producing extracellular keratinases showing dehairing activity has been described, and among
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bacteria, strains of Bacillus are the most studied. Proteolytic strains of Bacillus subtilis and Bacillus
amyloliquefaciens have been characterized, presenting desirable properties for leather processing
(George et al. 1995; Varela et al. 1997; Macedo et al. 2005; Giongo et al. 2007). The fact that these
keratinases can degrade keratin avoiding damage of other structural proteins like collagen, make
them exceptional candidates for use in leather industry.
Some keratinases do not hydrolyze gelatin and synthetic substrate for collagenase, and their
use for dehairing bovine pelts cause no collagen damage (Gradisar et al. 2000; Riffel et al. 2003b).
These enzymes have attractive characteristics for cosmetic and pharmaceutical purposes where
collagen should not be attacked. The use of keratinase for cosmetic application is described as an
ingredient in depilatory compositions for shaving (Neena 1993; Slavtcheff et al. 2004). Keratinases
may be also useful in topical formulations for the elimination of keratin in acne or psoriasis and
removal of human callus (Holland 1993; Vignardet et al. 2001).
Fig 3- application of keratinase enzyme
Recently, the use of keratinolytic enzymes to enhance drug delivery was investigated. The
effectiveness of topical therapy of nail diseases is usually limited by the very low permeability of
drugs through the nail plate. The presence of keratinase from Paecilomyces marquandii significantly
increases drug permeation through human nail clippings (Mohorcic et al. 2007). Detergent
applications for keratinases have been also suggested (Gupta and Ramnani 2006). These include
removal of keratinous dirt that are often encountered in the laundry, such as collars of shirts, and
used as additives for cleaning up drains clogged with keratinous waste. Keratinolytic enzymes may
have potential application in the woolen textiles industry as a method of shrink proofing wool and to
improve wool dyeing (Sousa et al. 2007).
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Therefore, innovative solution for waste disposal along with
biotechnological alternative for recycling of such wastes is of utmost importance. An alternative and
attractive method for improving the digestibility of feathers or feather meal is biodegradation by
keratinolytic microorganisms. Biotechnological processing of feathers for the production of feather
meal, instead of chemical processing is preferred as it preserves the essential amino acids
(Methionine, Lysine, and Histidine) (Riffel et al., 2003) and it can also be used as animal feed, this
can prevent accumulation of feather in the environment and decrease the development of pathogenic
strains. Keratinase are specific protease that degrades keratin specifically. It is produced by
Saprophytic and Dermatophytic Fungi and some Bacillus species. Keratinolytic fungi are an
ecologically important group of fungi that cycle one of the most abundant and highly stable animal
proteins on earth – Keratin. Keratinolytic enzymes have found important utilities in biotechnological
processes involving keratin-containing wastes from poultry and leather industries, through the
development of non-polluting processes.
The aim of the study is to isolate, screen the keratinolytic fungi from poultry waste
feathers which has the capacity to degrade poultry waste feathers using poultry feather keratin as the
sole carbon source and to characterize their keratinolytic activity.
The objectives of the present study were as followed:
Isolation of keratinolytic fungi from the poultry feathers waste.
Screening and preparation of pure cultures of efficient keratinolytic fungi.
Isolation of keratinases from the isolated fungi.
Estimation of keratinase activity by different methods.
Identification of high keratinolytic fungi.
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2. REVIEW OF LITERATURE
Yasmeen Faiz Kazi, et al., (2012) has been conducted the study on comparative
characterization of indigenous keratinase enzymes from district Khairpur, Sindh, Pakistan. To isolate
and characterize keratinolytic fungi and bacteria from indigenous soils, a total of 80 samples were
collected and these organisms were isolated using standard microbiological technique. The isolated
keratinolytic microorganisms comprised: Absidia sp., Chrysosporium asperatum, Chrysosporium
keratinophilum, Entomophthora coronata, Bacillus subtilis and Staphylococcus aureus and their
keratinolytic properties were distinguished from the production of keratinase by measurement of
zone of hydrolysis on skimmed milk agar (P < 0.05). C. keratinophylum and B. subtilis produced
largest zone among all the isolated species. The crude keratinase revealed that the optimum time for
production of the enzyme was seven days, optimum temperature 30°C and optimum pH 9 for C.
keratinophylum but for B. subtilis, the optimum time was three days, optimum temperature 37°C and
optimum pH 7. The enzyme activity of C. keratinophylum and B. subtilis were determined to be 220
U/ml and 260 U/ml respectively (P< 0.05).
Prerna Awasthi * and R. K. S. Kushwaha (2011) has been reported about the Keratinase
Activity of Some Hyphomycetous Fungi from Dropped off Chicken Feathers. Among 101
keratinolytic hyphomycetous fungi, 13 species belonging to six genera examined for keratinase
activity in submerged cultures using chicken feather as keratinous substrate. The highest
keratinolytic activities were recorded in Acremonium brunnescens MTCC 10376, (65.73± ku/ml),
Chrysosporium indicum MTCC 10377 (63.5±2.47 ku/ml), Acremonium chrysogenum NFCCI 1883
(45.11±1.59 ku/ml), Acremonium byssoides MTCC 9985 (41.66±0.75 ku/ml), Scopulariopsis
stercoraria NFCCI 1885 (34.6±3.69 ku/ml) Chrysosporium tropicum NFCCI 1884 (24.69±2.11
ku/ml), Fusarium culmorum GPCK 3204 (22.91±0.86 ku/ml) and Alternaria alternata NFCCI 1878
(20.8±3.69). These noval nondermatophytic keratinolytic fungi have potential use in
biotechnological processes involving keratin hydrolysis. The result of this work contributes to show
that keratinolytic activity is relatively widespread among common hyphomycetous fungi.
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Ana Maria Mazotto, et al., (2011) employed that Keratinase Production by Three Bacillus
spp. using Feather Meal and Whole Feather as Substrate in a Submerged Fermentation Three
Bacillus species (B. subtilis LFB-FIOCRUZ 1270, B. subtilis LFB-FIOCRUZ 1273, and B.
licheniformis LFB-FIOCRUZ 1274), isolated from the poultry industry, were evaluated for
keratinase production using feathers or feather meal as the sole carbon and nitrogen sources in a
submerged fermentation. The three Bacillus spp. produced extracellular keratinases and peptidases
after 7 days. Feather meal was the best substrate for keratinase and peptidase production in B.
subtilis 1273, with 412 U/ml and 463 U/ml. The three strains were able to degrade feather meal (62–
75%) and feather (40–95%) producing 3.9–4.4mg/ml of soluble protein in feather meal medium and
1.9–3.3 mg/ml when feather medium was used. The three strains produced serine peptidases with
keratinase and gelatinase activity. B. subtilis 1273 was the strain which exhibited the highest
enzymatic activity.
Mukesh Sharma, et al., (2011) investigated about In vitro biodegradation of keratin by
dermatophytes and some soil keratinophiles. Ten fungal species, out of which, six (Chrysosporium
indicum, Trichophyton mentagrophytes, Scopulariopsis sp., Aspergillus terreus, Microsporum
gypseum and Fusarium oxysporum) were isolated from soil and four clinical (Trichophyton rubrum,
Trichophyton verrucosum, Trichophyton tonsurans and Microsporum fulvum) were obtained from
human skin. The isolates were tested for their keratin degradation ability on human and animal (cow
and buffalo) hair baits. The rate of keratin degradation was expressed as weight loss over three
weeks of incubation. Human hair had the highest rate of keratin degradation (56.66%) by
colonization of C. indicum. whereas M. gypseum and T. verrucosum were highly degraded (49.34%)
to animal hairs. There was a significant difference (p < 0.05) in keratin substrate degradation rates by
the examined fungi. Human hair served as an excellent source for the biodegradation of keratin by
the isolated test fungi as compared to animal hair. This study reveals that, the isolated test fungi play
a significant impact on biodegradation of keratin substrates for betterment of environmental hazards
Thanaa h. Ali et al., (2011) has been conducted the studies on production, purification and
some properties of extracellular keratinase from feathers-degradation by aspergillus oryzae nrrl-447-
Extracellular keratinase was produced during submerged aerobic cultivation in a medium containing
chicken feather for enzyme synthesis. The enzyme was partially purified by acetone fractionation
and DEAE- Sephadex A-25 column chromatography. A purification fold about 20.7 with a yield of
37.9% as determined with keratin as substrate of the activity in crude extracts. Specific activity of
this partially purified enzyme is 2312.7 U/mg. The km and Vmax values were 7.15 mM and
Page 16 of 49
300U/ml respectively. The optimal pH and temperature for keratinolytic activity was approximately
7.0 and 70°C respectively. Essential amino acids like threonine, valine, methionine, isoleucine,
leucine, lysine, histidine and tyrosine as well as ammonia were produced when feathers were used as
substrates. Exposures of purified keratinase in absence of substrate at 80°C, for 60 minutes caused
lose about 56% of its activity. This keratinase was inhibited in a variable rates by addition of EDTA,
CuSO4, ZnCl2 MnCl2 and HgCl2 at a concentration of 15mM, where as iodoacetate and 2-
mercaptoethanol slightly activation at the same concentration. Strain Aspergillus oryzae, therefore,
shows great promise of finding potential applications in keratin hydrolysis and keratinase
production.
T. Jayalakshmi, et al., (2011) has been conducted the studies on Purification and
characterization of Keratinase enzyme from Streptomyces species JRS 19. A keratinase producing
enzyme bacterial culture JRS 19 was isolated from Soil samples were collected from 5 different
districts of Tamil Nadu and in addition, soil samples were also collected from prawn shell
decomposing area at Chennai. It was related to Streptomyces sp. on the basis of biochemical
properties and Screening for sensitivity and resistant to antibiotics. Determination of cell wall amino
acid and cell wall sugar techniques applied to identify the chemotaxonamy of Actinomycetes. The
purification of keratinase present in the culture medium was grown in a fermenter containing
optimized production medium for eight days and showed an optimal activity at 4ºC for 15 minutes.
The concentrated crude enzymes were analyzed extracellular protein profile and Keratinase
hydrolytic activity using SDS-PAGE and Native-PAGE. Keratinase activity of each fraction was
determined Diethyl Amino Ethyl Cellulose (DEAE) column chromatography Sephadex G-100 gel
filtration column chromatography Polyacrylamide gel electrophoresis of the Keratinase was carried
out to determine the protein profile of the enzyme and make this keratinase extremely useful.
Krishna Rayudu, et al., (2011) has been described about Isolation, Identification and
Characterization of a Novel Feather- Degrading Bacillus Species Ker 17 Strain. In this study, they
were isolated around 23 bacterial strains and were characterized through morphological and
biochemical studies; all of them belonged to the genus Bacillus. Among them one isolate, KER 17
was capable of producing high clearance zone on Skimmed Milk Agar at pH 9.0 and temperature
370C for 24 to 72 h incubation. Keratinolytic ability of the same strain was confirmed on Feather
Meal Agar medium. Initial pH and temperature played a vital role apart from the incubation time.
The strain KER17 has shown its maximum keratin degradation at 72 h incubation in 1% Feather
Meal Broth. The total soluble concentration of the cell free supernatant was observed as 25.96
Page 17 of 49
mg/ml at 72 h incubation. Under in situ-feather degradation studies, the strain KER17 grown in basal
mineral medium along with 1% chicken feather as carbon, nitrogen and energy source and exhibited
maximum keratin degradation at 72h incubation. However, it has taken 120 h for the complete
degradation of the feather along with the shaft. The strain Bacillus species KER17 can be employed
in poultry waste (feathers) management.
Mukesh Sharma, et al., (2010) reported that In vitro biodegradation of keratin by
dermatophytes and some soil keratinophiles. In that 10 fungal species, out of which, six
(Chrysosporium indicum, Trichophyton mentagrophytes, Scopulariopsis sp., Aspergillus terreus,
Microsporum gypseum and Fusarium oxysporum) were isolated from soil and four clinical
(Trichophyton rubrum, Trichophyton verrucosum, Trichophyton tonsurans and Microsporum
fulvum) were obtained from human skin. The isolates were tested for their keratin
degradation ability on human and animal (cow and buffalo) hair baits. The rate of keratin
degradation was expressed as weight loss over three weeks of incubation. Human hair had the
highest rate of keratin degradation (56.66%) by colonization of C. indicum. whereas M.
gypseum and T. verrucosum were highly degraded (49.34%) to animal hairs. It was reveals that, the
isolated test fungi play a significant impact on biodegradation of keratin.
M. M. Aly and S. Tork (2010) has been reported that Biochemical and Molecular
Characterization of a new local Keratinase Producing Pseudomonas sp. MS21. This study aimed to
isolate and identify a new local bacterial strain, able to completely degrade keratin-rich wastes into
soluble and useful materials which can be used in many proposes. Out of 23 bacterial isolates, 7
isolates were selected. The best keratinase producing bacterium kera MS21 was selected and
identified based on morphological, physiological and some biochemical characteristics. It was
recorded as a species belonging to the genus Pseudomonas spp. The results of identification were
confirmed by 16S rDNA studies. The enzyme molecular weight was determined to be of 30 KDa
using sodium dodecyl sulfate Polyacrylamide gel electrophoresis analysis. The optimum temperature
and pH were determined to be37°C and pH 8.0, respectively.
C. Vigneshwaran, et al., (2010) has been screened and characterized the keratinase from
bacillus licheniformis isolated from namakkal poultry farm. Keratinase (EC 3.4.4.25) belongs to the
class hydrolase which are able to hydrolyze insoluble keratins more efficient than other proteases.
The bacteria Bacillus licheniformis showing higher keratinase activity was screened out of the ten
different bacterial strains isolated. The ability of Bacillus licheniformis to utilize chicken feather
powder as a substrate was tested. It was found that maximum enzyme activity was 10.76U/ml.
Page 18 of 49
Similarly optimum temperature and pH for the enzyme activity was found to be 60_C and 7.0
respectively. It was found from this study, organism such as Bacillus licheniformis isolated from
poultry soil can be used as a potential candidate for degradation of feather and for dehairing process
in leather industry.
Amany L. Kansoh, et al., (2009) entitled that Keratinase Production From Feathers Wastes
Using Some Local Streptomyces Isolates. From various soil samples isolate different Streptomyces
spp. Only, 21 of these isolates showed quantitative protease activity. Under restricted medium
conditions that contain feather as a sole carbon and nitrogen source, eight isolate showed
keratinolytic activities. In that 2 isolates showed high keratinase activities were identified on the
bases of the International Streptomyces Project (ISP) and were designated as S.albidus E4 and S.
griseoaurantiacus E5. Some cultural conditions were tested to attain maximum keratinase
production. Ammonium nitrate was a good nitrogen source for the production of keratinase by S.
albidus E4. Maximum enzyme production was reached on the 5th day of incubation of the shaking
culture at 30oC and pH 8.0 by Streptomyces spp. E4 and E5.
Fuhong Xie, et al., (2009) reported that the process of Purification and characterization of
four keratinases produced by Streptomyces spp. strain 16 in native human foot skin medium. Four
extracellular keratinases (designated KI, KII, KIII, and KIV) were produced during submerged
aerobic cultivation in a medium containing native human foot skin (NHFS) for enzyme synthesis.
The molecular weights, determined by SDS–PAGE, were 25, 50, 34, and 19 kDa, respectively. All
four keratinases exhibited high activities at pH 8.0– 10.0 with an optimal pH of 9.0. The optimal
temperature for keratinolytic activity of KI, KII, and KIII was approximately 50oC and 60oC for
KIV. 1mM
Cheng-gang C, et al., (2008) has been reported that a new feather-degrading bacterium was
isolated from a local feather waste site and identified as Bacillus subtilis based on morphological,
physiochemical, and phylogenetic characteristics. Screening for mutants with elevated keratinolytic
activity using N-methyl-N′-nitro-N-nitrosoguanidine mutagenesis resulted in a mutant strain KD-N2
producing keratinolytic activity about 2.5 times that of the wild-type strain. The mutant strain
produced inducible keratinase in different substrates of feathers, hair, wool and silk under
submerged cultivation. Scanning electron microscopy studies showed the degradation of feathers,
hair and silk by the keratinase. The optimal conditions for keratinase production include initial pH of
7.5, inoculum size of 2% (v/v), age of inoculums of 16 h, and cultivation at 23 °C. The maximum
Page 19 of 49
keratinolytic activity of KD-N2 was achieved after 30 h. Strain KD-N2, therefore, shows great
promise of finding potential applications in keratin hydrolysis and keratinase production.
Helena Gradisar, et al., (2005) has been studied that screening of keratinolytic
nonpathogenic fungi, Paecilomyces marquandii and Doratomyces microsporus were selected for
production of potent keratinases. Studies of substrate specificity revealed that skin constituents, such
as the stratum corneum, and appendages such as nail but not hair, feather, and wool were efficiently
hydrolyzed by the P. marquandii keratinase and about 40% less by the D. microsporus keratinase.
Kinetic studies were performed on a synthetic substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide.
The keratinase of P. marquandii exhibited the lowest Km among microbial keratinases reported in
the literature, and its catalytic efficiency was high in comparison to that of D. microsporus
keratinase and proteinase K.
J. A. Scott, et al., (2004) has been determined that Azure dye-impregnated sheep’s wool
keratin (keratin azure) was incorporated in a high pH medium and overlaid on a keratin-free basal
medium. The release and diffusion of the azure dye into the lower layer indicated production of
keratinase. Fifty-eight fungal taxa, including 49 members of the Arthrodermataceae, Gymnoascaceae
and Onygenaceae (Order Onygenales), were assessed for keratin degradation using this method. The
results were comparable to measures of keratin utilization reported in studies using tests based on the
perforation or erosion of human hair in vitro.
Rahul Sharma and R C Rajak, (2003) has been described Keratinophilic Fungi as Nature’s
Keratin Degrading Machines and their Isolation, Identification and Ecological Role. Keratinophilic
fungi are an ecologically important group of fungi that cycle one of the most abundant and highly
stable animal proteins on earth – keratin. This article briefly explains how to isolate and identify
them, the process of keratin degradation, and the ecological role of this important but unnoticed
group of minute keratin cycling machines present in soil. We believe that Indian soil contains many
more such fungi which have not been isolated and we hope this article will create interest among
students to isolate and study these interesting fungi.
Page 20 of 49
3.MATERIALS AND METHODS
3.1 Materials required
• Decaying feather samples
• Raw chicken feathers
• Glassware
• Mortar and pestle
3.2 Chemical requirements:
• Agar-Agar
• Potato dextrose Agar
• Analytical and Inorganic chemicals
a. Dimethylsulfoxide
b. Acetone
c. Sodium nitrite
d. Sulphanilic acid
e. Hydrochloric acid
f. Sodium hydroxide
• Distilled Water
3.3 Buffers
Phosphate buffer(50mM, pH 7.5)
Tris-acetate buffer(0.2,pH 7.0)
Tris buffer(0.1M, pH 8.0)
3.4Staining solutions
Lactophenol plus cotton blue
Crystal violet
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3.5 EQUIPMENT
Analytical equipment
S. No Equipment Manufacturer
1 UV-Vis Spectrophotometer ELICO., SL-159
2 Refrigerator centrifuge REMI(C84)
3 Lab. centrifuge REMI(R-8C)
4 Research microscope OLYMPUS(BX51)
5 Incubator HECO
6 Laminar air flow chamber CLEAN AIR
Table2: Equipments required for the complete process
3.6 Sample collection
The source for this study is the decaying feathers collected from poultry waste dumb yard,
situated at Vidyanagar, Tirupathi. Different samples (3) were collected, depending upon the rate of
feathers decayed in the soil. The samples (decaying feathers) were collected in dry, sterile, plastic
boxes, which were stored in room temperature till the isolation of fungi.
3.7 Isolation of fungal strains
3.7.1 Serial dilution
1. With a glass marker, label five sterile test tubes as 1 to 5 containing 9ml of distilled
water.
2. In separate sterile test tube dissolve 1gm of decaying feather samples in 1ml of
distilled water (mixed culture).
3. Inoculate saline tube 1 with 1 ml of the mixed culture using aseptic technique (see
figure- 3) and mix thoroughly. This represents a 10‾1dilution.
4. Using aseptic technique, immediately inoculate tube 2 with 1 ml from tube 1; a 10–2
dilution.
5. Using aseptic technique, mix the contents of tube 2 and use it to inoculate tube 3 with
1 ml; a 10–3 dilution.
Page 22 of 49
6. Same as follows for 4th and 5th tube respectively.
Fig4: Serial dilution up to 10-5 concentration
3.7.2 Culturing
1. Inoculation of samples is to be done on potato dextrose agar (PDA) of commercial
quality.
2. Potato dextrose agar (Himedia) is to be dissolved in 250 ml of distilled water and it
should be sterilized by autoclaving at 121 Wc for 15 minute at 15lb pressure before
serial dilution (21gm per litre).
3. The laminar air flow chamber is to be sterilized by ethanol wiping and UV-light for
10 minutes for the inoculation of samples.
4. With a glass marker, the bottoms of five petri plates are to be labeled as 1 to 5, their
name and date.
5. In the laminar air flow chamber after all serial dilutions, the respective sample is to
be poured in their respective petri plates i.e. 1 to 5 aseptically.
6. After cooling the medium in the laminar air flow chamber the contents of the melted
potato dextrose agar is to be added by pouring them in to the petri plates. Gentle
mixing of each agar plate in a circular motion is to be done while keeping the plate
on the bench top. Do not allow any agar to splash over the side of the plate! Set the
plate aside to cool and harden.
7. The replica’s of each petri plate is to be maintained.
8. All the plates are to be incubated at 37°C for 5 to 7 days in an inverted position in
order to prevent condensing moisture from accumulating on the agar surfaces after
solidification.
Page 23 of 49
Fig 5: Culturing of samples on potato dextrose medium by culture method.
3.7.3 Preparation of chicken feather powder
Chicken feathers was cut in to small fragments and washed extensively with distilled water
and detergent and dried in a ventilated oven at 40 WC for 72 hrs. To prepare feather powder, the
feathers were milled in ball mill and passed through a small mesh grid to remove coarse particles.
The feather powder was used during the optimization of keratinase production.
Fig-6 preparation of feather powder by cutting feathers in to small-small pieces
3.7.4 Feather agar media preparation
1. The medium is supplemented with 1% chicken feather powder (Wawrzkiewicz et al.,)
as sole source of carbon and nitrogen along with g/l 0.5 NaCl, 0.4KH2PO4, 0.5MgSO4, 1.0
K2HPO4, 20.0 Agar.
Page 24 of 49
2. Medium was sterilized by autoclaving and transferred to petri plates after cooling in
the laminar air flow chamber.
3.7.5 Inoculum preparation
Spore suspension of the selected fungal isolates was prepared by adding 10 ml of distilled
water to 7 days old fungal isolates growing on the plates of potato dextrose agar.
3.7.6 Inoculation in feather agar plates
Spore suspension of isolated fungi was streaked on feather agar plates by streak plate method
and incubated at 37 °C for 5 to 7 days in an inverted position in order to prevent condensing
moisture from accumulating on the agar surfaces after solidification.
Fig 7 (a) Streak plate method Fig 7 (b) Streaking lines 1, 2, 3 end of streaking
3.7.7 Isolation of keratinolytic fungi
Keratinolytic activity of fungi was detected as clear zones around the colony after incubation
for up to 7 days at room temperature. The selected keratinolytic fungi spores were suspended in the
distilled water and stored for future use.
3.8 Sub-culturing of selected spores
Sub-culturing of the selected spores of keratinolytic fungi was carried out in potato dextrose
agar and feather powder (70:30) and incubated at 37 °C for 5 to 7 days in an inverted position in
order to prevent condensing moisture from accumulating on the agar surfaces after solidification.
3.9 Characterization of fungi
3.9.1 Preparation of Lacto phenol Cotton Blue (LPCB) Slide Mounts
Page 25 of 49
Lacto phenol Cotton Blue (LPCB) wet mount preparation is the most widely used method of
staining and observing fungi and is simple to prepare. It can be used to also look at filaments and
higher life forms with microscopic work. Just remember, the stain will slow down and or cause the
higher life forms to die. This is great for taking photomicrographs, but not for a wastewater biomass
analysis. Use a normal wet mount first for higher life form counts.
3.9.2 Staining procedure:
1. A drop of sample is to be placed on a microscope slide.
2. One or at most two drops of the Lacto phenol Cotton Blue stain is to be added over
the sample.
3. By holding the cover slip between forefinger and thumb, one edge of the drop of
sample is to be touched with the cover slip edge, and is to be lowered gently, avoiding air bubbles.
The preparation is now ready for examination.
4. If desired, the edges of the cover slip are to be sealed with nail polish or per mount to
preserve the mount as a reference slide.
3.10 ENZYME PRODUCTION
3.10.1 Preparation of feather basal broth medium
The feather basal broth medium contains same contents as in the feather agar medium except
that agar.
3.10.2 Inoculum Development
The selected fungal colony after its identification and characterization was inoculated into
feather basal broth medium. It was incubated in orbital shaker incubator at 30o C for 5-14 days at 150
rpm. The PH of medium should be 7 i.e. neutral.
3.10.3 Production of Enzyme
After seven days of incubation, 10 ml of culture medium was transferred to250 ml medium.
The medium was prepared similarly as previously described. All incubations were done at 30oC with
shaking at 150 rpm in a controlled environment shaker. This flask was incubated for 5-14 days. The
Page 26 of 49
purpose of this step is only large scale (i.e. in sufficient amount for biochemical testing) production
of keratinase enzyme.
Fig-8 Broth cultures of five types of fungi
3.11 EXTRACTION OF ENZYME
3.11.1 Filtration
The culture medium was filtered through Whatmann No.1. Filter paper to remove residual
undegraded feathers and mycelium. The filtrate was then subjected to centrifugation at 10,000 rpm
for 10 min to remove fungal residue. After centrifugation ammonium sulphate was added to the
supernatant to achieve 30% saturation, which gives the precipitation of enzyme in suspension, now
this crude enzyme then used for enzyme assay and characterization.
3.12 Assay for Keratinase Activity
Keratinolytic activity of culture filtrates was measured spectro-photometrically; the test
described below was developed in order to simplify analytical work on Keratinase. Azo-keratin
hydrolysis provides a colorimetric assay for enzymatic activity on keratin.
Page 27 of 49
3.12.1 Synthesis and Enzymatic Hydrolysis of Azo-keratin
1. Ball-milled feather powder was prepared by using a ball mill.
2. A 1gm portion of the feather powder (the keratin source) was placed in a 100-ml
round bottomed reaction flask with 20 ml of deionized water and the suspension was
mixed with a magnetic stirrer.
3. Two ml of 10% NaHCO3 (weight per volume) were mixed into the feather suspension
(Lin et al., 1992).
4. In a separate 10-ml tube, 174 mg of sulfanilic acid were dissolved in 5 ml of 0.2 N
NaOH.
5. 69 mg of NaNO2 were then added to the tube and dissolved.
6. The solution was acidified with 0.4 ml of 5 N HCl, mixed for 2 min and neutralized
by adding in 0.4 ml of 5 N NaOH. This solution was added to the feather keratin
suspension and mixed for 10 min.
7. The reaction mixture was filtered and the insoluble azo-keratin was rinsed thoroughly
with deionized water.
8. The azo-keratin was suspended in water and shaken at 50°C for 2 hrs and filtered
again.
9. This wash cycle was repeated until the pH of the filtrate reached 6.0-7.0.
10. Finally, the wash cycles were repeated twice using 50 mM potassium phosphate
buffer, pH 7.5.
11. The azo-keratin was washed once again with water and dried in vacuum overnight at
50°C.
12. The resulting product is a chromogenic substrate that was incubated with enzyme
solution to produce and release soluble peptide derivatives that cause an increase in
light absorbance of the solution.
3.12.2 Preparation of keratin solution
Keratinolytic activity was measured with soluble keratin (0.5%, w/v) as substrate.
Soluble keratin was prepared from white chicken feathers by the method of Wawrzkiewicz. Native
chicken feathers (10gm) in 500 ml of dimethyl sulfoxide were heated in a reflux condenser at 100°C
for 2 hrs. Soluble keratin was then precipitated by addition of cold acetone at −20°C for 2 hrs,
Page 28 of 49
followed by cooling centrifugation at 8050×g for 10 min. The resulting precipitate was washed twice
with distilled water and dried at 40 °C in a vacuum dryer.
3.12.3 Enzymatic hydrolysis of Azo-keratin
a) In phosphate buffer (50 mM, pH 7.5.)
This procedure was used for the testing of keratinolytic activity of keratinase on azo-
keratin. To begin the process; 5 mg of azo-keratin was added to a 1.5-ml centrifuge tube along with
0.8 ml of 50 mM potassium phosphate buffer, pH 7.5. This mixture was agitated till azo-keratin was
completely suspended. A 0.2-ml aliquot of supernatant of crude enzyme was added to the azo-
keratin, mixed and incubated for 15 min at 50°C with shaking. The reaction was terminated by
adding 0.2 ml of 10% trichloroacetic acid (TCA). The reaction mixture was filtered and its activity
was analyzed. The absorbance of the filtrate was measured at 450 nm with a UV-159
spectrophotometer. A control sample was prepared by adding the TCA to a reaction mixture before
the addition of enzyme solution. A unit of keratinase activity was defined as a 0.01 unit increase in
the absorbance at 450 nm as compared to the control after 15 min of reaction.
b) In Tris-acetate buffer (0.2 M, pH 7.0)
The keratinolytic activity was assayed as follows: 1.0 ml of crude enzyme was properly
diluted in Tris-acetate buffer (0.2 M, pH 7.0) and was incubated with 1 ml keratin solution at 50 °C
in a water bath for 30 min, and the reaction mixture was stopped by adding 2.0 ml trichloroacetic
acid (15%). After cooling centrifugation was done at 5000×g for 10 min, the absorbance of the
supernatant was determined at 280 nm against a control. The control was prepared by incubating the
enzyme solution with 2.0 ml TCA without the addition of keratin solution. One unit (U/ml) of
keratinolytic activity was defined as an increase of corrected absorbance of 280 nm (A280)
(modified method by Gradisar et al.,) with the control for 0.01 per minute.
c) In Tris buffer (0.1M, pH 8.0)
20 ml of 0.1M tris buffer (pH 8.0) containing 0.1% feather and 40 µl of enzyme solution and
was incubated for 30 minutes at 55 WC.The reaction was stopped with 500 µl of 0.1M trichloroacetic
acid (TCA) in 0.1 mol tris buffer, pH 8.0.The amino acids liberated were measured as the
Page 29 of 49
absorbance at 590 nm against reagent blank. One unit (U/ml) of keratinolytic activity was defined as
an increase of corrected absorbance of 590 nm (A 590) with the control for 0.01 per minute.
3.13 Staining of feathers
Some quantity of white feathers were stained by crystal violet dye and dried. These feathers
were inoculated by the spores of fungi in the sterile test tubes and incubated for 7 days at room
temperature.
Fig-9 slide with stained feathers by crystal violet dye
Page 30 of 49
4. RESULTS AND DISCUSSION
Mixed cultures of fungi on feather agar medium showing the number of colonies with
keratinolytic activity in pour plate method performed after the serial dilutions up to 10 -7. As feather
was supplemented as sole source of carbon and nitrogen, it allows the growth of only proteolytic
fungi.
Fig 10 – The above plates showing the mixed cultures of fungal mycelia on feather agar
medium through pour plate method.
A: Mixed culture at 10-2conc. B: Mixed culture at 10-3conc. C: Mixed culture at 10-4conc.
D: Mixed culture at 10-5 conc. E: Mixed culture at 10-6conc. F: Mixed culture at 10-7conc.
The petri plates S2 to S7 contains the colonies of fungi of serial dilutions from 10 -2 to 10-7
respectively which shows the decreasing number of colonies from S2 to S7. Among those plates 5
morphologically different fungi was selected for pure culture formation.
Page 31 of 49
A B C
D E F
Pure cultures were isolated by sub culturing the above selected spores on feather-potato
dextrose media (70:30). We identified 5 types of mycelia regarding their initial appearance in color.
Those are in black, white, pink, green and green-yellow.
Fig11 A: Isolated fungal mycelia appeared in black colour grown on feather- potato
dextrose agar media.
B: Microscopic image of isolated black colored fungi.
Page 32 of 49
A
B
The microscopic image has been shown the basic features of conidial heads was short
globose, black and biseriate. Mycelia are long and smooth-walled and hyaline. Conidia was globose
and rough-walled (David ellis et al., 2007) as follows this features we concluded this will belongs to
the Aspergillus spp.
Fig12A: Isolated fungal mycelia appeared in white colour grown on feather- potato
dextrose agar media.
B: Microscopic image of isolated white colored fungi.
The microscopic image has been shown the basic features of condial head smooth-walled,
simple white cottony at first becoming brownish grey to blackish-grey depending on the amount of
sporulation. Sporangia are globose, greyish black, powdery in appearance and many spored.
As follows this features we concluded this will belongs to the Rhizopus spp.
Page 33 of 49
B
A
Fig13: A: Isolated fungal mycelia appeared in light pink colour grown on feather-
potato dextrose agar media.
B: Microscopic image of isolated pink colored fungi.
The microscopic image has been shown the basic features of simple white cottony at first
becoming blackish-grey sprongia depending on the amount of sporulation. Conidia are globose,
greyish black and many spored.
This species was unidentified.
Page 34 of 49
A
B
Fig14: A: Isolated fungal mycelia appeared in green colour grown on feather- potato
dextrose agar media.
<
B: Microscopic image of isolated green colored fungi.
The microscopic image has been shown the basic features of colonies show typical blue-
green surface. Conidial heads was globose and greenish coloured. Conidia was globose to
subglobose and green colour.
As follows this features we concluded this will belongs to the Aspergillus spp.
Page 35 of 49
B
A
Fig15: A: Isolated fungal mycelia appeared in yellow-green colour grown on feather-
potato dextrose agar media.
B: Microscopic image of isolated yellow-green colored fungi.
The microscopic image has been shown the basic features of colonies show typical yellow-
green surface. Conidial heads was globose and greenish coloured. Spores were globose to
subglobose and green colour.
As follows this features we concluded this will belongs to the mucor spp.
Page 36 of 49
B
A
Screening for high keratinase activity:
Keratinase activity was measured using UV-Vis spectrophotometer studies. Here we
followed three methods:
1. Tris-buffer method
2. Tris-phosphate buffer method
3. Tris-acetate buffer method
S.No Colour of fungi Absorbance at
450 nm
1 White colour fungi 0.109
2 Black colour fungi 0.011
3 Yellow-green colour fungi 0.007
4 Light pink colour fungi 0.182
5 Green colour fungi 0.242
Table3: keratinase activity shown by isolated fungal mycelia by using Tris-buffer
method (yamamura et al.,2002)
As follows this method we got high keratinase activity for green(0.242) and light pink(0.182)
colored fungi and low keratinase activity for black(0.011) and yellow-green(0.007) colored fungi
after incubating Azo-keratin with crude enzyme samples at 550C , pH - 8.0.
S.No Colour of fungi Absorbance at
590 nm
1 White colour fungi 0.040
2 Black colour fungi 0.012
3 Yellow-green colour fungi 0.024
4 Light pink colour fungi 0.043
5 Green colour fungi 0.019
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Table4: keratinase activity shown by isolated fungal mycelia by using phosphate buffer
method (Yu et al., 2008)
As follows this method we got high keratinase activity for white (0.040) and light
pink(0.043) colored fungi and low keratinase activity for black(0.012) and green(0.019) colored
fungi after incubating Azo-keratin with crude enzyme samples at 500C , pH – 7.5 for 15min.
S.No Colour of fungi Absorbance
at 280 nm
1 White colour fungi 0.431
2 Black colour fungi 0.011
3 Yellow-green colour fungi 0.594
4 Light pink colour fungi 0.202
5 Green colour fungi 0.052
Table5: keratinase activity shown by isolated fungal mycelia by using Tris-acetate
buffer method (Gradiser et al., 2007)
As follows this method we got high keratinase activity for white (0.431) and yellow-
green(0.594) colored fungi and low keratinase activity for black (0.011) and green (0.052) colored
fungi after incubating Azo-keratin with crude enzyme samples at 500C , pH - 7.0 for 10min.
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Feather degradation:
Fig16: Control samples to show the degradation of feather
A) Stained feather of white colored fungi before degradation
B) Stained feather of green colored fungi before degradation
C) Stained feather of pink colored fungi before degradation
Fig16: Degradation of feathers by isolated keratinolytic fungi which showed most
activity
D) Stained feather of white colored fungi after degradation
E) Stained feather of green colored fungi after degradation
F) Stained feather of pink colored fungi after degradation
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A B
B C
D E F
The figure A,B,C(100X magnification)were the control undegraded feathers which used to
compare the degradation of feathers in the figures D,E,F(100X magnification). The red circles in
above figures show the degradation of feather.
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5. CONCLUSION
From the decaying feather samples five types of fungi were successfully isolated and their
activity was measured spectrophotometrically. Of these isolated five types of fungi, each of them
showed different enzymatic activity in different methods. In the tris-buffer method, we found the
green colored fungi with highest activity and yellow-green colored fungi with least activity. In the
tris-phosphate buffer method, we found white colored fungi with highest activity and light pink
colored fungi with least activity. In the tris-acetate buffer method, we found white colored fungi with
highest activity and yellow-green colored fungi with least activity. From the overall analysis, we
found white colored fungi with optimal activity in all the three methods.
Further study in this aspect would be helpful in the poultry waste management and even in
the production of the proteinaceous feed which will be used for the cattle as their feed.
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