A
well-known protein named as keratin is mostly found in skin, hair, wool and
feathers. They form major components of epidermal and skeletal tissues. They
are one of the most stable proteins because of disulfide and hydrogen bonds,
properties that contributes to their resistance against proteolytic hydrolysis
(Arai et al., 1996; Jones et al., 1999; Tamilkani.P. et al., 2017).

Due
to large number of disulphide bridges, Keratin is abundant in sulfur compounds,
which is responsible for its insoluble nature. Cystine, lysine, proline and
serine amino acids are present most dominantly. Humans and other higher
vertebrates are unable to digest keratinous material (R.Kanchana, 2012; SubashC.B.Gopinath
et al., 2015)

DETAILED STRUCTURE OF KERATIN

Keratin
a fibrous protein is included among intermediate filament protein that give
rise to ?-keratins and ?-keratins polypeptides of 40-70 kDa and 10-20 kDa
molecular masses respectively (Fraser and Parry, 2011; Saucedo-Rivalcoba et
al., 2011; Patrícia Aline Gröhs Ferrareze, 2016). It is unreactive against many
chemicals (R.Kanchana, 2012). Trypsin, pepsin and papain are some of the most
common proteases to which keratin is resistant and this is largely due to its
unique properties that include compact packaging of ?-helix (?-keratin) or
?-pleated (?-keratin) structures along with polypeptide chains folding which is
mainly the contribution of disulfide bonds, hydrogen bonds and hydrophobic interactions
(Brandelli, 2008; Patrícia Aline Gröhs Ferrareze, 2016).  Two types of keratins that is Light, mainly a
component of stratum corneum , and  Rigid
such as hair and feather are distinct from each other because of  the contribution of cysteine percentage and
consequent disulphide bond formation. Soft keratins can have up to 2% cysteine,
whereas hard keratins can constitute10% to 22%. Keratin preset in bird feathers
is considered light weight and resistant due to structural differences. (Brandelli,
2008; Korni??owicz-Kowalska and Bohacz, 2011; Patrícia Aline Gröhs Ferrareze,
2016).

 

Feathers
are made up of almost crude protein keratin (90% or more) and are the
byproducts of poultry processing plants (Sanaa Tork et al., 2013). In order to
cope up with the rising demand for cheap and safe supply of meat and eggs the poultry
industry has made some astounding adjustments. Over the past three decades, the
poultry sector has been rising at a rate of 5 percent per annum  (in comparison with pig and bovine meat) and
its contribution in world meat production increased from 15 percent three
decades ago to 30 percent currently (FAO, 2006a;  Viale delle Terme di Caracalla, 00153 Rome,
Italy). The increase use of poultry farm facilities means greater quantities of
feathers i.e increase environmental issues (Sanaa Tork et al., 2013; Viale
delle Terme di Caracalla, 00153 Rome, Italy; Tamilkani.P. et al., 2017).

 

The
industrial use of keratin as a raw material is the major source of its
accumulation in environment. Dumping of this feather waste is also carried out
by poultry farms. Such disposal along with natural falling of bird hairs and
feathers can lead to serious pollution problems (M.N.Acda, 2010; SubashC.B.Gopinath
et al., 2015). A rapid action is required to overcome this situation that must
be eco-friendly, can be done at lower cost and has easy processing set up.
Fortunately, microbial keratinase may prove to be a better option.

Keratin
is an animal protein that doesn’t degrade easily. This complexity and stability
is achieved by high cross linking disulfide bonds, hydrophobic interactions and
hydrogen bonds. Currently the most common treatment of feather includes its
conversion to feather meal by steam pressure and/or chemical treatment which
can unfortunately lead to environmental concerns as they contribute to
pollution and can even cause damage to some of the useful amino acids such as methionine,
tryptophan and lysine. Such an alarming problem appeals for an environmentally
friendly solution making microbial fermentation methods the most suitable
option (Onifade et al., 1998; Ramakrishna Reddy, 2017).

 

Among
the list of diverse proteases for feather degradation, Keratinases have
successfully gained special attention in last few years. They are extracellular
enzymes categorized as serine or metallo-endoproteases. They have molecular
weight ranging from 30-90kDa and can act as both protease as well as disulphide
reductase (Paul et al., 2016; Ramnani et al., 2005; Ramakrishna Reddy., 2017).

 

Keratin
substances can be broken down by enzymes known as keratinases. In the process
of degradation these enzymes have the capability to release free amino acids
from keratinous proteins. Some of their essential uses in biotechnological
field include dehairing, catalysis in leather and textile industries, low
release nitrogen fertilizer application, cosmetic, and production of biodegradable
films and foils. (Mohamed A.Abdel-Naby, 2017; SubashC.B.Gopinath et al., 2015). Regarding
keratinases, more researches are being done due to its large variety of
applications in different sectors. Animal feed industry, fertilizers,
pharmaceutical sector and as dehairing enzyme in leather industry being the
most prominent one (Allpress et al., 2002; Anbu et al., 2005; Syed et al.,
2009; Tamilkani.P. et al., 2017). The most important reason of considering the
use of enzyme in detergent industry is because of its non-toxic, eco-friendly
and biodegradable property in clearing proteinaceous stains from clothes (Paul
et al., 2016; Ramakrishna Reddy., 2017).

The
ability of keratinase to degrade feather by disintegration of barbs and shafts
without  any redox or pre-treatment makes
it a promising tool for prion degradation (Ekta Tiwas and Rani Gupta, 2010;
Tamilkani.P. et al., 2017).

 

MICROBIAL KERATINASES

Microbial
strain, medium composition, fermentation method, pH, temperature and aeration
are some of the key factors responsible for highly active keratinase enzyme
production (Verma et al , 2017; Ramakrishna Reddy., 2017)

 

High
amount of protein in poultry farm waste makes it a valuable source of protein
and amino acids that can be utilized in animal feed and many other
applications. Although, it’s greater stability makes it difficult to degrade
keratin but proteases such as keratinase can be a solution to this problem.
Recent studies are targeting bacterial origin keratinase for the industrial
treatment of keratin containing materials (Bharti Agrawal, 2015). This
degradation has gained importance from medical point of view too (Shih, 1993;
Matsumoto, 1996).

 

FUNGAL KERATINASES

Decomposition
of keratinic waste using keratin as a source of carbon and nitrogen is a
characteristic observed by keratinophillic fungi using a type of proteolytic
enzyme (M.A.Ganaie, 2010; SubashC.B.Gopinath et al., 2015). This kind of fungi
consumes keratin as a food material, multiply asexually producing conidia.
Boring hyphae that are formed during colonization get into the keratin
substrate in order to carry out degradation. Hyphomycetes including both
dermatophytic and non-dermatophytic fungi can have keratinophillic property. (R.Kumar,
2013; Subash C.B.Gopinath et al., 2015)

 

The
identification of kertaninophillic fungi is carried out by observing
morphological characters of macro and micro conidia, molecular techniques and
examining DNA sequences (K.Pakshir, 2013)

 

Enzymes
are considered as one of the potential virulence factor in causing
pathogenicity against host skin (Matsumata, 1996), Keratinases being the most
important one (Howard, 1983).  Keratinases produced by dermatophytes are more
particularly famous for their pathogenic character (Sohnle and Wagner, 2000).
Their role in biotechnological industry have recently been recognized because
of their keratin degrading property into energy and cost effective keratin end
products (Onifade et al. 1998; Lin et al. 1999; Riffel et al. 2003) that can
further be utilized in making of nitrogenous fertilizers, biodegradable films,
glues and foils (Friedrich and Antranikian, 1996; Schrooyen et al. 2001)

BACTERIAL ORIGIN KERATIN DEGRADATION

Among
bacteria, fungi and actinomycetes many microorganisms have been able to produce
keratinases but in comparison Bacillus strains have gained much more popularity
in degrading keratin effectively (Brandelli et al., 2010; Ramakrishna Reddy.,
2017).

 

Among
bacteria, genus Bacillus has been most popularly known regarding the production
of extracellular keratinases. Many researches have successfully demonstrated
the property of degrading keratin by Bacillus species by adjusting different
growth related parameters (Wojciech ?aba, 2010; Ramnani P 2005; Ghosh A, 2008;
Samuel Pandian 2012; SubashC.B.Gopinath et al., 2015)

 

Same
as fungi, number of bacterial strains have also been known to degrade keratin.
Due to their small and speedy reproduction cycle they are much more favorable
in industrial applications as compare to fungi, although easier colonization of
fungal hyphae into the harder keratin gives a plus point for fungi. The most
famous commonly known bacterial isolate, involved in keratin degradation or
keratinase production, is the genus Bacillus;
which includes B. subtilis and B. licheniformis (V.Matikevicien, 2009; SubashC.B.Gopinath
et al., 2015).

 

Numerous
researches have reported the genus Bacillus, which is one of the most commonly
available bacteria, as good keratin degrader. This study is carried out to
detect the presence of any such type of keratinophyllic bacteria that might be
of great use in biotechnological industry as well as for controlling
environment pollution.

 

The
Present study was carried out to detect and isolate keratnohillic bacteria by
using keratin baiting system in which raw chicken feather was used as a
substrate in order to be degraded by enzyme keratinases. The method consisted
of screening proteolytic and keratinophillic properties of the organism on skim
milk agar and modified basal liquid medium respectively. Three isolates that
formed clear zone on skim milk agar were considered positive for proteolytic
activity and were further screened for keratinase production. According to the
research performed in Tamil Nadu, India using the same method, isolates showed notable
keratin degrading property in 15-25 days (Tamilkani.P. et al., 2017). In
comparison, when this study was carried out in Karachi, Pakistan the results
were negative for keratinophillic activity.

 

Although
the results of this study showed negative results as no visible keratin
degradation was observed but there are variety of factors that need to be taken
into account. The environment, nutrients and growth condition vary for
different species. It has been suggested that microbial strain, medium
composition, fermentation method, pH, temperature and aeration are some of the
key factors responsible for highly active keratinase enzyme production (Verma
et al , 2017; Ramakrishna Reddy., 2017).

 

Soil
samples in this particular work were taken from different poultry farms of
Karachi and were processed under general conditions on room temperature,
depending upon the microbial species; it is possible that they might have
required different temperature and PH for keratinase production. To support
this idea, reference can be made with respect to temperature. (Worapot et al.,
2005 and Xiang et al., 1992) concluded from their research that thermophillic Bacillus licheniformis will degrade
feathers efficiently on high temperature only. Whereas other study involving
Bacillus specie showed degradation on 25 to 40?C that is the normal temperature
of poultry waste soil (Bharti Agrawal., 2015).

 

In
Karachi, the keratin waste is not dumped and maintained as properly as in other
countries. Either the waste is burned or is directly used as animal feed. In
reality, it has been known that keratin substrate is necessary as inducer of
extracellular keratinase production (Gradisar et al. 2000) and many other reports
state the same. In addition, microbes utilize keratin as a unique carbon and
nitrogen source (Tamilkani.P. et al., 2017). Failure in dumping of keratinic
waste means no keratin material available long enough for bacteria to produce
keratinase for degradation and hence none was observed.

 

There
are many more keratin containing compounds present in nature including hair,
nails, wool and horns (hard keratins) and stratum corneum (soft keratins) of
animals and birds (Karthikeyan et al., 2007). The keratinolytic proteases made
by microorganisms help in degradation process by targeting peptide bonds of
fibrous protein. Keratinases along with disulfide reductases break disulphide
bonds that form the backbone of peptide keratin (Gupta and Ramnani, 2006;
Prakash et al., 2010).

 

Primary
screening involved detection of proteolytic natire of isolates by observing
clear zones around colony in skim milk agar same in accordance with A.Riffel and
A.Brandelli who also used soil samples containing feather waste and milk agar for
primary isolation (A.Riffel., 2006).

 

Soil
samples from poultry processing plants were processed in this study but sewage
sludge samples have also been a source of keratinophyllic fungi and good amount
of degradation was observed with chicken feathers (Itisha Singh., 2015). This
means other sources should also be examined and fungi species should also be
considered for production of enzyme for future studies.

 

 

 

 

Although
this study demonstrates negative results but the importance of keratinases in
terms of removing keratin waste and in other industrial applications cannot be
ignored. Many researches have proved the production of microbial keratinases
and successful degradation of one of the most stable protein keratin. These
keratinophillic microbes utilize keratin as sole organic source of carbon,
sulphur and energy. The successful degradation of keratin has been achieved but
still no such keratinases exist that can break keratin completely (Ignatova et
al. 1999; Ramnani et al. 2005). Moreover, purification of enzyme is a costly
method resulting in low yields, so utilization of crude enzyme is more
preferable (Paul et al., 2016). To make this possible, such strains are needed
to be recognized that can show promising results in terms of insoluble keratin
degradation by producing keratinases that can be of economic use.