CHAPTER TWO:

REVIEW OF LITERATURE
 Chapter 2: Review of Literature

2.1 Major landmarks in keratinase research:

Feather degrading microorganisms and keratinases have acquired an important
position in the enzyme industry owing to their ability to degrade recalcitrant keratin
proteins. This ability provides an edge to keratinases, over conventional proteases such as
trypsin and pepsin which are not capable of keratin degradation (Papadopoulos et al.,
1985). By virtue of this property, keratinases find major applications in the environmental
waste management and industrial sector. In waste management they can carry out the
biodegradation of keratin containing wastes accumulating from poultry, leather and other
related industries, thus cleaning up the otherwise undegradable keratinous waste. In
industry, keratinases have found applications in various sectors ranging from feed and
cosmetics to detergent, textile and leather industries (Macedo et al., 2005; Brandelli,
2008; Fang et al., 2013). Recently, keratinases have also been reported to carry out
degradation of prion and prion proteins that are responsible for diseases that affect the
brain and the nervous system. It was reported that keratinase from B. licheniformis PWD-
1 degraded prion protein in brain, stem tissue from animals with bovine spongiform
encephalopathy and scrapie (Langeveld et al., 2003).
So far, extensive amount of research has been carried out in the field of feather
degrading bacteria and keratinases, which can be reflected in the exhaustive reviews
published so far. The first review by Onifade et al., published in 1998, explained the
diversity of keratin degrading microorganisms. The potential of using microbial
technology and keratinase enzyme, as low-energy consuming technology for upgrading
the nutritional value of feathers and other keratin containing material has been reviewed
(Onifade et al., 1998). Another review by Gupta and Ramnani was published in 2006,
which explained the prospects of keratinases in industrial applications in varied sectors
such as production of cost- effective animal feeds and fertilizers, detergent and leather
industry, wool and silk cleaning. The review also mentions about the robustness of
keratinases by virtue of their characteristics with respect to wide temperature and pH
range for activity (Gupta and Ramnani, 2006). Brandelli A, 2008 has reviewed
keratinases with respect to different sources of enzymes, the ecosystems from which they
have been obtained, the biochemical properties of the reported keratinases and their
potential applications. Thus, over the years, keratinase research has increased steadily

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 Chapter 2: Review of Literature

into a vast field, providing several cost-effective solutions to environmental problems due
to keratin wastes and also several industrial applications.
Keratinase research first initiated with fungi capable to carry out degradation of
keratin-containing material such as nails, claws, hair, etc. These are dermatophytic fungi,
the presence of which depends upon their availability of keratin especially where human
and animals exert a strong selective pressure on the environment (Ulfig K, 2000).
Dermatophytic fungi are associated with dermatomycosis, in which, fungi belonging to
the genus Aspergillus spp., Trichophyton spp., Microsporum sp., etc. were included (Yu
et al., 1968, Asahi et al., 1985). Most of these fungi and their dependency and utilization
of keratin had pathogenic implications. Thus, research related to keratinase from fungal
sources was mainly a medical field.
The biotechnological and environmental relevance of keratinase occurred, with
the first report of bacterial keratinase- involving isolation of feather degrading bacterium,
Bacillus licheniformis PWD-1 from poultry waste digester (Williams et al., 1990). This
study was a major turning point in the field of keratinase enzyme with respect to its
application for biodegradation of recalcitrant proteins. The keratinase enzyme of this
bacterium was purified and extensively studied for its biochemical properties by Lin et
al., 1992. Also, extensive research was carried out involving this isolate for the microbial
conversion of feather waste to feather meal.
Further studies on Ker A, gene encoding keratinase were carried out with respect
to determining the nucleotide sequence of the coding and flanking regions. From the
sequencing results, it was indicated that the Ker A gene shared 97% sequence identity
with the gene encoding subtilisin Carlsberg unit from B. licheniformis NCIMB 6816.
From the study, it was concluded that transcriptional regulation controls Ker A expression
on different growth media (Lin et al. 1995). While efforts were made to comprehend the
process of keratin degradation (Evans et al., 2000), simultaneously, another milestone
that was achieved in this field was the potential to degrade prion protein by keratinase.
This step further pushed forward keratinase research, and keratinases since then have
acquired major attention for their role in varied applications. Continuous studies are
being carried out, with a huge diversity of keratin degrading microorganisms being
reported, and studies being carried out on their enzymes and applications (Gupta and

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 Chapter 2: Review of Literature

Ramnani, 2006; Brandelli, 2008). Although the mentioned applications of keratinase are
still at a primary stage, one application has reached the commercial level- Verazyme, a
keratinase preparation which is used for feather meal production (Gregg, 2002).
2.2 Diversity of keratin degrading microorganisms:
Keratin degrading microorganisms are classified under protease producing
microbes that are capable of degrading keratin containing materials. Keratinase
producing microorganisms can belong to bacteria, fungi and actinomycetes. Of these,
keratinase producing bacteria are most numerous, followed by fungi and then
actinomycetes.
2.2.1 Keratinase producing bacteria:
Most keratinase producing bacteria utilize feather keratin as their substrate. A
major proportion of feather degrading bacteria is Gram positive and mainly belongs to
the genus Bacillus spp. (Williams et al., 1990, Lin et al., 1992, Cai et al., 2008). Other
Gram positive feather degrading bacteria belong to the genus Micrococcus spp.,
Nisternkonia spp., and Clostridium spp. (Coello and Vidal, 2002, Ionata et al., 2008). A
few Gram negative keratin degrading bacteria belong to Pseudomonas spp., Vibrio spp.,
Chryseobacterium spp., Xanthomonas spp., and Fervidobacterium spp. (Sangali and
Brandelli 2000; De Toni et al., 2002; Yamamura et al., 2002; Lucas et al., 2003).

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 Chapter 2: Review of Literature

Table 2.1: Keratinase producing Bacteria:

Microorganism Source of isolation Substrate Degradation conditions Reference

B. licheniformis PWD-1 Poultry waste Chicken feathers 6 days at 50oC, pH 7 Williams et al.,
digester 1990
o
B. licheniformis and B. pumilus, Plumage of birds Chicken feathers 7 days at 50 C, pH 7.5 Burtt and Ichida,
Bacillus spp. 1999

Vibrio spp. Kr2 Poultry industry 1% Chicken feathers 72 hours at 30 oC, pH 6.0, 180 Sangali and
waste rom Brandelli, 2000
B. subtilis, Poultry waste 1% Chicken feathers 40oC, pH 5-9 Kim et al., 2001
B. pumilus 40oC, pH 5-6
B. cereus 30oC, pH 7.0
Xanthomonas maltophilia strain Poultry waste Feather meal 72 hours De Toni CH et
(POA-1) al., 2002

Xanthomonas spp. P5 ----- 0.1% Feathers 7 days Jeong J, 2010

Fervidobacterium islandicum Geothermal hot Feathers ------- Nam et al., 2002
AW-1 stream
Kocuria rosea Soil 1% Chicken feathers 40oC, pH 10 Coello and
Vidal, 2002

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Stenotrophomonas spp.D-1 Soil containing deer 1% Keratin powder 20oC, 2.5 days, pH 7.0, 130 rpm Yamamura S. et
fur predominantly al., 2002
containing human hair
B. pseudofirmus, Alkaline soda lake, Chicken feathers ------- Gessesse et al.,
Nesterenkonia sp. Ehtiopian Rift valley 2003
area
Bacillus spp. Kr 16 Poultry feathers in 1% Chicken feathers 30-37 oC Werlang and
decomposition Brandelli, 2005
Chryseobacterium spp. Kr6. Poultry waste Feathers, Feather meal 30oC, pH 6-8 Riffel and
Brandelli, 2005
Microbacterium spp. Kr10 Decomposing 1.5% Whole feathers, 30oC , pH 7.0, 36 hours Thys R.C.S. et
feathers, local poultry feather meal al., 2004
farm
B.pumilus FH9 Soil 1% Chicken Feathers 37oC, pH 8.0, 48 hours, 200 rpm El- Refai H. A.
et al., 2005
Gram negative: Burkholderia Feather waste 1% Feather meal, raw 30 and 37ºC Brandelli and
sp., Chryseobacterium sp. and feathers, chicken nails, Riffel, 2006
Pseudomonas sp. hair and wool.
Gram positive:
Microbacterium sp.

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 Chapter 2: Review of Literature

B. subtilis, B. amyloliquefaciens Brazilian Amazon 1% Chicken feathers, 37 oC at 125 rpm Giongo et al.,
and B. velesensis. basin Feather meal 2007
B. subtilis KD-N2 Local poultry farm 1% Chicken feathers 30 hours, 28 oC, pH 7.2, 200 rpm Cai et al., 2008
B. amyloliquefaceans Poultry processing 1% Chicken feathers 50oC, pH 8.0 Cortezi et al.,
plant 2008
Clostridium sporogenes bv. Solfatric muds 1% Chicken feathers 42oC and pH 7.0, 7 days Ionata et al.,
Pennavorans bv. Nov. 2008
Bacillus subtilis P13 Hot springs Chicken feathers ------ Pillai and
Archana, 2008
Bacillus polymyxa Soil and keratinous 1% Chicken feathers 1% keratin, 30 oC, pH 7.2, 170 Laba and
Bacillus cereus waste rpm Rodziewics,
2010
Bacillus pumilus KS12 Garden soil 0.5% Chicken feathers 24 h at 37◦C, 200 rpm, pH 10 Rajput R. et al.,
2010
Bacillus halodurans Rice mill effluents Chicken feathers 48 hours, pH 10 Prakash et al.,
PPKS-2 2010
Pseudomonas aeruginosa C11 Feather dumping soil 2% Feather powder 30°C for 4 days on a rotary Han M. et al.,
shaker at 180 rpm 2012

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2.2.2 Keratinase producing Fungi :

Keratinolytic fungi are capable of utilizing varied keratinous substrates such as chicken feather, wool, human hair, etc. These
fungi belong to fungi imperfectii group which includes: Trichophyton, Aspergillus, Microsporum, Fusarium, Chrysosporium,
Alternaria, Cladosporium, Doratomyces, Scopulariosis, Penicillium, etc. As most of the fungi belonging to the above mentioned
genus are considered as pathogens, they do not have much commercial significance. (Gradisar et al. 2000, Muhsin and Hadi, 2002;
Gupta and Ramnani, 2006).
Table 2.2: Keratinase producing Fungi:
Organism Source Substrate Degradation conditions Reference
Chrysosporium indicum, C. Soil samples from Human and 27oC, three to four weeks Abdel-Fattah et al., 1982
tropicum, C. keratinophilum, various locations animal hairs and
and Microsporum gypseum pigeon feathers.
Chrysosporium, Malbranchea, Poultry farm soils Human hair 28oC, pH 7.8, 40 days, static Kaul and Sumbali, 1997
Scopulariopsis, Microascus, and conditions
Gliocladium
Chrysosporeum georgear Chicken feathers Chicken feathers 9 days, pH 6-8, 30 oC El-Naghy MA et al., 1998
Doratomyces microsporus ---- ---- pH 8-9 and 50 degrees C Gradisar H et al., 2000
Trichophyton sp. HA-2 Dumping soil ----- pH 8.0, 35 °C for a period of 5 Anbu et al., 2008
weeks.
Alternaria spp. and Aspergillus spp. Poultry wastes Feather powder 5-6 days at 35oC pH 7.5, Saber et al., 2010

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2.2.3 Keratinase producing Actinomycetes:

Keratin degrading actinomycetes that belong to the genus Streptomyces include. S. fradiae (Novel and Nickerson 1959),
Streptomyces. sp. A11 (Mukhopadhyay and Chandra 1990), S. pactum (Bockle et al., 1995), S. albidoflavus (Letourneau et al., 1998),
S. thermoviolaceus SD8 (Chitte et al,. 1999) and S. graminofaciens (Szabo et al. 2000). Certain keratinolytic thermoactinomycetes
include T. candidus (Ignatova et al., 1999) and Thermoactinomyces sp. studied by Gousterova et al., 2005.
Table 2.3: Keratinase producing Actinomycetes:
Organism Source Substrate Degradation conditions Reference
Streptomyces pactum DSM ------ 2.5% Chicken 28oC, 280 rpm, 4 days Böckle B et al., 1995
40530 feathers
Streptomyces spp. S.K1-02 Hen house soil Feather meal 30°C, 500 rpm, 30 h Bressollier P. et al, 1999
Thermoanaerobacter Geothermal vents Feathers 96 hours Riessen and
keratinophilus spp. Nov Antranikian, 2001

Nocardiopsis dassonvillei Marine environment 2% feathers 37°C, 150 rpm, 3 days Abdel-Fattah A et
NRC2aza al.,2013
Streptomyces albus AZA Mediterranean sea ----- 5 days Esawy 2007
Streptomyces sp. Slaughter house Chicken feathers 5 days Tatineni et al., 2008
Actinomyces fradiae JSC “Biocentras” 0.75% chicken 7.2, 34oC, 200 rev/min Matikevičienė et al.,
microorganism culture feather meal 2011
collection
Thermoactinomyces spp. Soil 0.5-0.75% feathers 55oC for 24h, 130 rpm Tonkova et al., 2009

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2.3 Optimum requirements for keratinase production by bacteria:

The constituents of a medium must be sufficient enough to fulfill the elemental
requirements for biomass production and metabolite production. There must be an
adequate supply of energy for biosynthesis and cell maintenance. A typical production
medium would contain certain nutrients which are required for biomass production, some
for enzyme production and certain nutrients for both. This would include Water, Carbon
source, Nitrogen source, trace elements and substrate (Stanburry and Whittaker, 2003).
The choice of these nutrients for designing a production medium depends upon the type
of microorganism used for the process and the target product of the fermentation process.
From the previous reports, a typical medium for keratinase production would contain
Keratinous substrate as a source of carbon and/or nitrogen, phosphate salts as buffer,
Magnesium and ammonium salts as trace elements. In some cases, where the
microorganisms are not capable of synthesizing specific nutrients required for their
proliferation; amino acids, vitamins and growth factors are added into the production
medium. Optimization of process parameters such as temperature and pH and that of
media components is carried out with the objective of obtaining high yields of enzyme.
While optimizing a production medium, certain requirements must be fulfilled for
the successful and feasible enzyme production (Stanburry and Whittaker, 2003):
 The production medium must be inexpensive and must allow rapid biomass
production
 Enzyme must be produced at a high concentration, thus achieving a high yield
 The content of undesirable products or metabolites must be low.
 In order to increase the fermenter enzyme productivity, the microorganism should
be able to grow on a concentrated medium in a dense culture.
 Harvesting of the enzyme or product of interest must be feasible
 The overall production process must be safe to the handler and to the environment
 The effluent released from the production process easily treatable, such that it can
be released to the environment

In order to accomplish the above mentioned criteria, strain properties and process
parameters need to be collectively optimized.

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 Chapter 2: Review of Literature

2.3.1 Carbon source

For microorganisms to release keratinase and exhibit keratinase activity, keratin
would be required as a source of carbon and nitrogen. The presence of keratin containing
material would induce the release of keratinase by the organism. Keratinolytic proteases
were largely inducible, requiring keratin as an exogenous inducer. Thus, in many cases,
keratin is preferred as a sole source of carbon and nitrogen for to enable maximum
keratinase production by the isolates (Kim et al., 2001). The presence of another simpler
carbon source would result in the suppression of enzyme production or inhibition, a result
of catabolite repression, a common control mechanism used for biosynthesis of microbial
proteases (Gupta et al., 2002). It would allow the isolate to preferably metabolize it
instead of the protein substrate which would otherwise be difficult to degrade. The
breakdown of the simpler carbon source for energy would not require the isolate to
release the specific protein degrading enzyme, thus suppressing the production of that
specific enzyme. The presence of glucose in the production medium had totally
suppressed keratinase production in case of B. licheniformis PWD-1 (Williams et al.,
1990). This phenomenon is also reported for keratinases by Santos et al. 1996, Wang and
Shih, 1999; Thys et al. 2004, Brandelli and Riffel, 2005, Laba and Rodziewics, 2009 and
Kainoor et al., 2010, in which presence of simpler carbon source like glucose, sucrose or
lactose in the production medium has resulted in suppression or complete inhibition of
keratinase production. Other studies have documented about simpler carbon source like
glucose to enhance keratinase production. The addition of glucose not only enhanced the
growth of the isolates but also resulted in increased enzyme production in the studies
carried out by El- Naghy et al., 1998, Cortezi et al., 2008. Thus, easily metabolizable
carbon source like glucose, if present in the production medium can also have appositive
effect on keratinase production. Other studies have also mentioned about the presence of
glucose accompanied with yeast extract resulting in increased keratinase production, in
which glucose would serve as a source of carbon while yeast extract would serve as a
source of nitrogen, growth factors and energy (Yamamura et al., 2002). However, for
isolate B. subtilis JB-99, there was increase in keratinase activity in the presence of 0.1%
yeast extract in the growth medium, while an increase in yeast extract concentration
resulted in suppression.

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Thus, in the presence of two nutrient sources, one which is compact and resistant
to degradation, while other which is easily accessible, microorganisms will preferentially
utilize the latter. This explains the resulting suppression of keratinase production in the
presence of additional carbon source.

2.3.2 Nitrogen Source:

Most keratinase producing media contain keratinous substrate as a sole source of
carbon and nitrogen as mentioned before. In some cases, additional nitrogen source is a
requirement for the microorganisms to perform keratinase activity and release keratinase.
Some examples of additional Nitrogen sources required for keratinase production
include- Yeast extract (Yamamura et al., 2002), (NH4)2SO4 (Matikevičienė V et al.
2011), NH4Cl (Mousavi et al., 2013), corn steep flour (Ni et al., 2011), peptone
(Ramnani and Gupta, 2004), soyabean meal (Laksmi et al., 2013), NH4Cl and yeast
extract (El- Refai et al., 2005, Lin and Yin, 2010).
Yamamura et al., 2002, which have reported increase in microbial keratinolytic
activity due to addition of yeast extract in the production medium suggested that
increased enzyme activity is associated with increased microbial growth as a result of
yeast extract being an easily metabolizable nutrient source. The enzyme production was
two-fold higher as compared to that observed in a simple keratin-containing medium. For
Bacillus spp. JB-99, 0.1% (w/v) resulted in increased keratinase production; however,
there was a decrease in enzyme production when yeast extract concentration was
increased to 1% (Kainoor et al., 2010). Kim et al., 1999, have reported about a feather
degrading B. subtilis, for which, the presence of casein greatly increased the production
of the enzyme while B. pumilis did not produce the enzyme in the presence of casein. A
low level of enzyme production by B. cereus in the presence of casein was observed.
Thus, keratin powder was shown to be a poor substrate for enzyme production in B.
subtilis and B. cereus, but a good substrate in B. pumilis. The study suggested that the
synthesis of keratinolytic protease by B. pumilis and B. cereus is inducible. However, the
enzyme in B. subtilis was constitutively produced in the presence of proteins such as
casein, feather and BSA (Bovine Serum Albumin). For isolate B. amyloleiquefaceans,

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 Chapter 2: Review of Literature

addition of casein in the production medium resulted in decreased proteolytic and

keratinolytic activity.
2.3.3 Temperature and pH
Temperature and pH have a strong influence on microbial growth and therefore
enzyme production. Physical parameters for keratinase production are species-specific
and vary with respect to microorganism (Gupta and Ramnani, 2006). For example,
Williams et al., 1990 have reported B. licheniformis which carried out keratinase
production at 50oC. Feather degrading isolate Frevidobacterium pennavorans carried out
feather degradation at 70oC. (Friedrich and Antranikian, 1996). Sangali and Brandelli,
2000 have reported a feather degrading Vibrio spp. Kr2 performing feather degradation at
30oC. Vidal et al., 2000 have reported degradation of feathers by Kocuria rosea at 40oC.
For isolate Chryseobacterium spp. Kr6, the preferred temperature for keratinase
production is 25-30oC (Brandelli and Riffel, 2003). Thys et al., 2004 have documented a
Microbacterium spp. carrying out optimum keratinase production at 30oC. Thus,
Temperature for keratinase production ranges from 28 to 50°C for most bacteria. Certain
actinomycetes and fungi also prefer temperatures as high as 70°C for keratinase
production- Thermoanaerobacter and Fervidobacterium spp. (Friedrich and Antranikian
1996; Rissen and Antranikian 2001; Nam et al., 2002).
pH range of 6- 9 supports Keratinase production and most microorganisms carry
out keratinase production at alkaline pH range. The possible explanation for this could be
that alkaline pH modifies cysteine residues to lanthionine (Friedrich and Antranikian
1996), making it available for keratinase action. alkaliphilic strains Nesternkonia spp.
AL-20 (Gessesse et al., 2003) and Nocardiopsis sp. TOA-1 (Mitsuiki et al., 2004) have
been reported to show keratinase activity. Alkalophilic microbes have also been reported
to show keratinase activities at elevated pH- Bacillus halodurans carrying out feather
degradation at pH 10 (Prakash et al., 2010), Streptomyces albus showing keratinase
production at pH 10, (Esawy et al., 2007).

2.3.4 Types of substrates and their concentrations:

The production of any enzyme by a microorganism would dependent upon the

substrate. In case of keratinase production, presence of a keratin containing substrate
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would be vital, since it would induce the production of enzyme. Most feather degrading
microorganisms are capable of keratin utilization as a sole source of carbon and nitrogen
(Williams et al., 1990; El-Naghy et al., 1998; Lin et al., 1999; Szabo et al., 2000;
Gousterova et al., 2005). However, the type of keratin being varied in different substrates
(alpha keratin or beta keratin), may result in different microbes preferring different type
of keratin. The type of exogenous keratin inducer may range from whole chicken feather,
feather powder, wool, horns, nails, and stratum corneum to hair. In most cases, the
presence of keratin induces the production and release of keratinase from microbes
(Gradisar et al., 2000). Keratinases are therefore, classified as inducible enzymes, in
general. However, certain inducible keratinases have also been reported (Gessesse et al.,
2003; Manczinger et al., 2003). Further, as mentioned above, simple sugars such as
glucose have been reported to suppress keratinase production due to catabolite repression
(Santos et al., 1996; Ignatova et al., 1999; Wang and Shih 1999; Yamamura et al., 2002;
Bernal et al., 2003; Gessesse et al., 2003; Suntornsuk and Suntornsuk 2003; Thys et al.,
2004. However, it is difficult to compare the keratinolytic potential of microorganisms in
terms of keratinolytic activities, due to the wide variety of keratin substrates and the
varied definitions of keratinase units employed.
Microorganisms prefer a moderate range of keratin containing substrates. Chicken
feathers are the most preferred substrate, followed by feather meal, hair and wool
(Williams et al., 1990, Brandelli and Riffel, 2006). Since enzyme production is
concentration dependent, for the optimum release of keratinase, however, the appropriate
concentration of substrate is of paramount importance.

2.3.5 Trace elements:

All micro-organisms require certain mineral elements for growth and metabolism.
In many media, magnesium, phosphorus, potassium, sulphur, calcium and chlorine are
essential components, and because of the concentrations required, they must be added as
distinct components. (Stanburry and Whittaker, 2003). For keratinase production, one of
the most vital trace elements required to be added is Magnesium. A divalent ion,
magnesium acts as a co-factor for several enzymes, and is present in cell walls and
membranes, thus playing an important role for cell mass build-up and enzyme activity

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and stability. Magnesium should be present in the media composition either in the form
of MgCl2 (Kim et al., 2001; El-Refai et al., 2005; Lin and Yin, 2010; Mousavi et al.,
2012) or MgSO4 (Yamamura et al., 2002; Kainoor et al., 2010; Laba and Rodziewics,
2010).
Thus, a production medium must contain optimum concentrations of nutrients to
fulfill maximum enzyme production. In case of keratinase production, the necessary
nutrients include a source of keratin, an additional carbon source (optional), an additional
nitrogen source (optional) and certain trace elements. Along with this, optimum pH and
temperature are a requirement for biomass production and therefore enzyme production.

2.4 Type of substrates and degradation efficiency:

Keratin is a material found in the exo-skeleton of animals and human, thus being
a major component of skin, hair nail, horn, claws, wool and feathers (Martinez-
Hernandez and Velasco- Santos, 2012). These keratin containing materials are therefore
used as substrates for testing the keratinolytic potential of microorganisms. The most
widely used keratinous substrate is chicken feathers as indicated in table 2.1, 2.2 and 2.3
by Kertinolytic bacteria and actinomycetes. As for the keratinolytic fungi, hair is a more
common substrate as compared to feathers. Many organisms are also tested for degrading
a wider variety of substrates. For example, Brandelli and Riffel, 2006 tested the
keratinolytic ability of Gram negative: Burkholderia sp., Chryseobacterium sp. and
Pseudomonas sp. and Gram positive: Microbacterium sp using 1% Feather meal, raw
feathers, chicken nails, hair and wool.
For quantitative estimation of the keratinolytic potential of the enzyme in terms of
enzyme units, the keratinase enzyme is reacted with a keratin substrates which includes
azo-keratin (Lin et al., 1992; Kim et al., 2001; Riffel et al., 2003; Lin and Yin, 2010),
keratin azure (Bressollier et al., 1999) , keratin powder (Yamamura et al., 2002), feather
powder (Ni et al., 2011), DMSO- solubilized feather keratin, etc. (Wawrzkiewicz et al.,
1987). There is lack of uniformity in the unit definitions for keratinases however, the
most commonly used method defines one unit of keratinase in terms of an increase of
0.01 absorbance unit at 280 nm using chicken feather as substrate (Cai et al., 2008).

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2.5 Biochemical properties of keratinases:

Keratinases [EC 3.4.21/24/99.11] are extracellular or intracellular proteases
generated by microorganisms, which catalyze the specific breakdown of recalcitrant
proteins, keratins, which are resistant to other proteases and peptidases (Onifade et al.,
1998). Till date, keratinases have played the prime role in valorization of the sizeable
amount of keratinous waste that is continuously generated (Onifade et al., 1998, Gupta
and Ramnani, 2006). Largely, keratinases are extracellular enzymes, released externally
by microbes and induced by the presence of keratin. However, there are few reports about
intracellular or cell-bound keratinases (El-Naghy et al., 1998; Onifade et al., 1998). The
intracellular fraction in most of these reports mainly assists to disulfide reductases, sulfite
or thiosulfate that synergistically assists the extracellular keratinases to degrade keratin
by reducing the disulfide bonds of keratin (Gupta and Ramnani, 2006). To be more
specific, Keratin degradation required two steps: reduction in the disulfide bonds or
sulfitolysis, and proteolysis (Bockle and Muller 1997; Ramnani et al., 2005). However,
the order of these events and their exact nature are still being evaluated.
Keratinases are majorly inducible proteases, released by microorganisms capable
of utilizing keratin as a sole source of carbon and nitrogen (Williams et al., 1990; El-
Naghy et al., 1998; Lin et al., 1999). That is, the presence of keratin activates the release
of keratinase, keratin thus playing an inducer (Kim et al., 2001; Laba et al., 2014).
Although most keratinases have been categorized as inducible, certain studies on
constitutive keratinases have been reported (Gessesse et al., 2003; Manczinger et al.,
2003). Another reason for this is that production of keratinases have been either
suppressed or inhibited by the presence of a simpler carbon source such as glucose,
sucrose and lactose in addition to keratin, indicating catabolite repression (Santos et al.,
1996; Ignatova et al., 1999; Wang and Shih 1999; Yamamura et al., 2002a; Bernal et al.,
2003; Gessesse et al., 2003; Suntornsuk and Suntornsuk 2003; Thys et al., 2004).
By catalytic nature, most keratinases, being inhibited by phenyl methyl sulfonyl
fluoride (PMSF) belong to the serine protease category (Lin et al., 1992; Böckle et al.,
1995; Friedrich and Antranikian 1996; Bressolier et al., 1999; Suh and Lee 2001; Nam et
al., 2002), while some are metalloproteases (Allpress et al., 2002; Farag and Hassan
2004). Another trend observed is that keratinases which belong to the serine protease

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family have been generally isolated from Gram positive bacteria. The metalloproteases
reported are keratinases from the Gram negative family.
Keratinases have been characterized from several microorganisms and their
biochemical properties have been assessed with respect to their molecular weight, pH and
temperature optima, effect of metal ions and chemicals, substrate specificity, etc. A brief
review on biochemical properties of reported keratinases has been presented below:

2.5.1 Molecular Weight:

Keratinases have molecular weights ranging from 18 KDa to 200 KDa. The
keratinase with the lowest molecular weight belongs to the actinomycete S. albidoflavus
SK 1–02 (Chitte et al., 1999). The highest of 200 kDa for Kocuria rosea and F.
islandicum have been reported (Nam et al., 2002; Bernal et al., 2006). The molecular
weights of certain reported keratinases have been represented in the below table:

Table 2.4: Molecular weights of keratinases characterized from various organisms

Microorganism Molecular weight (KDa) Reference

B. licheniformis PWD-1 33 Lin et al., 1992
B. subtilis KS-1 25.4 Suh and Lee 2001
B. pseudofirmus FA30-10 27.5 Kojima et al., 2006
S. pactum DSM40530 30 Böckle et al., 1995
S. albidoflavus K1-02 18 Bressolier et al., 1999
F. pennavorans 130 Friedrich and Antranikian 1996
X. maltophilia 36.8 De Toni et al., 2002
Vibrio sp. kr2 30.8 Sangali and Brandelli 2000a
Chryseobacterium sp. kr6 64 Riffel et al., 2007
Microbacterium spp. kr10 42.7 Thys and Brandelli 2006
Kocuria rosea LPB-3 240 Bernal et al. 2006

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2.5.2 pH and temperature optima:

Keratinases from most organisms are best active within the pH optima of neutral
to alkaline range i.e. pH 7.5 onwards (Friedrich and Antranikian 1996; Letourneau et al.,
1998; Bressollier et al., 1999; Ignatova et al., 1999; Gradisar et al., 2000; Sangali and
Brandelli 2000; Rissen and Antranikian 2001; Yamamura et al., 2002; Riffel et al., 2003;
Farag and Hassan 2004; Thys et al., 2004; Anbu et al., 2005, Ionata et al., 2008).
Keratinase of isolate Streptomyces albus AZA is active within the pH range of 6.5 to 11,
with the optimum pH being pH 10 (Esawy, 2007). A few keratinases have the optimum
pH requirement in the extreme alkaline range i.e. > pH 12 (Takami et al., 1999; Mitsuiki
et al., 2004). As for enzyme stability, keratinases are generally active and stable over a
wide range of pH from 5 to 13 (Dozie et al., 1994; Bockle et al., 1995; Bressollier et al.,
1999; Nam et al., 2002; Farag and Hassan 2004). Keratinase characterized from S.
pactum DSM40530 (Bockle et al., 1995) has shown stability at acidic pH of 4, while that
of isolate Bacillus halodurans AH-101 (Takami et al., 1999) showed stability at alkaline
range of pH 13.
The optimum catalytic activity of keratinases occurs within the temperature range
of from 30 to 80°C (Lin et al., 1992, Lee et al., 2002, Gessesse et al., 2003, Riffel and
Brandelli, 2006, Thys et al., 2006, Esawy, 2007, Cai et al., 2008, Ionata et al., 2008, Han
et al., 2012, Brandelli 2005). Certain thermophilic keratinases have also been reported i.e.
the enzyme from Chrysosporium keratinophilum (Dozie et al., 1994) and thermophile
Fervidobacterium islandicum AW-1 (Nam et al., 2002) showed exceptionally high
temperature optima of 90 and 100°C. Thermostability of a particular keratinase depends
upon the source from which it is isolated. Most keratinases are stable over a wide
temperature range i.e. 30 to 60 oC, while the keratinase of Bacillus halodurans PPKS-2 is
found to be stable for 3 hours at 70oC and Bacillus pumilus KS 12 keratinase which is
found to be stable at 80oC (Prakash et al., 2010; Rajput et al., 2010).

2.5.3 Effect of inhibitors, metal ions, and other chemicals

Determined by their activity in the presence of inhibitor, keratinases are classified
into two categories- Serine proteases and metalloproteases. Largely, keratinases isolated
from Gram positive bacteria are serine proteases, while those obtained from Gram

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negative bacteria are metalloproteases (Brandelli, 2008). Most of the keratinases which
have been characterized are found to be serine proteases (Lin et al., 1992; Böckle et al.,
1995; Friedrich and Antranikian 1996; Bressolier et al., 1999; Suh and Lee 2001; Nam et
al., 2002; Bernal et al., 2006; Cortezi et al., 2008; Cai et al., 2008). A few keratinases
from Gram negative isolates have been reported to be metalloproteases (Allpress et al.
2002; Farag and Hassan 2004, Thys et al., 2006; Esawy, 2007; Han et al., 2012).
From the overall review of effect of metal ions on keratinase activity, it can be
noted that most keratinases are activated in the presence of metal ions- Ca2+, Mn2+, Mg2+,
(Rissen and Antranikian, 2001; Nam et al., 2002; Esway, 2007; Riffel et al., 2007;
Macedo et al., 2008). Such divalent cations must be promoting the active conformation of
the keratinase enzyme thus increasing the catalytic rate. However some studies have
reprted keratinase inhibition by Ca+2, Mn2+, Mg2+ (Cai et al., 2008; Cortezi et al., 2008).
Most keratinases are inhibited by Cu2+ (Nam et al., 2002; Riffel et al., 2003; Thys
et al., 2004), and Zn2+ (Riffel et al., 2003; Thys et al., 2004) and Hg2+ (Thys et al., 2004).
Keratinase of Kocuria rosea was reported to have no effect from divalent cations like
Zn2+, Co2+, Mg2+ Ca2+ Sr2+, Mn2+ Ba2+ and Hg2+ in concentrations as high as 10 mM
(Bernal et al., 2006). For B. subtilis KD-N2, all metal ions have shown an inhibitory
effect on keratinase (Cai et al., 2008)

2.6 Applications of keratinases:

2.6.1 Recycling of feather waste:
Keratinases have been majorly looked into for recycling of keratin containing
wastes such as chicken feathers. Being generated in large amounts, and having a
tendency to accumulate if untreated, treatment and disposal of poultry feather waste is of
major concern (Onifade et al., 1998). Since traditional methods like hydrothermal
treatment and incineration have proved to be harmful to the environment and have
ineffective utilization of the valuable feather protein keratin, alternative method of
treatment was required (Wang and Parsons, 1997). The use of microbial keratinases is a
viable alternative to this existing problem. Microbial treatment of feathers results in the
production of a feather hydrolysate or a feather meal which is rich in amino- acids and
has high nutritive value. Feather meal being nitrogen rich, inexpensive and readily

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available source serves as a potential additive for organic farming (Choi and Nelson,
1996)The hydrolysate obtained can also find application as a biofertilizer (Vasileva-
Tonkova et al., 2009).

2.6.2 Industries:
The use of keratinases would be a “greener” approach for de-hairing of leather,
and it can replace sodium sulfide for the same process. Enzymatic de-hairing will have
advantages over chemical de-hairing, since it would be a hair- saving process and it can
reduce the pollution caused by harmful chemicals like sodium sulfide (Giongo et al.,
2007; Wang et al., 2007; Pillai and Archana, 2008).
The application of keratinases can also be extended to the detergent industry just
as how other proteases play a major role in enzyme based detergents for stain removal
(Rai et al., 2009). Keratinases can also play a major role in clean-up of clogged drains by
removal of keratinous waste (Chitte et al., 1999).

2.6.3 Degradation of prion protein

Prions are highly aggregated, recalcitrant proteins causing severe
neurodegenerative disorders such as Transmissible Spongiform Encephalopathies
(TSEs).Recently, keratinases have also been reported to carry out degradation of prion
and prion proteins that are responsible for diseases that affect the brain and the nervous
system. It was reported that keratinase from B. licheniformis PWD-1 degraded prion
protein in brain, stem tissue from animals with bovine spongiform encephalopathy and
scrapie. (Langeveld et al., 2003).

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2.7 RATIONALE:
Keratin, a major component of poultry feathers, is a resistant protein that results
in the accumulation of poultry feathers, if not treated. The current methods that are
employed for the waste management of poultry feathers include hydrothermal treatment,
which involves high temperature and pressure resulting in the conversion of indigestible
poultry feathers to an easily metabolizable feather-meal. This treatment results in the loss
of certain valuable amino acids, due to high temperature. Another method employed is
incineration, which is burning off the feather waste. Both the methods consume enormous
amount of energy and they add up to environmental pollution. The presence of
keratinolytic microorganisms in nature provides an alternative method for treatment of
poultry feather waste. Microorganisms, possessing the enzyme keratinase are capable of
utilizing keratin, thus carrying out degradation of poultry feathers. Microbial treatment of
poultry feather waste would be an eco-friendly approach to combat the problem of
poultry waste. This treatment would not only result in the bio-degradation of poultry
feather, but also would generate amino acids which are useful by-products of keratin
degradation. The feather hydrolysate obtained at the end of microbial treatment, would be
enriched with amino acids and will have the potential to be used as an animal feed.

2.8 AIM:
The aim of the study is to isolate and identify feather degrading bacteria from poultry
farm soil, and to study the characteristics of the keratinolytic enzyme.

2.9 OBJECTIVES:
1. Isolation of feather degrading organism from poultry farm soil, which would involve
selective enrichment of soil microflora, followed by screening, to select feather
degrading isolates
2. Biochemical identification of the feather degrading isolates followed by 16SrRNA
sequencing to confirm the results of biochemical identification
3. Determination of keratinolytic activity of the isolates which would involve
standardization of the enzyme assay

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4. Optimization of process parameters and media components to attain maximum

keratinase production
5. To purify the keratinase using Ammonium Sulfate Precipitation and DEAE-Cellulose
Column chromatography and to study characteristics of purified enzyme
6. Detection of released amino acids, the by-products of feather degradation using TLC

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