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Earthworm- a potential source for stable and potent antimicrobial compounds- isolation and purification study

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In the current work, we have isolated and purified an antimicrobial peptide from celomic fluid of Indian earthworm Pheretima posthumous. The purified peptide was characterized as serine protease and possesses broad range of antimicrobial activity. Another important finding was stability of antimicrobial peptide towards higher temperature, different pH and various inhibitors. The celomic fluid of earthworm was subjected to series of purification step such as ammonium sulphate precipitation, dialysis followed by size exclusion chromatography and ion exchange chromatography. The average molecular weight of peptide was found 20KD by SDS PAGE analysis. The peptide was evaluated for antibacterial and antifungal activity in various temperatures, pH and in the presence of different inhibitors. We have evaluated antibacterial activity of purified peptides among physchrophiles, mesophiles and thermophiles.
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Research Article
EARTHWORM- A POTENTIAL SOURCE FOR STABLE AND POTENT ANTIMICROBIAL
COMPOUNDS- ISOLATION AND PURIFICATION STUDY
1
YOGENDRA KUMAR VERMA,
2*
MAHENDRA KUMAR VERMA
1
Research Fellow, Department of Microbiology, All India Institute of Medical Sciences, New Delhi 110 028, India,
2
Department of
Biotechnology, R.V.R & J.C. College of Engineering, Chowdavaram, Guntur 522019, Andhra Pradesh, India.
Email: mahendra.sonal1983@gmail.com
Received: 1 Aug 2012, Revised and Accepted: 10 Sep 2012
ABSTRACT
In the current work, we have isolated and purified an antimicrobial peptide from celomic fluid of Indian earthworm Pheretima posthumous. The
purified peptide was characterized as serine protease and possesses broad range of antimicrobial activity. Another important finding was stability
of antimicrobial peptide towards higher temperature, different pH and various inhibitors. The celomic fluid of earthworm was subjected to series of
purification step such as ammonium sulphate precipitation, dialysis followed by size exclusion chromatography and ion exchange chromatography.
The average molecular weight of peptide was found 20KD by SDS PAGE analysis. The peptide was evaluated for antibacterial and antifungal activity
in various temperatures, pH and in the presence of different inhibitors. We have evaluated antibacterial activity of purified peptides among
physchrophiles, mesophiles and thermophiles.
Keyword: Celomic fluid, Antimicrobial activity, Purification, Earthworm, Electrophoresis and chromatography.
INTRODUCTION
Since ancient time, earthworm was known and used for its therapeutic
potential
1
. Various therapeutic molecules have been isolated and
characterized such as fibrinolytic, antiviral, antitumor, hepato-
protective, cytotoxic and antioxidant in different species of
earthworms around the world. In last few decades, the fibrinolytic
property has been explored tremendously
2,3.
Earthworm has
recognized as potential source of therapeutics not only because of
availability of various molecules but also stability and potency of these
isolates. The antimicrobial property (antibacterial and antifungal) of
earthworm have been evaluated by many researchers in last
decade
4,5,6
. The entire earthworms as paste has used for treatment of
wounds and tropical ulcer
7
. Such potent molecules naturally reside
predominantly in intestinal fluids and subsequently in tissue fluid of
earthworm. Lumbricin I and II are peptides, which have been reported
in Eisenia. fetida and Lumbricus rubellu
8,9
. These molecules
characterized as glycoprotein, which offers strong antimicrobial
activity
10
. The molecular mechanism and biophysical characterization
of these molecules have not completely explored yet.
The question often arises that why earthworm possesses such
potent molecules in their intestinal fluid and tissue fluid and their
pharmacological significance in earthworm physiology. The most
accepted theory concluded its habitat where earthworm encounters
various microorganism including bacteria fungi and viruses
11
.
Besides advanced animal’s immune system such as humoral and cell
mediated immunity, lower organism’s immunity constituted with
such type of molecules, which offer first line defense mechanism.
Earthworm essentially requires such potent molecules as it feeds on
organic matter with soil
12
. These glycoproteins are characterized as
protease (especially serine protease) offer proteolytic degradation
of microbial population and provide first line defense system
13,14
.
In this study, we have isolated and purified protein offering
antimicrobial property of celomic fluids from Indian Earthworm
Pheretima posthumous. Further, we are looking for the complete
molecular mechanism of these molecules, so these can be used for
treatment of various bacterial and fungal borne diseases.
MATERIALS AND METHODS
The chemicals and consumables used in the following study
purchased form Hi-Media, Sigma and GE healthcare. All the
chemicals were molecular biology grade and prepared freshly at the
time of use. For determination of antimicrobial activity, used
bacterial and fungal strains were purchased from MTCC Chandigarh,
India. Bacterial and fungal strains used were wild type and
nonpathogenic for following study.
Preparation of Extract
For the current work, Indian Earthworm Pheretima posthumous was
selected as source of antimicrobial molecules. The earthworms were
collected from vermicomposting unit at Vijayawada, Andhra Pradesh,
India. Fully-grown healthy Earthworms were subjected to autolysis
and further homogenization in 20mM phosphate buffer pH 7.5 for one
week at 50
0
C with 0.02% sodium azide as bacteriostatic. Autolysed
earthworms were further subjected to high-speed centrifugation at
20,000rpm for 30 minute and soup was collected in separate sterile
tubes. The clear soup was filtered by membrane filters (0.4µm, Hi-
Media) and stored at 4
0
C for purification cascade
15,16
.
Purification of Peptide
The crude celomic filtrate was further processed by series of
chromatographic step for purification of desired peptide.
Ammonium Sulphate Precipitation
The salt precipitation is ideal method to recover total protein
content of crude extract. The precipitation of proteins via
ammonium sulphate does not hamper protein activity. Ammonium
sulphate was used to precipitate total protein from crude filtrate.
More than 90% of total protein was precipitated at 60% of
ammonium sulphate. The precipitate was separated from soup by
high speed centrifugation 20,000 rpm at 4
0
C for 30 minute. The
precipitate was suspended in 20mM phosphate buffer pH 7.5 and
further filtered with membrane filter of pore size 0.4µm.
Dialysis
To remove the salt proportion from protein approximately 12 hour
Dialysis was performed to remove salt from filtrate. Dialysis
membrane possessing Molecular Weight Cut Off (MWCO) of 12KD
purchased from Hi Media was preferred for desalting. The 2ml of
ammonium sulphate precipitate was loaded into dialysis bag and
desalted against 20mM phosphate buffer pH 7.5. The buffer was
replaced in each 4 hour and dialysis was run for 12 hour. After 12
hour, soup was collected in sterile tube and preserved at 4
0
C.
Size exclusion chromatography
The soup collected after dialysis was subjected to size based
separation of proteins and peptides from crude filtrate. In this study,
we have used Sephadex G 50. The Sephadex G 50 (GE Healthcare)
beads were allowed for complete swelling in 20mM phosphate
buffer pH 7.5 for 48 hours at room temperature. The swelled beads
were sonicated to remove traces of entrapped air. Further complete
swelled beads were loaded in glass column (60cm*1.5cm) up to
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491 Vol 4, Issue 4, 2012
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Verma et al.
Int J Pharm Pharm Sci, Vol 4, Issue 4, 540-543
541
paced bed length 30cm. 2 ml of dialyzed sample was loaded in the
Sephadex G-50 column and eluted with excess of 20mM phosphate
buffer pH 7.5 in automated fraction collector with 2ml per minute
speed
17
. Total 30 fractions were collected (3ml each) and each of
fractions was assessed for antimicrobial and protease activity. The
fractions shown maximum activity were pooled together and
subjected to further purification by charge-based separation. The
total protein estimation of each fraction was carried out by Lowery
method to calculate concentration.
Ion Exchange Chromatography
Pooled fractions possessing higher antimicrobial activity were
further fractionated by ion exchange chromatography where DEAE
Cellulose (GE Healthcare) was used for column preparation. The
DEAE resin was charged by treatment with acid (HCl) pH (2-3),
alkali (NaOH) (pH10-11), distilled water and finally in phosphate
buffer 20mM, pH 7.5. The charged DEAE Cellulose resin was loaded
on the glass column (60cm*1.5cm) with 20 cm packed bed length.
2ml of pooled fraction was loaded in column and eluted with excess
of phosphate buffer and different concentration of Sodium Chloride
(0.1% - 0.5%)
18
. A total 20 fractions were collected and each fraction
was subjected to the antimicrobial and protease activity analysis.
SDSPAGE Analysis
The average molecular weight of purified peptide possessing
antimicrobial activity was determined by SDS-PAGE. A 15% of
acrylamide gel was prepared, 15µl of purified sample was loaded
and electrophoresis was run for 3 hour at 50 milli-volts with 0.5 X
tris glycine electrophoresis buffer. With purified sample protein
marker was loaded to determine average molecular weight. After
completion of electrophoresis gel was stained with
Coomassie Brilliant Blue R-250 and destained subsequently with
excess of methanol in destaining solution.
Determination of Caseinolytic Activity
The purified fractions were analyzed for the proteolytic activity by using
casein as substrate. Casein agar plates were prepared with 2% casein
(Hi-Media) with agar and phosphate buffer 20mM pH 7.5. The
caseinolytic activity of purified fractions was assessed by well diffusion
method where casein agar plates incubated overnight at 37
0
C
19
.
Assessment of antimicrobial activity
Purified fractions were evalu ated for antib acter ial and
antifungal activity by well diffusion method. Different wild type
bacterial and fu ngal strai ns including phy schro philes,
mesophiles and thermophi les were used for assessing the
antimicrobial activity. Both Gram positive a nd Gram negative
bacterial strains were used in th e study included Escherichia
coli, Pseudomona s putida, Streptococcus aureus, Azotobactor,
Bacillus streothermophilous and Pectobacteri um carotovorum.
For the determination of antibacteria l activity, well diffusion
metho d was used with sli ght modifications where n utrie nt Broth
used for growth of bacterial cultures and nutrient agar for plate
preparation. The 6 mm diamete r wells were punched into
nutrient agar plates and plates were left for solidification. The
purified fraction of celomic fluid was filled (20µl) in each well
with ampicillin (100µg/µl) positive control. The plates were
incubated at 37oC for 14hours and antibacterial activit y was
deter mined by measuring the diameter of zone of inhibition.
Further, we have incubated purified fractio ns in various
tempe rature 200C-500C and variou s pH 4- 12 fur ther then
analy zed for t heir antimicrobial acti vity.
For the determination of antifungal activity, we have used
Sabouraud’s dextrose/agar (SDA) for the growth of fungal culture.
The protocol runs same as that for assaying, the antibacterial
activity was evaluated and fungal cultures were kept for 48 h to
determine the diameter of zone of inhibition. Here we have used
ketoconazole (1mg/ml) as positive control. For the accessing
antifungal activity Penicillium and candida, species have been used
20
.
RESULT
Isolation and Purification
The crude celomic fluid was purified by successive
chromatographic matrix. The total protein content of celomic fluid
was precipitated by 60% ammonium sulphate and recovered more
than 90% of total protein. Further precipitate was dialyzed against
20mM phosphate buffer pH 7.5 with dialysis membrane molecular
weight cut off (MWCO) 12KD procured from HiMedia. In Gel
filtration chromatography, we have collected 30 fractions and
fraction number 12,13,14,15 shown maximum antimicrobial
activities, which were further, pooled and loaded in run in ion
exchange chromatography using DEAE Cellulose column. In 20
eluted fractions from Ion exchange chromatography 9 and 10
fraction were selected for antimicrobial activity as shown
enormous activity for antibacterial and antifungal property and
SDSPAGE study.
Graph 1: Sephadex G 50 eluted fractions and their absorbance at 280nm for dialyzed celomic fluid
Graph 2: DEAE Cellulose eluted fractions and their absorbance at 280nm for Sephadex G 50 fractions have shown maximum antimicrobial activity
Verma et al.
Int J Pharm Pharm Sci, Vol 4, Issue 4, 540-543
542
Proteolytic Activity
The crude as well as purified fractions have shown proteolytic
activity on casein agar plate as zone of clearance after incubation for
14 hour at 20
0
C-50
0
C. The activity was accessed in different
temperature and various pH. The proteolytic activity was
constrained for purified fraction, which was found maximum at 40
0
C
at pH 8.0 while least in 20
0
C at pH 4.0.
Fig. 1: Caseinolytic activity of celomic fluid while purification
Dialyzed (1 & 2), Sephadex G 50 fraction (3, 4, 5 & 6) and
DEAE Cellulose fractions (7 & 8), while A, B and C are Phosphate
Buffer, distilled water and native Protease
Molecular weight determination
The average molecular weight for purified fraction was determined
by SDS-PAGE. The 15% acrylamide gel was prepared and purified
fraction was resolved with standard protein marker. After staining
with Coomassie Brilliant Blue R-250 in both two fractions after
DEAE Cellulose column separation a thick band was observed
corresponding to 20KD of standard protein marker.
Fig. 2: SDSPAGE analysis of purified fraction on 15% acrylamide
with standard protein ladder
Antimicrobial Activity
The antimicrobial activity of purified fractions was evaluated in the
context with zone of inhibition on nutrient agar plate after overnight
incubation. The following data is collected while accessing activity
with different microbial population, which is shown in table-
Table 1: Assessment of antimicrobial activity of purified
peptide by well diffusion method
Bacterial strain
s
Zone of Inhibition
(mm)
Escherichia
coli
19
P
actobacterium
.
carotovorum
14
P
seudomonas
putida
16
S
treptoc
occus.
aureus
17
B
acillus
streothermophilous
14
Azotobactor
16
Penicillium sp
.
14
Candida sp
.
13
Fig. 3: Zone of clearance after incubation of purified fraction on
nutrient agar plate plated with Escherichia coli and
Pseudomonas putida
CONCLUSION
In the current work, we have collected Indian earthworms and
processed mature worms for collection of intestinal fluid. The
intestinal fluid was further processed and purified by different
chromatographic techniques including size based and charged based
separation
21,22
. The purified fractions were analyzed for apparent
molecular weight of purified protein was found 20KD. Further
purified fractions were accessed proteolytic activity and found
serine protease by casein agar plate as zone of clearance after
overnight incubation with casein as substrate. Further purified
fractions were evaluated for antimicrobial activity (antibacterial and
antifungal activity) by well diffusion method and zone of inhibition
was measured
31,24&25
.
Many of facts about the molecules possessing antimicrobial property
of earthworm’s intestinal and tissue fluids need more attention in
order to explore at molecular level
26,27
The biophysical
characterization of such potent molecules led to understand precise
molecular mechanisms and mode of action that further can be tuned
to cure for various microbes borne diseases. Further, molecular
characterization can lead to identification of DNA coding sequence of
these molecules in earthworm genome
28
. That can provide a
platform for production of antimicrobial peptides by r-DNA
technology, which is economical and does not violate bioethics.
Currently our team is working for finding exact molecular weight
and N-terminal protein sequence
29
. That will be useful not only to
understand molecular properties of antimicrobial molecules but also
to predict evolution and biological inter-relation of biomolecules
after generating phylogenetic tree in various species of earthworm
.We are also looking for crystallography of purified peptide in order
to generate pdb, essentially useful for insilico analysis and system
biology for new generation antimicrobial compounds.
ACKNOWLEDGEMENT
I would like to thank Management and Principal, R.V.R. & J.C. College
of Engineering, Chowdavaram, Guntur, Andhra Pradesh, India for
providing laboratory facility to carry out research and manuscript
preparation.
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... Cooper et al. [46] illustrated the presence of antimicrobial and anticancer molecules in the earthworms. Similarly, several species of earthworms were screened for antimicrobial activities by Kathireswari et al. [47], Istiqumah et al. [48], Verma and Verma [49], and Chauhan et al. [50]. ...
... e outcomes of the current research are consistent with the findings of previous literature as Vasanthi et al. [76] presented the antimicrobial activity of Eudrilus eugeniae against S. aureus; Kathirewari et al. [47] found the antimicrobial effect of coelomic fluid of earthworm against microbes; Istiqumah et al. [48] studied the antibacterial activity of Lumbricus rubellus extracts against E. coli, S. aureus, Salmonella pullorum, and P. aeruginosa; Verma and Verma [49] found that coelomic fluid of earthworm P. posthumous had maximum antibacterial activity against E. coli (19.00 mm); Chauhan et al. [50] illustrated the antibacterial and antifungal activity of Eudrilus eugeniae; and Bhorgin and Uma [77] showed that ethanolic extract of earthworm powder possessed maximum antibacterial activity in comparison with petroleum ether and aqueous extract against A. hydrophila. ...
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Humans evolved with a unique immune system consist of innate and adaptive immunity. Both innate and adaptive immunity comprised of different types of cells, immune players, and associated with different functions. In higher animals, including humans, innate immunity derived from birth hence called inbuilt immunity. On the contrary, adaptive immunity called acquired immunity is gain due to the environment. Vertebrates are ranked as superior over invertebrates on the classification table due to many reasons, including the presence of vertebra and spinal cord. Invertebrates often considered as lower animals due to poorly developed organs, lack of vertebra and spinal cord. Similarly, the immune system and set of immune organs in a lower organism are entirely different and primitive on the basis of the organization. Like humans and other vertebrates, the invertebrates do not have a distinct clas-sification/setup of innate and adaptive immunity. In human and higher vertebrates, innate and adaptive immune systems are consisting of different cells; immune players such as cytokines, anti-bodies, and chemokines, etc. enable crosstalk between innate and adaptive immune systems. On the contrary, invertebrates' immune system is consists of unique cells and potent biomolecules; enzyme , peptides, and proteins. These molecules provide a complete defense to the animal via several mechanisms. The question we raise here does the classification superiority of humans, and other vertebrates over in vertebrates are justified in terms of immunity. How a complex and evolved immunity system mostly in human often fail to fight against infections while lower animals with the primitive immune set can survive even in hearse conditions. Apart from organization setup in humans and higher verte-brate's immunity can be further classified based on function such as cell-mediated and Humoral immunity. Cell-mediated immunity provides protection using different cells such as white blood cells (WBC), Macrophage, Phagocytes, Dendritic cells and T cell (CD4 and CD8), etc. Humoral immunity is a function of B cells, memory cells and antibodies. The immune response in human and other higher vertebrates consist of activity of different cells and immune players for identification of the foreign substance, presentation to immune system followed by killing/removal. The effective identification and clearance of foreign objects depend on synchronization between immune cells and players of both innate and adaptive immunity. On the contrary, lower animals, i.e. invertebrates, do have a more straightforward mechanism of identification and clearance of foreign objects that ultimately depends on endogenous short peptides, proteins, and enzymes. The more straightforward sets often provide ease in synchronization of immune players in lower animals reported more effective and robust. There is growing evidence that enzyme and protein as part of the immune system in the lower animal are highly dynamic and do possess promiscuity at the substrate and catalytic level. Enzyme promiscuity is capacity often describe for broad substrate affinity and catalytic diversity. The short peptides, proteins, and enzymes present in lower animals; invertebrates are highly promiscuous and capable of catalyzing multiple biochemical reactions. Earthworm, a classic invertebrate model for immune comparison , studied tremendously in the last couple of decades. The animal habitat is an environment rich in infections and invading elements. The presence of promiscuous and potent biomolecules including enzyme; serine protease provides a complete defense to the animal. The coelomic cavity of animals is rich in many short peptides, and other bioactive molecules offer extended protection to a wide range of infections. The key molecules are Fetidin 1, Fetidin 2, Lysenin, Eiseniapore, Coelomic Cytolytic Factor (CCF), and Lumbricin I. These molecules exist in isoforms and vary in size among different species of earthworm. The isoforms of these molecules provide extended support in defense mechanisms. There are growing research findings suggested protease present in coelome of earthworm are generally exist in isoforms. Each isoforms possesses a broad substrate affinity and catalytic diversity. This can be compared with VDJ recombination in B cell for the diversity of anti-bodies. There is another advantage in having promiscuous and iso-forms of series protease in coelome of animal, i.e. a large number Citation: Mahendra Kumar Verma and MV Raghavendra Rao. "Mapping Immunity; Vertebrates Versus Invertebrates". Acta Scientific Biotechnology 1.8 (2020): 42-43.
... Natural extracts are advantageous in this regard as their therapeutic properties can be exploited with minimal adverse effects. The medicinal benefits of earthworm extracts have been explored by researchers in the past few decades with notable antimicrobial, antipyretic, antiinflammatory, fibrinolytic and antioxidant properties [15][16][17]. The earthworms possess innate and adaptive immunity properties as a wide range of immunoprotective leukocytes are synthesized and secreted [18,19]. ...
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... Serine protease has been produced by many organisms, including plants, animals and microbes (Liu et al. 2010;Sakanari et al. 1989). Verma and Verma (2012) reported the isolation and purification of antimicrobial peptide from celomic fluid of Indian earthworm Pheretima posthumous. They identified the isolated peptide as serine protease that possesses a broad range of antimicrobial activity. ...
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... The salt water in which the earthworms are soaked is often given to manage postpartum complications in women [4] . Additionally, earthworm paste, since ancient times, is used clinically for wound heal and as antimicrobial in surgeries [5] . In Chinese Materia Medica earthworm is considered as complete medicine and used for various therapeutic purposes such as blood purification, blood disorders, against jaundice, wound healing, and as antimicrobial, anti-inflammatory and antioxidant agent [6] . ...
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