Detection of Keratinolytic Actinobacteria and evaluation of Bioprocess for Production of Alkaline

12 Pages·2002·0.74 MB·English

Int.J.Curr.Microbiol.App.Sci (2015) 4(7): 907-918
ISSN: 2319-7706 Volume 4 Number 7 (2015) pp. 907-918
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Original Research Article
Detection of Keratinolytic Actinobacteria and evaluation of
Bioprocess for Production of Alkaline Keratinase
Nandini Allure, Madhusudhan D.N. and Dayanand Agsar*
A-DBT Research Laboratory, Department of Microbiology,
Gulbarga University, Kalaburagi 585106, India
*Corresponding author
A B S T R A C T
A large number of actinobctria were reported from the regional soil around poultry
Keywords farms. Few isolates of actinobactria could reveal the keratinolytic activity and an
actinobactrium DNA38 showed higher keratinolytic activity on starch feather meal
Streptomyces; agar. This potential isolate of actinobactrium DNA38 was identified as
Molecular Streptomyces sp. based on morphological, biochemical and physiological
analysis; properties, and further characterized as Streptomyces minutiscleroticus DNA38 by
Submerged 16S rRNA analysis. Submerged bioprocess was found to be a suitable process for
bioprocess; the production of higher amount (122.1 IU) of keratinase, when compared to solid
Solidstate state bioprocess with lesser amount (34.9 IU) of keratinase. High titer production of
bioprocess; keratinase by Streptomyces minutiscleroticus DNA38 was achieved successfully
Alkaline under submerged bioprocess using chicken feathers as substrate, along with starch
keratinase as source of carbon and few mineral salts. Alkaline pH (9.0) of the medium and
higher incubation temperature (40 °C) reveals the production of thermophilic
alkaline keratinase, which can be further explored for enhanced production aiming
at myriad applications.
Introduction
and hydrophobic interactions (Parry and
Keratin is an insoluble, high stable protein
North, 1998) . The enzyme has received
found mostly in feathers, wool, nails and
particular attention for its relevant
hair of vertebrates (Shih, 1993). Keratin is
applications in various types of agro and
resistant to the common proteolytic
biotechnological industries. Certain
enzymes, papain, pepsin and trypsin
microorganisms hydrolyze the keratin by
(Papadopoulos et al., 1986). The high
synthesizing specific class of enzymes,
resistance of keratin to proteases may be
which degrade keratin in to small peptides
attributed to the molecular conformation
that can be utilized further by the cells.
of their structural amino acids, that is tightly
Several feather and hair degrading
packed in the -helices (hairs) and -sheets
Streptomyces have been isolated from
(feather) in the presence of cystine
soil, poultry wastes, hair, debris and
disulfide bonds, hydrogen bonds
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Int.J.Curr.Microbiol.App.Sci (2015) 4(7): 907-918
animal skin. Keratinases, a group of been done on production of proteolytic
serine metallo proteases, release the free enzymes, relatively little information is
amino acids from keratinous proteins. available on keratinases. Upto now, a
After treatment with keratinase, feather limited number of studies have been
can be used as feeders, fertilizers and reported on the isolation of thermophiles,
insoluble polymers (Yamauchi et al., in particular thermophilic actinobacteria
1996). Feathers consist of about 5-7% of with the ability to hydrolyse feathers and
total weight of mature chicken from poultry other keratinous wastes. Enzyme
processing plants, approximated by about production has been extensively studied by
million tons produced annually, worldwide. submerged and solid state bioprocesses.
Feather from the poultry processing plant Submerged system is usually implemented
is the common source for the in case of bacterial enzyme production, due
accumulation of more than 90% of to the requirement of higher water potential
keratinous proteins in the environment, (Chahal, 1983). Solid state system is
causing pollution (Onifade et al., 1998).A preferred when process require lesser water
current value-added use for feathers is the potential (Troller and Christian, 1978). The
conversion to feather meal, adigestible main reason can be attributed to the
dietary protein for animal feed, using metabolic differences of water requirement.
physical and chemical treatments. These The metabolic differences of
methods can destroy certain aminoacids and microorganisms involved in the submerged
decrease protein quality and digestibility and solid state systems have a direct impact
(Moritz and Latshaw, 2001; Anbu et al., on the quantity of the product. Many
2005). Keratinolytic microorganisms and actinobacteria are also reported to produce
their enzymes may be used to enhance the important commercially viable enzymes by
digestibility of feather keratin. They may either submerged or solid state bioprocesses.
have important applications in processing Normally, keratinase has been produced by
keratin-containing wastes from poultry and submerged bioprocess, but in recent years, it
leather industries through the development is also being produced under solid state
of non-polluting methods (Onifade et al., bioprocess. With regard to productivity
1998). Keratinous wastes represent a source aspect, it can be inferred that, both the
of valuable proteins and amino acids and systems are equally emerging popularly as
could find application as a fodder additive field of choice for the production of
for animals or source of nitrogen for plants. keratinases. The aim of this study was to
screen some actinobacteria for their ability
Biodegradation by microorganisms to degrade native feather followed by
possessing keratinolytic activity represents identification of a new feather-degrading
an alternative attractive method for Streptomyces sp. and evaluation of suitable
improving thenutritional value of keratin bioprocess for the production of keratinase.
wastes, as it offers cheap and mild reaction
conditions for the production of valuable Materials and Methods
products (Kim et al., 2001). Streptomyces
keratinases are of particular interest because Isolation and screening of keratinolytic
of their action on insoluble keratin substrates actinobacteria
and generally on a broad range of protein
substrates. These enzymes have been studied Unique ecological sites of poultry farms
for de-hairing processes in the leather were identified for collection of soil samples
industry.Despite all the work that has around Kalaburagi region to isolate
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Int.J.Curr.Microbiol.App.Sci (2015) 4(7): 907-918
actinobacteria. The soil samples were used for amplification of 16S rRNA gene.
collected from the selected spots as per the 16S rRNA amplification of cultures was
standard procedures (Skinner, 1951). The done using universal F27
top layer of the soil was removed for about 5 (5 AGAGTTTGATCMTGGCTCAG-) and
to 6 cm and with a clean spatula or scoop the R1525 (5 TACGG(C/T)TACCTTGTT
soil was collected in sterile airtight ACGACTT) primer (Weisburg et al, 1991).
polythene bags. The collected soil samples PCR master mix was prepared containing
were enriched for isolating actinobacteria by 100ng (1µL) DNA, 1.25 µL of both primers
different methods (Agate and Bhat, 1967; F27 and R1525, 2.5 µL each of 10X PCR
Pridham et al., 1956/57). One gram of soil buffer and 1mM dNTPs, 0.25 µL Taq
suspension was added to 100ml of 1.4 % polymerase and 17 µL nuclease free PCR
phenol solution and kept at room grade water to make up a volume of 25 µL.
temperature for 10 minutes. The mixture PCR master mix is a premixed ready to use
was diluted further (Lawrence, 1956) and solution containing Taq DNA polymerase,
used to isolate actinobacteria by following reaction buffers containing MgCl2, dNTPs
standard serial dilution plate culture method at ideal concentrations for efficient
(El-Nakeeb and Lechevalier, 1963). 0.1 ml amplification of templates by PCR. The
of the sample from the respective dilutions program was set with the initiation
was plated on starch casein agar, where in temperature of 94°C for 3 minute, followed
casein of SCA was replaced with chicken by 34 cycles of denaturation at 94°C for 30
feather meal(Soluble Starch - 10.0 g; sec, annealing at 55°C for 30 sec and
K HPO - 2.0 g; KNO - 2.0 g; NaCl - 2.0 g; extension at 72°C for 1.30 minute. The final
2 4 3
chicken feather meal - 5 g; MgSO - 0.05 g; extension after these 34 cycles is at 72°C for
4
CaCO - 0.02 g; FeSO .H O - 0.01 g; Agar - 7 minute followed by hold at 4°C. Accuracy
3 4 2
20 g; Distilled water - 1000 ml; pH - 8.5). of PCR product was visualized on agarose
The inoculated plates were incubated at 40 gel. The PCR product was purified using
ºC for 120 h. Based on the growth on feather Genei pureTM Quick PCR Purification Kit
medium, the actinomycete colonies were and were then sequenced using a Big Dye
selected and sub-cultured on the skimmed Terminator kit, version 3.1, on an automatic
milk agar (Ronald, 2010). Based on zone of ABI 3100 sequencer (Applied Biosystems
hydrolysis on skimmed milk agar the Inc.). The Sequences were compared using
isolates were selected and assessed for the 16S rRNA gene sequences from EzTaxon to
keratinolytic activity using starch feather restore closest relatives. The entire work of
meal agar by plate culture method. Selected molecular characterization was facilitated at
potential isolates of actinobacteria were Genomics Services Xcelris Labs Ltd,
identified up to the level of genus by Ahmedabad.
morphological (Shirling and Gottlieb, 1976),
biochemical and physiological properties Evaluation of bioprocess for production
(Williams et al., 1989). of keratinase
Molecular characterization of Submerged and solid state bioprocesses
keratinolytic Streptomyces were evaluated in empirical conditions for
the production of keratinase by the potential
Chromosomal DNA was extracted by using isolate of Streptomyces DNA38, explained
Chelex 100 (Sigma-Aldrich, USA) chelating in brief as follows.
ion exchange resin method (Laurent et al,
1999). Around 100 nanogram DNA was Submerged bioprocess: The bioprocess
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(Lerch and Ettlinger et al., 1972) was M Na 2CO and 0.5 ml of Folin-
3
carried out using StreptomycesDNA38 in a Ciocalteau s phenol reagent. The absorbance
250 ml Erlenmeyer flask containing 100 ml was measured at 660 nm against blank after
of starch feather medium with pH 9.0. After 30 min. All assays were carried out in
sterilization of the medium at 121 ºC for 15 triplicate. One unit of keratinase activity was
min, 1 ml suspension of three days old test defined as the amount of enzyme that
isolate with spore count 1x108 spores/ml was released one microgram of tyrosine per
inoculated and kept for incubation at 40 ºC minute under the standard assay conditions.
for week in shaker incubator at 180 rpm.
The amount of keratinase produced was Results and Discussion
estimated at every 24 h.
Isolation and Screening of Actinobacteria
Solidstate bioprocess: 25 g of substrate
(chicken feathers) was taken in 250 ml More than hundred colonies of
Erlenmeyer flask and rehydrated with actinobacteria were observed on the plates
mineral salt solution (K HPO - 2.0 g; KNO of starch feather meal agar with typical
2 4 3
- 2.0 g; NaCl - 2.0 g; chicken feathers - 5 g; colony characters (Figure 1). Forty two
MgSO - 0.05 g; CaCO - 0.02 g; colonies of actinobacteria were selected
4 3
FeSO .H O - 0.01 g; Distilled water - 1000 randomly based on prominent colony
4 2
ml; pH 9.0) to achieve 65% moisture characters and screened for the synthesis of
content. Initial pH was adjusted to 8.0. The protease on skimmed milk agar. Among
cotton plugged flasks were autoclaved at forty two isolates, five isolates formed the
121ºC for 15 min and allowed to cool at zone of catalysis (Figure 2) indicating the
room temperature. The contents of flask production of protease. The four prominent
were inoculated with 1 ml of spore inoculum proteolytic isolates of actinobacteria were
(spore count 1x108 spores/ml), mixed gently further inoculated on the starch feather meal
and incubated in a slant position at 40 ºC in agar and incubated. Based on keratinolytic
a humidity chamber at 65-70% relative zone, the isolate DNA38 was emerged as a
humidity. The substrates were analyzed for potential strain for the synthesis of
the production of keratinase at an interval of kertainase (Figure 3).
every24 h.
Potential isolate of keratinolytic
Estimation of keratinase actinobacterium on starch feather meal agar
isolated from the soils from surroundings of
The keratinase activity of the fermented poultry farm wasidentified based on
broth or extract of both the bioprocess was standard colony characters and microscopic
estimated by modified method of Cheng et features (Table 1). Pigmentation pattern
al. (1995) using keratin as a substrate. The (Shirling and Gottlieb, 1976)of
reaction mixture containing 1ml of 1% aerial/substrate mycelium and diffusible
keratin in phosphate buffer (pH 8.0) and 0.5 pigment is an important attribute for the
ml of fermented broth or extract was identification of anactinobacterium. Gram s
incubated at 30 ºC for 30 min. After positive property, high mycelial branching
incubation, the reaction was terminated by and sporulation feature confirms the isolate
adding 2 ml of 10% trichloroacetic acid of actinobacterium as belonging to the genus
(TCA). After the separation of untreated Streptomyces. Important biochemical
keratin as pellet by centrifugation, 1ml of properties such as catalase production, no
clear supernatant was mixed with 5ml of 0.4 H S production and nitrate reduction were
2
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also recorded (Williams et al., 1989). The Bioprocess for the production of
growth of actinobacterium at higher range of keratinase
temperature, sodium chloride and pH were
assessed for the better understanding of its The efficient isolate Streptomyces
physiological adaptability and tolerance. minutiscleroticus DNA38was considered for
Several isolates of actinobacteria were the quantitative production of keratinase
reported (Shiveerakumar et al., 2013; under submerged bioprocess using starch
Madhusudhan et al., 2014; Mazhari et al., chicken feathers broth and solidstate
2014) earlier from the regional alkaline soils bioprocess under chicken feathers mineral
for the production of various enzymes at our salt substrate over a period of 168 h and 192
A-DBT (Actinobacteria- Diversity and h respectively. The isolate DNA38 showed
Bioprocess Technology) research laboratory 122.1 IU of keratinase activity (Figure 5)
and explored for various biotechnological under submerged process and 34.9 IU of
applications. keratinase activity (Figure 6)under solid
state process at 144 h and at 168 h of
Molecular characterization of
incubation respectively. Quantitative
Streptomyces DNA38
estimation for the production of keratinase is
most important criteria to select the suitable
Actinobacteria can be analyzed at various
bioprocess for the optimized enhanced
molecular levels to gain information suitable
production of keratinase.
for constructing databases and effective
identification. Sequence analysis of various
Production of keratinase has been achieved
genes provides a stable classification and
in liquid cultures of various microorganisms
accurate identification, which has become
and least from fungi. Bacteria like
the corner stone of modern phylogenetic
Microbacterium sp.- 10.5 U/mL (Riffel
taxonomy (Muyzer et al., 1996). The region
Alessandro and Adriano Brandelli 2006),
of 16S rRNA gene are highly variable and
Pseudomonas aurogenosa- 35.25 U/mL (Li
differ significantly between species where as
Jung Yin et al., 2006), Bacillus sp. JB99 -
other areas are more conserved and suitable
35.0 µg/mL/min (Pushpalata and Naik
for identification at the generic level
2010), B. subtilis- 463 U/mL (Ana Maria
(Amann and Ludwig, 2000).
Mazotto 2011), Bacillus sp. - 10 KU/mL
(Jeevana Lakshmi et al., 2013) have been
Apartial 16S rRNA gene sequence of isolate
reported for the production of keratinase.
DNA38 (566 nucleotides) was determined
Among Actinobacteria, Streptomyces sp.
(Genbank, NCBI Accession number:
MS-2 - 9.11 U/ml (Mona E. M. Mabrouk
KP419934). A phylogenetic tree was
2008), Streptomyces gulbargensis 1.39
constructed based on 16S rRNA gene
U/mL (Dastager et al., 2009), Streptomyces
sequence to show the comparative
albogriseolus NGP 71.43 U/mL (Selvam et
relationship between isolate DNA38 and
al., 2013) and Saccharothrix xinjiangensis
other related Streptomyces species (Figure
92.81 U/mL (Shilpa Ashok Jani et al.,
4). The comparative analysis of 16S rRNA
2014)and many more were reported for the
gene sequence and phylogenetic relationship
synthesis of keratinase. Several investigators
reveals that isolate DNA38 lies in a subclade
have reported on the occurrence of a variety
with Streptomyces minutiscleroticus, sharing
of keratiophilic fungi including
99.7 % of 16S rRNA gene sequence
dermatophytes in soils of varying habitats.
similarity.
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Int.J.Curr.Microbiol.App.Sci (2015) 4(7): 907-918
bioprocess and maximum of 172.7 U/mL of
The predominant keratinophilic fungi keratinase activity was achieved by Ana
reported in most studies include Maria et al. (2013) using Endophytic
Chrysosporium spp (mainly C. indicum, C. Penicillium sp. Bacillus pumilus GHD could
tropicum and C. keratiophilum) and the able to produce 73 U/mg of keratinase under
dermatophyte M. gypseum (Hoog et al., solid state bioprocess using sugar cane
2000). Production of keratinase have also bagasse as substrate (Ghada et al., 2011).
been achieved in solid state culture system. The present investigation reveals relatively a
Fungi are the favorite for the production of higher amount keratinase production by an
keratinase under solid state bioprocess efficient isolate of an actinobacterium,
followed by bacteria. Reports on the Streptomyces minutiscleroticus DNA38,
production of keratinase under solid state under submerged bioprocess with 9.0 pH of
bioprocess using actinobacteria are very the medium. Thus produced alkaline
scanty. Mervat et al. (2010) reported the protease can be explored further for various
production of keratinase using Aspergillus applications.
niger (160 U/mg) under solid state
Table.1 Morphological, biochemical and physiological characters of actinobacterium
Characters DNA38
Colony
Aerial mycelium Grey
Substrate mycelium White
Microscopic
Gram s staining + ve
Mycelium branching Poor
Sporulation level Scanty
Biochemical
Catalase production - ve
H S production + ve
2
Nitrate reduction + ve
*Growth at
Temperature
400/450 /500 C ++ / + / +
Sodium chloride
1 % / 2% / 3 % ++ / ++ / +
pH
8.0 / 9.0 / 10.0 ++ /+++ /
++
*+: Poor growth, ++: Moderate growth, +++: Maximum growth
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Int.J.Curr.Microbiol.App.Sci (2015) 4(7): 907-918
Figure.1 A representative plate showing colonies of actinobacteria on starch casein agar
Rear View Front View
Figure.2 Zone of catalysis by actinobacteria on skimmed milk agar
Rear View Front View
Figure.3 Keratinolytic zone by prominent isolates of actinobacteria on starch feather meal agar
Rear View Front View
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Figure.4 Phylogenetic tree indicating the systematic position of Streptomyces DNA38
Figure.5 Keratinase produced under submerged bioprocess using chicken feathers in starch and
mineral brothby Streptomyces minutiscleroticus DNA38
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Figure.6 Keratinase produced under solid state bioprocess on chicken feathers with starch and
mineral broth as moisture by Streptomyces minutiscleroticus DNA38
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