project major1234

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

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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

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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.

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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

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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.

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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.

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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.

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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.

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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

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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

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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.

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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,

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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

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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

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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.

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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.

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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


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.

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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.

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A

B

Fig14: A: Isolated fungal mycelia appeared in green colour grown on feather- potato


<

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.

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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.

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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






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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






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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