Contents

1.Introduction
1.1. History
1.2.Need for enzyme
1.3.Importance
1.4.Sources of keratinase

2.Production
2.1.Polymerase chain reaction
History
Principle
Components of PCR
a)
Genomic DNA
b)
Primers
c)
Taq DNA polymerase
d)
Taq buffer
e)
Barrier tips for automatic pipettes
f) Positive displacement pipettes
g)
Thermal cycler
Steps involved in PCR:
a) Initialization
b) Denaturation
c) Annealing
d) Extension/Elongation
Procedure

2.2.Gel Electrophoresis:
a)
b)
c)

Principle
Components of Agarose gel electrophoresis
Procedure

2.3.Purification of Recombinant Keratinase

2.4. Digestion with restriction enzymes:
Restriction enzymes or DNA cutters:
Naming
History
Principle
1

 Restriction digestion
a)Digestion of gDNA:
Genomic DNA
Reaction mixture
b) Digestion of plasmid DNA:
Plasmid
Reaction mixture
c)Procedure

2.5.Ligation:
DNA ligase
Steps involved in DNA ligation
Ligation Reaction mixture
Procedure

2.6.Transformation
Methods of making host cell competent:
Chemical transformation
Heat shock
Particle bombardment

DNA transformation procedure

Enzyme Assay:
Keratinase Assay:

3.Industrial Applications

Bioprocessing keratin rich wastes

Leather Industry
Textile Industry
Detergent Application
Removal of Earwax
Pearl Bleaching
Cosmetics or personal care products
Processing of Edible Birds Nest
Keratinases for transungual drug delivery
Removal of Corn and Callus
Acne treatment

1.Introduction
The keratinases are protein degrading enzymes present in nature.
Their mechanisms were not clearly defined when they were
3

discovered. Nomenclature committee gave them the name of serine

proteases as they show much of sequence homologies to alkaline
protease. Besides, inhibitors used to inhibit serine proteases can
also prevent keratinases. Any substrate containing keratin can be
used to produce keratinases. The main attack by keratinases is on
the disulfide bonds in keratin substrate. Many microorganisms are
able to produce keratinases as reported by many researchers. This
enzyme may be produced by some fungi and bacteria preferably in
the thermophilic conditions and at basic pH. It can act on a variety if
substrates. It may result in the breakdown of fibrous proteins as well
like fibrin, collagen and elastin. It also has the ability to degrade
casein, albumin, bovine serum etc. that are non fibrous proteins
(Brandelli et al., 2010).

1.1.History
It was first isolated from bacteria that could degrade keratin. That
organism

was

isolated

from

contents

of

dermoid

cysts

(experimentally induced) that were taken from middle lateral part of

sheep. When wool sample was examined, it showed degradation of
wool by many cyticular and corticle cells. The wool fiber was
disrupted both in vitro and in vivo. The isolated organisms were
shown to belong to Bacillus genus and it can breakdown protein in
wool. Streptomyces fradiae can also enzymatically degrade native
keratin. This bacterium produces extracellular enzymes that was
able to degrade human hair. Some keratinophilic fungi were also
observed

that

could

produce

keratinase

enzyme.

Some

Streptomyces species can also produce keratinolytic protein. An

experiment

was

performed

for

this

purpose.

An

inducible

homogenous extracellular enzyme was isolated. This enzyme

showed 7.5 times more activity after column chromatography. The
activity of enzyme was slowed down by mercaptaethanol. The work
was continued to be performed to get pure culture so that organism
4

might be identified to species level. The bacteria were shown to be

Bacillus licheniformis in 1990s. Other techniques were used to purify
and characterize keratinase from pure bacterial strain that was
degrading feather. Techniques included gel chromatography and
ultra filtration. The purified enzyme had 70 times more activity than
the previous one. Its molecular weight was determined as 33 kilo
Daltons. Molecular technique like SDS-PAGE made it easy to
determine weight. Hence purified enzyme had characters like
thermostablility, activity in alkaline conditions and this enzyme
could also solubilize keratin even in media having lactose or mineral
salts. However the best conditions for activity were at pH 9 and at
temperature of 90 C. A fermentor was used to increase the
fermenting conditions of keratinases. The production of enzyme was
increased to 10-fold level by optimizing the fermentation (Lin et al.,
1995).

1.2.Need for enzyme

Keratin is one of those biopolymers present abundantly throughout
the world. This is a fibrous material that is tough and insoluble. It
acts as an outer covering of many of organs for the prevention of
body fluids loss. It is present in tissues of birds, humans and other
animals. Keratin made the structural component of many of body
parts like hair and nail. Keratins are of two types i.e. alpha keratins
(found in mammals) and beta keratins (present in birds). Keratin is
also found in the digestive organs as a part of epithelial cells.
Keratin is insoluble due o presence of sulfur compound with disulfide
bonds. Many amino acids are also present in keratins. It is unreactive and is not degraded by other protein degrading enzymes
like pepsin. Materials having keratins cannot be digested by most
vertebrates. It is associated with liver diseases in humans. Keratin is
used as a raw material in many industries which may result in its
accumulation causing environmental problems (Farag and Hassan,
5

2003). Feather wastes (that may include keratins) are also dumped
by poultry farms. These feathers contain large keratin quantity and
large amount of these feathers are dumped. Here the need arose
that could reduce the increasing amount of keratin. To avoid
accumulation of keratin in environment, a method was developed
that was easy to handle and less expensive. Keratinases are keratin
degrading enzymes that could counter such problems. These
enzymes target disulfide bridges and convert keratin from complex
compounds to simpler forms. Keratinase production by microbes is
considered a good source. Microorganisms are present on keratin
wastes having the ability to produce keratinase.

1.3.Importance of enzyme
Keratinase produced by microbes are becoming very important
biotechnologically. These enzymes result in hydrolysis of keratin that
has strong bonds and is a structural rigid polypeptide. This cross
linked polypeptide cannot be degraded by the other protein
degrading

enzymes

like

pepsin,

trypsin

and

papain.

When

keratinous substrates are present, these enzymes produce by

degrading them. It has a quite complex mechanism which involves
the cooperative proteolytic and sulfitolytic activity. Keratinases are
very energetic enzymes that have wide temperature range and wide
pH range. They are considered proteases because they target the
keratin residues and so they are important in developing the byproducts of feathers for fertilizers and feed. They are also useful in
leather industries and in detergents. In addition to this, they also
find applications in cleaning of silk and wool. These enzymes also
have better dahairing property which cause them to serve in hair
saving technology. The enzyme can dehair hides of goat in a short

time
without
using

other

chemicals

(Macedo

et al., 2005).

Mechanical methods and electron microscopy of goat skins show

comparison between enzymes treated and chemically treated skins.
When enzyme is used to dehair, it show improved results without
causing harm to collagen layer. So it can be safely used in leather
industries to avoid problems that were associated with using
chemicals for the same purpose. Moreover, they also have role in
degrading prions which may change the world of protease in future.
Uses of Keratinase in Pharmaceutical industry:
Pharmaceutical industry is involved in the
discovering, production and development of procedures for the synthesis of
pharmaceutical products that are used in medicine for the treatment of various
diseases. Development of pharmaceutical products is a long task and subjected to a
long process of approval. Pharmaceutical industry is also involved in the marketing of
drugs. Pharmaceutical industries approve their drugs from FDA in long period of time
and then launched it in the market (Brandelli et al., 2010).

a) Keratinase as biomaterials:
Keratinase is used as a biomaterial in tissue engineering. Tissue engineering is a
combination of cellular biology, engineering and biomaterials, and biochemicalphysiochemical factors to repair or replace the biologically important tissues. Most
tissues that are repaired or replaced with biomaterials during tissue engineering are as
follows:

Bone
Cartilage
Blood vessels
Bladder
Skin
Muscles
They must have biocompatibility (Brandelli, 2008).

The following features of keratinase makes it suitable for use as biomaterial:

Keratinase has high biocompatibility.
It has cell adhesion sequences which are normally present in fibronectin.

Fibronectin is a glycoprotein that bind to extracellular components like collagen.

It has cellular binding motifs that makes it suitable for the attachment to biological

tissues.
b) Keratinase as nourishing agent:
A nourishing agent can be defined as a substance that
provides nutrients to the body in addition to hydration, that are considered to promote
the health, life and growth of particular body tissues. The following characteristics of
keratinase makes it suitable nourishing agent:
It is considered good for skin and hair because of its hydrophilic nature.

t has good penetrating power so it can penetrate the skin and hair and helps to
nourish them.
Keratinase is non-toxic and it does not have any side effects.
It has high biological compatibility.
c) Keratinase as wound healing agent:
Healing agents are actually the topical agents that promote healing of wounds and
effective granulation. It is an excellent healing agent because it cleaves the keratinous
material in the body by breaking down or through the oxidation of disulfide bonds.
9

The oxidation of these disulfide bonds results in the production of water soluble
peptides that activates the healing of wounds (Gupta and Ramnani, 2006).
Keratinase in bioprocessing of poultry (keratin rich) waste:
A bioprocess refers to a specific process which utilizes complete living cells or their
components

(e.g., enzymes,

bacteria, chloroplasts)

to

get

desired

products.

In agriculture, poultry litter is a mixture of feathers, poultry excreta, spilled feed etc.
The dominant constituent of feather is keratin and shows 90% of feather weight that
make up to 10% of chicken weight (Harrap and Woods, 1964; Cherry et al., 1975).
The large amount of feathers produced as a result of Poultry processing on
commercial scales may cause a pollutant problem and requires proper management
(Shih, 1993).

Feather meal:
The conversion of feathers into feather meal until recent years has been accomplished
at high pressure and temperature which means it requires high input of energy. To
replace the high energy requirements, another option for feather meal production is
enzymatic hydrolysis of feathers by keratinase (Onifade et al., 1998; Grazziotin et al.,
2006).

10

Analysis of feather meal:

Contents
Protein
Fat
Moisture
Ash
Calcium
Phosphorus

Percentage
Min. 80
Max. 10
Max. 10
Max. 5
Max. 5
Max. 5

Applications:
a) Animal Feed:

The use of keratinase in the production of feather enhances the digestibility and
nutritional value of protein enriched products. This high potential of digestibility
and nutritional value of feather meal is due to modification in keratin structure
produced by keratinase(Lee et al., 1991; Odetallah et al., 2003).Keratinase

utilization also replaces the hydrothermal process that needs high energy value.
It is inexpensive and is superior to soybean meal with reference to

cysteine,threonine, valine and 7% dietary contents.

The crude enzyme is also used as a nutraceutical product that improves broiler

performance significantly (Odetallah et al., 2003).

On one side, fermentation secures essential amino acids (arginine, lysine and
methionine) level, and on other hand microbial biomass contributes in the high

protein level.
Moreover, overexpression, cloning and bio-immobilization of crude enzyme have
been effectively carried out to fulfill the requirements of animal feed industries.
The technology for crude keratinase PWD1 production is authorized to BRI and is
being formed trade name as Versazyme.

b) Biodiesel production:
11

An alternative fuel that is similar as 'fossil' or conventional diesel is known as

biodiesel.Keratinases and Keratinolytic microorganisms act on keratinous byproducts
and can be used for production of natural gas, gas fuel pellets, methane and
biohydrogen production. This conversion fuels may meet the hot issues for recycling
and energy conservation. Poultry waste can be used for biodiesel generation besides
effective animal feed.

c) Nitrogen fertilizer:
The feather meal is protein-rich concentrate so can also be used for the organic
farming as N-fertilizer (Hadas and Kautsky, 1994; Choi and Nelson, 1996). Feather
meal as nitrogen rich compound (15% N) is very good alternative of high expensive
guano fertilizer which has been frequently used in organic farming in the past

Feather meal has following advantages:

Feather meal increases retention capacity of water
Maintains soil structure
Inexpensive and reliable source of nitrogen
Accelerates microbial activities in soil
Improves plant growth
Its production is economic feasible
It has high nutritional value (Hadas and Kautsky, 1994).

12

1.4.Sources of enzymes
Keratinases are essential in many industries. So it is needed to
develop keratinases from sources that can be easily developed and
managed for the continuous production of enzyme. Conventional
methods of keratinase production are comparatively difficult as
compared to obtain keratinase from microbes. Keratinases can be
isolated from bacteria, fungi and actinomycetes (Fellahi et al.,
2016).

Keratinolytic fungi
Many keratinophilic fungi are present that produce proteolytic
enzymes capable of degrading the waste materials containing
keratin. These keratins are used by keratinophilic fungi living as the
parasites on keratin material. These fungi use such materials as
their source of nitrogen and carbon which help them to reproduce
asexually

and

produce

conidium.

When

fungi

colonize

such

substrates, they put their hyphae in such materials like a drill.

Examples of such fungi are hyphomycetes and many others.
Hyphomycetes

may

comprise

both

dermatophytic

and

non

dermatophytic. Other keratinophilic fungi are Mirosporum species,

Chrysosporium species, Trichophyton and Epidermophyton. These
keratin degrading species were identified after many experiments
and due to their morphological characters. Shapes and size of
conidia helped in their identification. Besides, molecular methods
also made it easy to identify them like DNA sequences.
Keratinolytic fungi has ability to produce sulfide for breaking it and
during sulfitolysis, disulfide bridges present in cysteine are broken.
Cysteine in one of important amino acid present in keratin material.
After sulfide bonds are broken down, many of protein degrading
enzymes are released by fungi which can break down the keratin
13

more easily. During this breakdown process, many products are

released into the surroundings. These products include amino acids
like cysteine, cysteine acid and sulfate. These products are released
in culture medium during process of degradation. The presence of
keratinophilic fungi thus can be identified by the occurrence of these
products in the medium. If no such behavior is shown by fungi and
there is no such products presence in medium, it means the fungi
are non keratinophilic. Fungi may differ according to their nature of
keratin degradation and varies according to the type of substrate.
Such fungi can either be animal loving (zoophilic) or human loving
(anthropophilic). Many soil samples were collected having a variety
of keratinophilic fungi. These soils contain such fungi due to the
presence of keratin wastes in that soil. Soils having keratin wastes
are geophilic. Geophilic habitats like beaches, agriculture, parks and
other such visiting places were used as a source for collecting soil
samples rich in keratin wastes and hence keratinolytic fungi. Keratin
baiting is a technique for isolation of samples. In this technique, hair
and feathers are used as source of keratinolytic fungi. These fungi
are found in different countries if world including Australia, Kuwait,
Malaysia, India, Egypt and many others. Different experiments were
performed for isolation of fungi. 330 different samples were
collected from 13 schools and 7 different public places. These
samples contained 214 species among which the most abundant
were Keratinophilum and Chrysosporium. They were present more
than half of the total species. Other species included Microsporium
species, Trichophyton species and other Chrysosporium species. In
another experiment, twelve soil samples were collected from poultry
industry where the feathers were dumped as a waste. Thirty four
different fungal species were observed that were all keratinophilic.
They belonged to nineteen different genera. Most of the species
were dermatophytes.
800 different soil samples were collected from a province in Iran. It
was found that among them 588 species belong to keratinophilic
14

fungi. In the similar manner, large number of soil isolates was

collected from different places considered to be rich in keratin
source. Each of these samples showed different species of
keratinophilic fungi as can be visualized due to their morphological
features and other molecular diagnostic techniques. Chrysosporium
species were identifies in almost all cases. Keratinophilic species
were obtained from visiting places like zoos, public parks, gardens,
elementary schools, rivers and the sites where dumping of garbage
is observed. Many of keratinophilic fungi were obtained from
samples taken from gardens. Soil sample taken from rivers showed
minimum

presence

of

keratinophilic

fungi.

After

all

these

experiments and studies, it can be said that fungi degrading keratin

are abundant and occur in different types of soils. They are most
abundant in places where most of animals and humans visit (Rapli,
2000).

Keratin degrading bacteria

A large number of bacterial isolates are present that can degrade
keratin. Bacteria have ability to grow rapidly as compared to fungi
and hence bacterial isolates are preferred in industries. But this is
not always the case. Some fungi are preferred for use in industries
as it is easy for fungi to penetrate keratin hard waste substance
when compared with bacteria. This ease of colonization is due to
presence of hyphae that are found only in fungi. Soil samples were
taken and bacterial strains were observed in the presence of keratin
wastes. These isolates mostly included Bacillus species, Vibrio
species, Mycrobacterium and Xanthomonas. Other Gram positive
and Gram negative bacteria were also present in different samples.
All of them can degrade keratin easily. In an experiment, feather
wastes were taken as sample which indicated four isolates by
growing on agar plates. Three of them were Gram negative bacteria

15

and the one was Gram positive strain. Many of Streptomyces

species also can produce keratin degrading enzyme.

Secretion of enzyme
Proteases that can degrade keratinases are called keratinases which
can easily be obtained from microorganisms. Keratinases from fungi
can be obtained through secretion and it is also inexpensive method
as compared to obtaining keratinases from bacteria but fungal
growth rate is slow and it is time consuming to obtain keratinase
from fungi. As many strains are available that can produce enzyme
so it is crucial to select the ones giving better and easy results
(VInsze at al., 2003).

16

2.Part A. Production
2.1.Polymerase
chain reaction:
PCR/ polymerase chain reaction is a molecular biological technique that is
used to amplify a particular DNA segment. It produces thousands to millions of
copies of a specific DNA segment. It is very affective to amplify small DNA
fragment that is degraded as well.
History:
This technique was developed by Kary Mullis in 1983 and received Nobel Prize for
his work on PCR. He used this method in the diagnostics of influenza virus but now
it is used in the following applications as:

Gene cloning for sequences

DNA based phylogeny
Genes functional analysis
DNA fingerprinting
Diagnostics of hereditary diseases
Diagnosis of infectious diseases(Lin et al., 1995).

Principle:
PCR is based upon the principle of DNA replication in which thermostable DNA
polymerase in which DNA polymerase utilizes the primer-template junction as a
substrate to carry out the process of replication. PCR consists of repeated cycling of
heating and cooling. Heating denatures the double stranded DNA. Primers (forward
and reverse) containing the sequences complementary to the target gene bind to their
target strands at

17

comparatively low temperatures. Primertemplate junction can serve as template for

thermostable.
DNA polymerases that adds new nucleotides to the primer-template junction resulting
in the synthesis of Deoxyribonucleic acid. The whole process is carried out in the
automatic machine called thermal cycler. This machine automatically controls the
temperature during each step of PCR. Taq DNA polymerase has optimum temperature
of about 78-80 Celsius with a half-life that is usually greater than two hours at 92
Celsius. Taq DNA polymerase 3-5 end proofreading activity. The error rate of
TaqDNA polymerase is very low about 1 in 9,000 nucleotides. It was first isolated
from Thermus aquaticus that live in hot springs and live in high salt concentrations.
Other thermostable DNA polymerases have also been isolated from other
thermophilic species and archaea. Whatever the source may be, all DNA polymerases
perform similar function that is the replication of Deoxyribonucleic acid(Lin et al.,
1997).

Components of PCR:
18

The components that are used to carry out the amplification of

keratinase gene are as follows:
a) Genomic DNA:
Genomic DNA is isolated from the microorganism that produce keratinase enzyme.
Genomic DNA contains the target gene sequence. Sequence of target gene can be
determined by using genome databases like GenBank and NCBI.
b) Primers:
Two primers are synthesized for two strands of DNA named as forward and
reverse primer. Primer must have a sequence complementary to the target sequence.
Primers can be synthesized manually by adjusting their melting temperature by using
the following formula as:
Tm (Tm = {4 [G + C]} + {2 [A + C]} 0C)
They can also be synthesized by using the Bioinformatics tools like primer 3/ primer
3plus.
Primers must have following characteristics as:
Melting temperature of primers must be 55 to 65 Celsius.
They must lack dimerization capability.
Lack of secondary priming sites in the templates.
They must have low specific binding at 3-end to avoid mispriming.
Usually a 20 to 24 nucleotide primer works well (Radha and Gunasekaran, 2008).
c) Taq DNA polymerase:
This enzyme is required for the replication of Deoxyribonucleic
acid.
d) Taq buffer:
Taq buffer consists of following components as:
Tris-HCl

KCl

19

MgCl2
Gelatin

Tween-20

NP-40

e) dNTPs:
Deoxyribonucleosides serve as building blocks through which DNA polymerase

synthesizes new DNA strand.

f) Barrier tips for automatic pipettes
g) Microfuge tubes
h) Positive-displacement pipette
i) Thermal cycler(Brouta et al., 2002).
Steps involved in PCR:
Typically, a PCR consists of 20-30 repeated cycles, and each cycle consists of
following steps as:
a) Initialization:
This step involves heating PCR reaction mixture at 94-96 Celsius for 1 to 9 minutes.
This step is usually employed when extremely thermostable DNA polymerases are
used in PCR.
b) Denaturation:
The main purpose of this step is to heat the double-stranded Deoxyribonucleic acid in
order to convert it into single strands. It is a first regular cycling event that consists of
heating the reaction mixture to 94-98 Celsius for 20 to 30 seconds. This high
temperature causes the DNA to melt by disrupting the hydrogen bonds between
complementary bases in double-stranded DNA yielding two single stranded DNA
molecules.
c) Annealing:
At this step, temperature of 50-65 Celsius is used to allow the binding of primers to
target sequence. This temperature must be low enough to allow the annealing of
primers but must be high enough to allow the specific binding of primers to template
DNA strands (Fellahi et al., 2016).
d) Extension/Elongation :
Temperature at this step depends upon Taq DNA polymerase used. Taq DNA
polymerase has optimum activity at 70-80 Celsius and temperature of 72 Celsius is
most commonly used with this enzyme. At this step, DNA polymerase synthesizes a
new strand complementary strand by adding nucleotides at 3 end of the primer by
condensing the phosphate group and hydroxyl group of nucleotides(Decamps et al.,

20

2002).Steps involved in basic PCR protocol can be represented by the following

diagram as:

Procedure:
21

a) In a sterile microfuge tube, following ingredients are mixed in the following

concentrations as:
10X amplification buffer
20mM four dNTPs solution
20M forward primer
20M reverse primer
1-5 units/l of Taq DNA polymerase
Template DNA
Nuclease free distilled water
Total volume

5l
1l
2.5l
2.5l
1-2 units
5-10l
28-33l
50l

b) After adding all the above ingredients to microfuge tube, microfuge tube is placed
in thermal cycler (Gu et al., 2016).
c) Nucleic acid is then amplified using the denaturation, annealing and
polymerization times and temperatures as
Cycle #
30 cycles
Last cycle

Denaturation
30 sec at 94 C
1min at 94C
1.5min at 94C
d) Test reaction mixture of about 5-10l is

Annealing
30sec at 55C
30sec at 55C

Extension
1 min at 72C
1 min at 72C

withdrawn from the microfuge tube and

analyzed by Gel electrophoresis by using the DNA markers of appropriate size

The whole process can be represented by the following diagrams as:

2.2.Gel Electrophoresis:

22

Gel electrophoresis is a molecular method that is used to separate the macromolecules

like DNA, RNA and proteins. These macromolecules are separated
on the basis of their size and charge on the gel by the application of electrical current.
In present case, agarose gel electrophoresis is used to analyze the product (keratinase
gene) produced by PCR (Liu et al., 2013).
a) Principle:
DNA fragments are separated on the basis of their size and charge in gel
electrophoresis. They are separated on the gel by the application of electric current. As
electric current is passed through the gel, negatively charged particles move towards
anode. Their migration is primarily determined by their molecular weight. Low
molecular weight macromolecules move more quickly than high molecular weight
macromolecules. When separated, DNA fragments can be visualized by staining the
gel with ethidium bromide and SYBR green (Rapley, 2000).After staining, DNA
bands can be visualized and illuminated under 300nm UV light.

23

24

b) Components of Agarose gel Electrophoresis:

The components most commonly used in agarose gel electrophoresis are as

follows:
Agarose gel (0.8g in 100 ml 0f distilled water).
Amplified fragment of DNA that contains keratinase gene.
DNA staining solution like ethidium bromide and SYBR green.
Electrophoresis buffer like TAE buffer (Tris acetate EDTA).
Gel loading buffer that consists of following ingredients as:
0.25% bromophenol blue
0.25% xylene cyanol FF

Procedure:
Agarose gel electrophoresis which is used for the quantification and

analysis of DNA can be performed as:

0.8g Agarose is weighed on weighing balance.
Agarose is then added to 100ml of TAE buffer in a flask that can be placed in
microwave oven.
25

When agarose is added to water, its mouth is covered with aluminum foil and

placed in microwave oven for just 1-2 minutes.

After 1-2 minutes, the flask is removed from microwave oven and cooled down

for 5 minutes.
Then, 5g/ml of ethidium bromide is added to agarose solution. Ethidium bromide
reacts with DNA and illuminates when placed in UV lamp. If ethidium bromide is
not added at this step then it can also be added at the end by staining the DNA

fragments.
Finally, the contents of above flask are added to casting tray.
Combs are placed in agarose gel. The size of combs is determined by the number
and size of DAN fragments. These combs are produced in order to load PCR

product on agarose gel.

After placing the comb in the agarose gel, it is placed undisturbed at 4C for 5

minutes or at room temperature for 20-30 minutes.

Then, 6l loading buffer containing bromophenol blue, xylene cyanol FF,

and sucrose is added to each sample.

Electrophoretic tank is set up.
Gel box of Electrophoretic Tank is then filled with 1x TAE buffer until it is

completely covered with buffer.

Known molecular weight marker is then loaded into one lane.
PCR product containing the Keratinase gene is then loaded on the other

lanes.
Electrophoretic tank is then covered and connected to electricity source.

Electrophoretic tank is allowed to run at 1000volts for 90 minutes.

After 90 minutes, the electrophoretic tank is plugged off and agarose gel

containing the DNA fragments is removed from the tank.

At this stage, DNA fragments are not visible. In order to visualize them UV

lamp is required.
Agarose gel containing the DNA fragments is placed under the UV lamp in
order to visualize the DNA fragments. Ethidium bromide that was added to
the gel illuminates under UV light. Ethidium bromide is carcinogenic so
great care is required to use it. SYBR green can also be used in place of

ethidium bromide but it is very expensive.

The size of PCR product containing Keratinase gene is determined by

comparing it with DNA marker of known molecular weight and size.

The size of Keratinase gene is very close to the size that was calculated from the

size of base pairs (Brady and Kern, 2004).

The whole procedure can be summarized by the following diagram as:
26

The final PCR product containing the keratinase gene is run on agarose gel along
with the marker of know molecular weight marker. On the application of electric
current, PCR product runs on the gel and it is visualized by using the UV lamp.
The results of this gel electrophoresis can be represented by the following diagram
which is an actual result of Saied Alinezhad and Amir Mirabdollah:

1440 bp

27

The size of Keratinase gene amplified from PCR is almost similar to the
theoretically predicted size (Alinezhad and Mirabdollah).

2.3.Purification of Recombinant Keratinase:

Keratinase enzyme that was produced by recombinant DNA technology is purified by
using the Ion-exchange chromatography. Ion-exchange chromatography is usually
considered as a kind of column chromatography. In ion-exchange chromatography,
polar molecules like proteins and nucleic acids are separated on the basis of their
affinity to ion-exchanger. Purification of keratinase enzyme can be performed in the
following steps as:

Supernatant is collected after centrifuging the culture containing keratinase

enzyme.

Supernatant
is

then

filtered
by using
the filter
paper
having
the pore
size

of

0.45m.
The
filtrate is
then
concentrated to approximately 10 fold with a spiral cartridge concentrator. This
28

concentrator is especially designed for the concentration of proteins. It consists of

a sandwich of two membrane layers that are wrapped spirally around a permeate
collection tube. In this, edges of membranes are fused together. The fluid
containing the proteins to be separated is passed through the membranes into the
central collection tube and then passed through the header assembly(Farag and
Hassan, 2003).
Retentate, which came out from the cartridge concentrator, is dissolved in 20mili
molar Tris-HCl. The resulting solution is then added to a column containing cotton
wool at the bottom and DEAE (Diethyl amino-ethyl cellulose) is added to the top of
cotton wool.

DEAE-C is a resin that contains positive charge and used in column

chromatography for the purification of proteins. Gel matrix beads containing
DEAE helps to capture the negatively charged proteins into the matrix. The
proteins are then separated by using the eluent containing both cations and anions
and passed through the column at very high concentrations. Thus, column

chromatography is a technique that is used to separate the compounds on the basis

of their specific charge/polarity and hydrophobicity(Brassdli, 2010).

Then, above the DEAE cellulose beads, the fluid containing the protein to be

separated is added.
Eluent containing Tris-HCl is passed through the column at a rate of 5ml per minute
which is then followed by washing with 1M NaCl in the same buffer. This results in
the elution (separation) of proteins from DEAE-C beads.
29

The resultant filtrate is finally exposed to enzyme activity assay in order to determine
the presence of keratinase enzyme (Bressolieret al., 1999).

2.4.Digestion with restriction enzymes:

Restriction enzymes or DNA cutters:
Restriction enzymes are manufactured in bacteria and isolated from them for
utilization.Oftenly, they are known as restriction endonucleasesas they cause
cleavage within molecule.Specific DNA sites at which they perform their activity are
called restriction sites. More than 400 REs have been purified from bacteria that form
them (Lin, X.,1997).
Naming:
The name of enzyme depicts the organism from which it was first isolated. For
example:

EcoRI is obtained from Escherichia coli strain RY13. Here, Eco indicates genus and
species ( E is first letter of genus and co first two letters of specific epithet); R refers
to strain of Escherichia coli; I is the Roman numeral suggests it was the first enzyme

of that nature obtained from E. coli RY13

Sau3A origin is Staphylococcus aureas strain 3A.
BamHI origin is Bacillus amyloliquefaciens strain H
And so on (Roberts,R.J., 1976).
History:

Arber and Meselson: isolated type I restriction enzymes i.e. EcoB and EcoK. Type I

enzymes cut DNA randomly distant from recognition site.

Hamilton O. Smith,Kent WilcoxandThomas Kelly: isolated type II restriction enzyme
i.e. HindII obtained from Haemophilus influenzae bacterium.Such type of enzymes
are more beneficial for lab work because they cut DNA at site of their particular

recognition sequence.
Daniel Nathans and Kathleen Danna declared that REs can be used for DNA mapping
after cleaving SV40 (simian virus 40) DNA by restriction enzymes and separating the
cleaved fragments using gel electrophoresis.

30

Werner Arber, Hamilton O. Smith andDaniel Nathans got nobel prize in 1978 due
their work on discovery and characterization of Res (Smith and Wilcox,1970).
Principle:
Sequencespecific restriction enzymes recognize their own restriction sites. e.g.
GAATTC is the restriction site for EcoR1. After binding to their sites, REs cleave
sugar phosphate bonds present in backbones of DNA strands. All REs produce sticky
ends after cleaving except two or three enzymes which produce blend ends. Later on,
these restricted ends are joined by another enzyme called DNA ligase (Vincze, et
al.,2003).

Fig:(a) EcoR1 recognition site is GAATTC

(b) Binding of EcoR1 to GAATTC (c) Sticky
Restriction digestion
ends are produced by EcoR1 (d) Rejoining
restrictedby
ends
by DNA ligase.
A method of cleaving DNA molecules into small of
fragments
usingrestriction
enzymes at restriction sites where the desired genes can be inserted. For proper
ligation, same types of restriction enzymes are used to restrict gDNA and plasmid. It
is necessary so that resulting products of both DNA have complementary ends for
each other after restriction (Laemmli, U.K., 1970).
a) Digestion of gDNA:

Genomic DNA:
Genomic DNA is chromosomal DNA and abbreviated as gDNA oftenly. Single or
couple set of REs can be used to restrict gDNA or PCR product e.g. EcoRI and Hind
III or NdeI. The restrictions sites of these enzymes are checked either these are

31

present or not before restriction, for this purpose NEB cutter software is used (Vincze,
et al., 2003).

Reaction mixture:
Following ingredients are required in reaction mixture tube for digestion of gDNA.

Reagents

Volume

gDNA or PCR product

2l

Restriction enzymes

1l

10x RE buffer (suggested by the supplier)

2l

Sterile water

15l

Total volume

20l

b) Digestion of plasmid DNA:

Plasmid:
Plasmid is extra chromosomal DNA which is used as vector and carried new DNA to
a new cell for the purpose of isolation,multiplication and expression of genetic
material in host cell. All plasmids have following few similar features; origin of
replication (Ori) , multicloning site (MCS) ,promoter, M13 and antibiotic resistant
gene. MCS is small region in plasmid which holds sites for different restriction
enzymes. Plasmid is restricted by the same types of enzymes used for gDNA
restriction (Biedendieck, 2007).

32

Fig showssimilar features of all plasmids.

Reaction mixture:
Reagents required for restriction of plasmid DNA are listed below:

Reagents

Volume

Plasmid DNA

2l

Restriction enzymes

1l

10x RE buffer (suggested by the supplier)

2l

Sterile water

15l

Total volume

20l

c) Procedure:
Two microfuge tubes; one having gDNA and second one containing plasmid DNA are
taken. The final volume of reaction mixture tubes is 20 l by adding similar reagent
described above in table 1 and 2.

33

Reagents in above two reaction tubes are gently mingled by pipetting and then

incubated at optimal temperature 370C for 1-3 hours to ensure complete digestion.
Prior to ligation process, these reaction mixtures are heated (to inactivate the
restriction enzymes) for 10 minutes at 70 0C. Later on, these mixtures are utilized for
proper ligation (Lin,X.,1997).

2.5.Ligation:
It is a process through which two fragments of nucleic acids are joined to form a
recombinant DNA (a plasmid carrying a foreign DNA) with the favour of DNA ligase
(Lohmanet al.,2011).

DNA ligase:
T4 DNA ligase is used to ligate the DNA fragment having cohesive ends or sticky
ends as in the example of EcoR1- this is same as repairing of "nicks" present in DNA
duplex (Fig: ). T4 DNA ligase is isolated from T4 bacteriophage. DNA fragments
with blunt ends are also ligated by this enzyme though enzyme in higher
concentrations is suggested for this purpose (Western and Rose, 1991).
Steps involved in DNA ligation:
DNA ligation process comprising two principle steps:

34

At first, the DNA ends collide by chance and stay jointly long enough wait for ligase
which joins them. This inefficient part of ligation reaction takes place at low
temperature. Two reasons support the process to be happened at low temperature:

The molecular movement through the solution is slow at low temperature so it is easy
for DNA ends to collide and stay jointly at this temperature

Lower

temperature

is

helpful

in

stabilizing

hydrogen

bonding

between

complementary nucleotides; no doubt it is really favourable to keep the things in

place.

In second step of reaction that is enzymatic in nature, DNA ligase joins 3-OH to the
5-OH phosphate through two step mechanism. AMP nucleotide that is attached with
lysine residue of ligase active site is moved to 5-OH phosphate in first step. Then this
AMP phosphate bond is invaded by 3-OH results in formation of covalent bond and
AMP is freed. AMP in ligase active site must be replenished by Adenosine
triphosphate to permit DNA ligase for carrying out further reactions (Rossi et
al.,2013).

Ligation Reaction mixture:

Table: Ligation reaction mixture contains
Reagents

Volume

Restricted PCR product

9l

Restricted plasmid DNA

1l

DNA ligase

0.5l

10x ligase buffer

2 l

35

Sterile water

7.5

Total volume

20l

Procedure:
The ligation mixture is prepared by using reagents listed in above table. Sterile water

is used to make up final volume i.e. 20 l.

Then the reaction tube is placed for 24hours at 4 oC,16oC or 22oC not at 37oC whichis
not suitable for enzymatic activity.Temperature selection depends on desired efficiency
of ligation (Laemmli, 1970).

2.6.Transformation
A process via exogenous DNA or foreign DNA is introduced into host cell by force is
familiar as Plasmid or vector transformation.Most of the cells are unable to take up
DNA effectively without special electrical or chemical treatments

to build up

competence. However, some bacterial types are instinctively transformable means

they can accept DNA from their environment without applying special treatment
(Chen and Dubnau, 2004).It is necessary that cells must be competent if not then cells
36

are made competent artificially by following different methods.Competent cells are

those which has ability to take up foreign free genetic material and this ability is
considered as competence (Lorenz and Wackernagel, 1994).
Methods of making host cell competent:
Methods like chemical transformation, particle bombardment or electroporation is
used to introduce DNA in host cell.

Chemical transformation:

In it, cells are soaked in calcium chloride (CaCl 2) solution which has high
concentrations of divalent calcium cations. The permeability of bacterial cell wall is
enhanced by creating pores in it and cells are made competent.

Heat shock:

Heat shock is utilized to create temporary pores in bacterial cell membrane,

permitting the transfer of foreign DNA into the host cell. A short electrical pulse is
applied to make the cells competent (Froger and James, 2007).

Fig: Overview of CaCl2 treatment and heat shock method to produce competence in
cells.

Particle bombardment:

It is specifically used to make plant cells transformed. Tungsten or gold particles

coated DNA physically forced into the host cell using gene gun.

37

DNA transformation protocol

Reagents

Volume

Component cells

20-50 l

Ligation reaction

1l

LB media

250-1000 l

Competent cells are taken out of -80C and thaw them on ice for 20-30 minutes.
1 L of ligation reaction is added to 20-50 l thawed competent cells in a falcon tube

or microcentrifuge.
The mixture is gently mixed by tapping the tube bottom a few times with finger.
Reaction mixture is incubated for 20-30 minutes on ice.
Mixture is exposed to heat shock by putting the bottom (1/2 to 2/3 ) of tube in water
bath at 42 C for 30 to 60 seconds (usually 45seconds is recommended but it depends

on the competent cells which are used in transformation) for incubation.

Tubes are placed on ice in order to incubate for 2 minutes.
250-1000 L of LB media is added to the mixture, placed at 37C for 60 minutes
and shaken vigorously (250 rpm) or rotate.

Cells are thoroughly mixed by flicking and inverting the tube, then several 10-fold
serial dilutions in LB are prepared.

Spread 10, 50, and 100 L of transformed cells on selection platesto obtain

transformants.
Incubate plates at 30 C overnight.
Colonies that formed clearing zones in the medium are selected. Cells are harvested
by performing centrifugation using microfuge (9000 g at 40C for 5 minutes) and
supernatant is taken for enzyme assay (Radha and Gunasekaran, 2007).

38

Fig: transformed cell is shown above.

Enzyme Assay:
39

Enzyme assay

refers

enzyme activity.

This

to laboratory method
assay

measures

for

either

evaluating

the

substrate

consumption or product yield over time.There are basically two

types of enzyme assays:Continuous assays:Spectrophotometric,
calorimetric,

chemiluminescent,

thermophoresis,

light

Discontinuous

scattering,

microscale

assays:Radiometric

and

chromatographic assays. Continuous assays are convenient, with

one assay which gives the reaction rate with no further work
essential. Discontinuous assays are when samples are taken from an
enzyme

reaction

consumption

or

at

intervals

product

and

production

the
is

amount

of

calculated

substrate
in

these

samples.There are various methods for performing enzyme assay;

the choice of method depends on its feasibility and possibility in lab
(Bissanger, 2014).
Keratinase Assay:
Keratinase activity is monitered with azokeratin used as substrate(Sangali and
Brandelli, 2000).The reaction mixture tube carried the enzyme preparation that is
100 mL and azokeratin (500 mL of 10 g L-1)in tris buffer (50mM, pH 8). The mixture
is incubated for 15 min at 45C and the reaction is terminated by adding
trichloroacetic acid (TCA) and its final concentration is 60 g L -1. After centrifugation
at 10,000 g for 5 minutes the absorbance of supernatant is examined at 440 nm using
spectrophothometer. A control sample is arranged by adding TCA to a reaction
mixture before adding the enzyme solution. One unit of keratinase activity is the
amount of enzyme that produces an increase in absorbance (A 440) of 0.01 for 15
minutes at 45C.Keratinase activity is expressed by KU.A similar protocol is used to
check keratinase activity on azocasein as substrate (Riffel et al., 2003).

40

3.PART B: INDUSTRIAL
APPLICATIONS
Recombinant keratinases can be extensively used in the areas which require the
degradation of various tough proteins such as hair, feather, hair and nails that
cannotbe targeted byconventional proteases and other chemical procedures. After the
process of recombination and its higher yield, keratinases are used in wide range of
areas such as:

Bioprocessing keratin rich wastes

Leather Industry
Textile Industry
Detergent Application
Removal of Earwax
Pearl Bleaching
Cosmetics or personal care products
Processing of Edible Birds Nest
Keratinases for transungual drug delivery
Removal of Corn and Callus
Acne treatment

Bioprocessing Keratin Rich Wastes:

Wastes which are keratin rich in the form of nails, horn, hair and feathers are
available in high amount in the form of byproducts of agro- industrial processing. The
highly increased needs for the energy recycling and serving, summed with the result
of large increase in poultry industry, have powerfullystirred the search of the
alternatives to control the enormous recalcitrant keratinous wastes. The use of
keratinolytic microorganisms ascended as an important substitute for recycling keratin
containing byproducts, specifically from the leather and poultry industries. The
advancements in bioprocesses that can change the large amounts of these byproducts
into highly valueable products have been examined (Zaghloul et al. 2004; Bertsch and
Coello 2005; Grazziotin et al. 2007). The capacity of most of the keratinase enzymes
to hydrolyze various substrates (Lin et al. 1992; Bckle et al. 1995; Brandelli 2005)
shows the capability of such enzymes for the bioconversion of keratin containing
wastes to use in feed or other valueable products. Keratin signifies nearly 90% of the
41

total weight of feather, which makes up to 10% of the total weight of the chicken. The
high amountoffeathers produced bypoultry processing commercially may characterize
a pollutant addition in environment thus needs satisfactoryadministration (Shih 1993).

Fig: Chicken wastes such as feathers and nails

Presently, feathers are converted into feather meal by the process of steam
pressure cooking, which requires the input of high-energy. Feather meal has been used
in a limited amount as aconstituent in the animal feed, as it contains methionine,
histidine, and tryptophan in a very low amount (Papadopoulos et al. 1986; Wang and
Parsons 1997). The consumption of keratinase enzyme to increase the nutritional
value in feathers or in feather meal has been recorded (Onifade et al. 1998; Grazziotin
et al. 2006). Comparable rate of growth was also observed between the chickens
which fed with only soybean and those which fed with the feather meal fermented
with the Streptomyces fradiae plus the supplementation of methionine (Elmayergi and
Smith 1971). The utilization of the crude keratinase enzymeconsiderably increased the
digestibility of amino acid of the commercial feather and raw feathers meal (Lee et al.
1991; Odetallah et al. 2003). The optimization of the conditions of experiment for the
hydrolysis of keratin by the keratinase ofDoratomycesmicrosporum was studied using
the twoorder experimental design. Abetter quantity of the soluble protein was attained
when thioglycolatedhooves or nails were treated with the enzymekeratinase
(Vignardet et al. 2001). The protein hydrolysatesfound from the submerged
cultivation of keratinolytic bacteria on the feathers of poultry show increased
nutritional value in feather keratin (Bertsch and Coello 2005; Grazziotinet al.2006).
42

The use of keratinolytic bacteria to produce the feather hydrolysates has been the
subject of some of the patented processes (Shih and Williams 1990; Burtt and Ichida
1999), and the keratinase from B. licheniformis PWD-1 is produced under the trade
name Versazyme commercially. The production of keratinase enzymes using the raw
feathers or feather meal as basis for the culture media has been described, and various
factors such as feather concentration, pH, temperature and inoculum can affect the
enzyme yield results (Wang and Shih 1999; Brandelli and Riffel 2005). Recently, the
use of statistical optimization by the response surface methodology have been used
for the production of bacterial keratinases during the growth on raw feathers
(Ramnani and Gupta 2004; Thys et al. 2006). Factors such as media composition and
temperature considerablyeffect the production of the keratinase enzymes, which could
be improved after optimization upto nearly 40-fold. Production of leather yields the
substantialamounts of organic wastes, a major portion of which comes from the
degraded keratin. Biotechnological possibilities are available for handling the
effluents and the proteinaceous solid waste (Thanikaivelan et al. 2004).
Biodegradation of the waste from the leather industry has been described, as the
ability of Bacillus and Streptomycesin order to hydrolyze wool and hair keratins
which has been already described (Mukhopadhyay and Chandra 1990; Takami et al.
1992b; Lal et al.1996). The bacterium S. fradiaesignificantly cause the degradation of
the complex morphological structure of hair, feathers and wool substrates by the
combination of enzymatic and mechanical and activity, resulting in the release of the
soluble protein during the process of fermentation (Hood and Healy 1994). Shama
and Berwick (1991) have described the production of bioreactor of a rotating frame in
which wool substrate was totally solubilized by S. fradiae. Now adays, the industry of
animal feed is the main consumer for the keratin hydrolysates from agro-industrial byproducts. The recycling of feathers is a matter of great interest for the nutrition of
animal because of its potential as well as an alternative and inexpensive protein
source. Despite of the limited nutritional values of the keratin, both the balance of
feather protein and digestibility of amino acid might be improved by the process of
microbial fermentation (Williams et al. 1991; Grazziotin et al. 2006). Keratin rich
wastes have also been considered as the soil fertilizers. Several organic materials for
the animal origin, such as hooves, feathers, horn, manure, bones and blood meal have
been evaluated as slow release nitrogen fertilizers. However, about 70% of the
nitrogen is released during the first 30 days in the field condition, and except for the
43

chicken feathers, total release of nitrogen occurs between the 6 and 7 weeks (Williams
and Nelson 1992). In case of feather meal, which suffers anwide-ranging thermal
treatment, the total release of nitrogen also occurs at 67 weeks (Hadasand Kautsky
1994). Feathers containalmost15% of nitrogen, possessing high potential to be used as
slow-release of nitrogen fertilizer. The slow-release of the nitrogen from raw feathers
specifies that the microorganisms of soil could not digest the keratin structure easily.
However, if the keratin structure is further modified by the disturbance of chemical
bonds, the rate of mineralization increased. The modification in keratin structure can
be achieved by the enzymatic hydrolysis and thermal treatments with the cleavage of
peptide bonds and disulfide bridges (Williams et al. 1990; Kim et al. 2005). An
additional substitute is the formation of Schiff base between the amino groups of
keratin with the aldehydes (Means and Feeney 1998), resulting in the new chemical
bonds that subsidize a slower release of nitrogen (Choi and Nelson 1996). In this case,
the use of keratinolytic microorganisms may characterize an alternative for the
development of nitrogen sources for the utilization of fertilizers. The featherdegrading bacteria enzymatic capability to increase the composting of feather waste
or dead chickens could be an environmentally safe and economical method of the
recycling of these organic materials into fertilizers containing high nitrogens (Ichida
et al. 2001).

Leather Industry:
Leather processing consists of four major steps

Soaking
Dehairing
Bating
tanning

Conventionally, soaking is performed by using alkaline conditions; dehairing is

based on sulfide, while bating and tanning includes the use of solvents, salt and lime.
The use of these harsh chemicals makes the leather industry highly polluted that
creates the disposal problems of major effluents (Madhavi et al. 2011). The use of
various enzymes is considered as a greener substitute leading to the reduction in the
pollution of environment and improved the quality ofleather. Among the different
enzymes, protease has been familiar as a potential dehairing enzyme. A number of

44

various commercial proteases such as, Pyrase, NUE,Aquaderm and Clarizyme

are available, which are mainly used in soaking, bating and dehairing.

Fig: Dehairing of leather during processing

However, the complete alternative of chemical processes by the enzymes is noteasy
step due to economic strategy of the enzyme based processes. This issue can be
addressed by the consumption of those proteases which have improved specificity
towards hair, and are catalytically more proficient, and will be required in lesser
amount.

Moreover,

keratinase

enzymes

missingcollagenolytic,

and

having

lowelastolytic activities are progressively being discovered for the dehairing process.
Such enzymes could help in the breakdown of keratin tissues in the follicle
selectively, thus, pulling out integral hair without the alteration in the tensile strength
of leather (Macedo et al. 2005). As for the industry of leather, keratinase enzymes
from Bacillus subtilisS14 (Macedo et al. 2005), B. subtilisKD-N2 (Cheng- gang et al.
2011; Cheng-gang et al. 2008), Bacillus sp. PPKS-2 (Prakash et al. 2010),
Trichodermaharzianum MH-20 (Ismail et al. 2012), Aspergillusnidulans (Nashy and
Ahmady 2012), Paenibacilluswoosongensis TKB2 (Paul et al.2013) shows the
remarkable dehairingability without the degradation of the collagen molecule.
Thedehairing which is enzyme-based thus, completely removes the need of toxic
sodium sulfide chemical during the processing of leather. This eco-friendly, enzymebaseddehairingapproach has various advantages, mostly, on the final quality of leather
product, also eliminating the problems of environmental pollution caused due to
traditional chemical processes, and their waste by-products.

Textile Industry:
Wool is a structural protein fibrous material characterized by high degree of
various cross-linked disulfide bridges (SS) which confer the resistance to
degradation by different proteases and mechanical strength. This is also the same case
45

of the overlapping layers of the cuticle characterized by epicuticle, exocuticle and

endocuticlethat cover the outer surface of fibers. Epicuticle is rich in lipids while
exocuticle and endocuticleconsist of keratin which is rich in disulfide bonds. The
structure comprises the cuticle plays a vital role in felting the shrinkage of fibers
during washing process and also alters their property of dyeing. Chlorine-Hercosett
process involves the use of the organic chlorides which are absorbable (AOX), has
been used in wool processing since the last 30 years in order to control the felting
shrinkage process of wool fibers. Bu this treatment leads to the loss of weight of the
fibers along with the disposal of chemicals in the environment which are hazardous to
our health. Other chemical based fundamental finishing processes, including
bleaching, dyeing or cleaning also become the cause of the discharge of many toxic
chemicals. In addition, these processes are not only time consuming and energy
intensive, but also responsible to degrade the basic material. Use of enzyme based
process is considered as an ecologically safe substitute and many protease-based
commercial products such as:

Alcalase (from B. licheniformis; Novozymes)

PeriZyme Tuggummi (Enzymatica AB)
Savinase (from Bacillus sp.;Novozymes)
Type EX (Bacterial subtilisin; Novozymes)
Esperase (from Bacillus sp.; Novozymes)

These enzyme based processes have been discovered in many textile finishing
processes since the last decade. On the other hand, in addition to removing of the
cuticle, proteases are able to penetrate deeply inside the wool fiber thus damaging it
and causing the loss of tensile strength and weight of the fiber (Shen et al. 2007).
Upto some degree, this problem has been enhanced by increasing the molecular
weight of protease enzymes by the modifications in the chemical structure using
polymers like PEG (Silva et al. 2005),Eudragit S100 (Silva et al. 2006; Shen et
al.2007) orglutaraldehyde (Silva et al. 2004). By using such keratinases that could
specificallytarget the scaly keratinous layer of the wool without the damageof other
parts of the fiber may be a substitute of our choice. Several keratinase enzymes from
B. thuringiensis L11 (Infante et al. 2010),Pseudomonas sp. (Cai et al.2011),B.
licheniformis (Liu et al. 2013), ChryseobacteriumL99 (Lv et al. 2010), Fusarium
(Noriyuki

et

al.

2003)Bacillus

cereus

(Sousa

et

al.

2007)

andStenotrophomonasmaltophilia DHHJ (Cai et al. 2008) have been confirmed to

46

enhance dyeing property and felt-shrink resistance without the loss of weight of fiber.
The action of keratinase enzymes has been enhanced further by combining
themwithorlipase (Hossain et al. 2008; Wang et al.2010) orcutinase (Wang etal. 2009;
2011a, b). The strength of fiber has also been enhanced by using transglutaminase
(Cortez et al.2002;Cardamone 2007). Thus, keratinase enzymes alone or in
combination form with other different enzymes can help in the development of
desirable formulations for the improved processing of wool.
Raw silk needs the process of degumming to remove fibrous proteins, sericin
thatcements the fibroin fibers with each other, soastoprovide the fibers actual luster
and softfeel. This process isalso vital for successive dyeing. The conventional
degumming methods are the treatment with oxidizing agents,alkali and soap at higher
temperature under the process of agitation. These conditions, on one side are
environmentally unfriendly, and on the other side change the chemical and physical
properties of the fibers which leads to the degradation of basic material of the silk
(Freddi et al. 2003). Enzymatic treatments such as by the way of proteases such as:

Alcalase
Degummase
Papain
Trysin
Pepsin
Alcalase
Savinase
Protease A Amano
Protease N Amano
Protease M Amano
Palkobate

They are now in main focus over the old methods (More et al. 2013; Sumanaet
al.2013). However, most of protease enzymes are characterized by the low degree of
specificity towards the sericin (Freddiet al. 2003). Thus, enzymes which have better
specificity are now the need of hour and keratinase enzymes with their wide range
substrate may prove to be feasible substitutes.

Detergent Applications:
Proteases have been of much importance to the detergent industry from a very
long time in order to assist in the removal of proteinaceous stains. Though, keratinase
proteases are supposed to have better property of detergency due to the fact that they
47

are wide range proteases with improved specificity of substrate for both insoluble and
soluble protein substrates. They can easily do the hydrolysis of the fixed proteins
present on the surface, and also can remove stains which includethe keratinous blood
stains, cuffs and soiled collars(Gupta and Ramnani, 2006). For this purpose,keratinase
enzymes

fromBacillus

thuringiensis,Paecilomyceslilacinus,

Bacillus

pumilus

andBrevibacillushave been discovered already as effective detergent additives (Rajput

et al. 2010; Sivakumar et al. 2013; Rai and Mukherjee, 2011; Cavello et al. 2012).
Another approaching application of keratinase enzymes is in the region of
cleaning up drain pipes and outlets which are clogged with hair and various other
keratinous materials. Researchers all over the world have shown their interest in the
discovery and development of this application. Itsune et al. (2002) have established a
composition of cleaning agent which is prepared by compounding a keratinase with a
non-sulfur reducing agent which has ablility to clean slimy nature matter attached to a
drain outlet of bathroom or dirt due to the pollutants such as hair and scales, with high
effectiveness in a safe manner. A commercial product, BioGuard Plus is also
available which incorporates a mixture of various enzymes including keratinases for
the cleaning of drain pipes and tanks.

Fig: Keratinase containing Detergent: BioGaurd Plus

Removal Of Earwax:
Earwax which is also known in a medical term ascerumen, is a hydrophobic
protective covering in the ear canal of humans and other mammals. Earwax basically
constitutes various shed layers of skin, in which 60 % of the earwax made up of
keratin. Cerumen build-up material is mainly treated by softeners having
cerumenolytic

agents

consisting

of

glycerine,carbamide

peroxide,

sodium

bicarbonate, turpentine,arachis oil, hydrogen peroxide, urea anddichloroben- zene.

48

Fig: Earwax also known as Cerumen

Cagleet al. (2001) have revealed the composition for the removal of human
cerumen that consists of bicarbonate in union withcombination of various enzymes
including amylase, keratinase and lipase. Among keratinases, they have discovered
the possibility of using various enzymes such as:

Pancreatin
Trypsin
Subtilisin
Collagenase
Keratinase
Carboxypeptidase
Papain
Bromelain
Aminopeptidase
Elastase

The enzyme-based cerumenolytic compositions are viable, effective and safe on

commercial scale to remove thecerumen from the region of external ear canal. The
high keratin concentration in cerumen makes keratinase or keratinolytic protease very
probable candidate to be discovered further for this and other applications.

Pearl Bleaching:
Pearls are formed as a result of defense mechanism action against potentially
aggressive irritants in the living shelled molluscs such as the oysters. Nacre or
Mother of pearl is an organic substance secreted in the result over the obtrusive
irritant. Nacre is primarily composed of conchiolin which is dark colored organic
protein and crystallized form of calcium carbonate. During the formation of pearl,
49

organic impurities like free cells of pearl oysters of mother, pieces of necrotic and
mucilage part of mantle tissue are thus trapped in the forming pearls and they need to
be processed in order to enhance the quality of gem.For this purpose, pearls are
mainly subjected to bleaching treatment process that aids in their whitening;
diminishes their color irregularities and disabling the brown color of conchiolin
protein. The different forms of bleach used are very mild in their naturelike hydrogen
peroxide which allows the gentle lightening of pearl nacre without causing the
damage to its quality as the pearl surface is contaminated with organic impurities like
mucilage, tissues and cells. Zhang et al. (2010) have discovered the use of keratinase
enzymes during the process of pearl bleaching. Keratinases can also be used during
the initial treatment in order to remove impurities of keratin which is present on the
surface of the pearl followed by the conventional processing such as bleaching
methods.

Fig: Process of Pearl Bleaching

Cosmetics or Personal Care Products:

Keratinases have been adminsterred in various cosmetic formulations for hair
and skin. For skin, keratinases have been added in the compositions for following
purposes:

Skin whitening
Freckle dispelling
Bleaching (Yang 2012).

50

Keratinasescanalsobe mainly usedfor the removal and exfoliation of stratum

corneum as described by Yongetal. (2009) and Ding and Sun (2009).
For hair, keratinaseshavebeen mainly addedto the hair compositions such as hair
gel, shampoo and conditioner where they play dual functional role in improving hair
quality, luster and color with instantaneous removal of impurities adhered on hair
surface and cleaning (Mo and Sook, 2010).

Fig: keratinase containing hair treatment product

Processing of Edible Birds Nest:

Nests which are built by few species of swiftlets are used by humans all over
the world, as a medicinal food and as high-value fragility. These nests are made up of
gelatinous substances; contain interwoven feather material and plumage in the form of
impurities.Before consumption, they are cleaned to remove the impurities by
processes such as hand picking, warm water treatmenr and sieving that are often
ineffective and time consuming (Marcone, 2005). The extraordinary costant demands
from public for these nests have also urge their manufacturers to adopt some unsafe
practice measures such as treatment with silicates and peroxides. Sinceplumage and
feather are major impurities, preparations based on the specificity of enzymes such as

51

keratinases, that can selectively attack on insoluble proteins may also prove to be an
efficient way in the processing of these nests. Although, large amount of research is
needed conduct in order tovalidate this procedure, and these enzymes have to acquire
GRAS (Generally Regarded As Safe) status to be used for food application.

Fig: Processing of Edible Birds Nest

Keratinases for Transungual Drug Delivery:

There are various nail disorders ranging from relatively mild conditions such
as pigmentation, to debilitating and painful states in which the nail unit can be
hypertrophied, infected, inflames anddystrophied (Kumar and Raju, 2013).Themost of
the common nail fungalinfection isonychomycosis, which has a very high prevalence
rate with nearlyupto 700 million people suffering from this disease all over the world.
Many of the nail diseases are muchdifficult to cure especially those in which infection
happen underneath of the nail plate.

52

Fig: Fungal infection of Nails such as Onychomycosis

These type of infections require a long period for treatment and also face a
main problem of reoccurrence.At this time, available treatment administrations for
nail infections include topical and oral medications. Oral therapy has the characteristic
disadvantage of various side effects ranging from mild such as pain, skin rashes to
extreme such as liver damage, and furthermore, interaction of drugs can also happen
(Murdan, 2007;Mohorcicetal, 2007). On the other side, topical application of the
drugs is a more beneficial option because of theirnon-invasiveness property,
elimination side effects, increased patient compliance and drug specificity (Rajendra
et al. 2012). Unfortunately, many of the topical nail drugs have limited effectiveness
because of poor permeability of drug across the nail plate. Currently, different
chemical, physical and mechanical methods are being used for trans-ungual delivery
of the drugs for topical infections (Rajendra et al. 2012).

Mechanical methods,such as nail avulsion and nail abrasion are painful and

invasive.
Physical methods include carbon dioxide laser, etching, occlusion

andhydration.
Chemical methods include the use of various keratolytic agents like urea,
salicylic acid, papain and thioglycolic acid in the combination with oxidizing
agent such as hydrogen peroxide.Chemicalssuchas thioglycolicacid, N-acetyl
cysteine, N-2 mercaptoethanol, mercaptoethanol are used at different times in
53

order to damage the nail surface and increasing the permeation of nail plate
(Malhotra and Zatz, 2002).
These chemicals are acidic in nature, have a potential to react with certain drug
combinations and have pungent odor. The disadvantages of already present methods
can be efficiently decreased by the use of various keratinases. Keratinase enzymes can
act as molecular scissors which cleavethe tough keratin protein: the major part of nail
plate, thus loosening the plate toughness and increasing trans-ungual permeability of
drug (Gradisar et al. 2005;Mohorcicetal, 2007). Keratinases act on boththe dorsal nail
corneocytes by the mechanism of corroding their surface as well as the intercellular
matrix which holds the cells of the nail plate together (Rajendra et al.
2012).Keratinasefrom Paecilomycesmarquandii was revealed to disturb the nail plates
partially and also increase the permeability of drug (Mohorcic et al. 2007).
Acomplexof subtilisin--glutamyltranspeptidase such askeratinaseKerN has also been
shown to increase the drug delivery through nails (Tiwary and Gupta
2010).Moreover,afewkeratinasebased commercial preparations such asPure100
Keratinase,Kernail-Soft PB andFixaFungus TM, are offered in the market for the
treatment of various nail infections.
In addition to the treatment of nail disorders, keratinases can also be used for
permeabilization of skin tissues to increase drugdelivery through various surfaces of
skin. It has been reported that thekeratinolytic agents are responsible to remove
hyperkeratolic lesions, thus, improving the exposure of infected skin surface to the
various topical drugs (Aly, 1996). Susumu et. al., (1998)establishedaskinagent
through a process of immobilizingkeratinasetoward a porous sheet that loosens the
skin, thus, improves permeability of drug administerred. To achieve the actual
potential of keratinases as effective skin tissue permeabilizers and ungual enhancers,
extensive research is needed to be done. Keratinases has become an essential additive
of existing nail lacquers. Absence of proper in-vitro procedures to measure the degree
of permeation of drug is the major challenge in this field. Much strategic methods
along with animal and human trials are needed to be carried out for the
commercialization of more keratinase based drug delivery protocols.

54

Fig: Medicinal use for the treatment of skin Fungal infections

Removal of corns and calluses:

Corn and calluses which are also referred as hyperkeratosis are painful
thickenings of the dead skin that mainly form on the dorsal surface of fingers and
toes.

Fig: Corn and Callus disease

For the treatment of these thickenings, podiatrists usually prescribe the use of
a keratinolytic agent like salicylic acid. It mainly dissolves the keratin layers that
usually constitutes the major part of corn and thick layer of the dead skin. The use of
keratinase enzymesis a natural and greener substitute to the use of chemical
treatmenti.e. salicylic acid. Different research groups have demonstrated the use of
keratinases in order to remove the horny layer of skin (Encarna and Elena,
2011;GuptaandRamnani, 2006).Inaddition to these, Proteos Biotech has launched
55

two keratinase-based cosmetic products such asPure100 Keratinase and Keratoclean

Hydra PB that have proved their effectiveness in the treatment of corns and calluses.

Fig: Treatment for the removal of Corn and Callus

Acne treatment:
Acneisa very commonskinproblem thatoccursdue to the blockage of sebaceous
gland because of the presence of excessive keratin on skin surface (Selvamand
Vishnupriya, 2012).As we know, keratinase enzymes have the ability to dissolve dead
cells and keratin fibers which cause the cloggingof sebaceous glands, they can be
easily applied for the treatment of acne. A keratinase based product which can be an
effective adjuvant in the therapy of acne has been patented in the year of 2001
(Spyros, 2003).Keratopeel PB and Keratoclean Sensitive PB are commercial
products which are available for gentle enzymatic peeling. Inthesamelines, keratinases
can also able to find applications in the areas of epithelium regeneration and scar
removal.

56

Fig: Acne and its treatment

57

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