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ENCLOSURE-I
6.0 BRIEF RESUME OF THE INTENDED WORK:
6.1 GENERAL DISCUSSION:
Coagulation is a complex process by which blood forms clots. It is an important
part of hemostasis , where in a damaged blood vessel wall is covered by a platelet and fibrincontaining clot to stop bleeding and begin repair of the damaged vessel. Coagulation begins
almost instantly after an injury to the blood vessel has damaged the endothelium (lining of the
vessel). Exposure of the blood to proteins such as tissue factor initiates changes to blood
platelets and the plasma protein fibrinogen, a clotting factor. Platelets immediately form a
plug at the site of injury; this is called primary hemostasis. Secondary hemostasis occurs
simultaneously. The coagulation cascade of secondary hemostasis has two pathways which
lead to fibrin formation. Those are intrinsic pathway, and the tissue factor pathway (extrinsic
pathway). It is known that the primary pathway for the initiation of blood coagulation is the
tissue factor pathway. The pathways are a series of reactions, in which a zymogen (inactive
enzyme precursor) of a serine protease and its glycoprotein co-factor are activated to become
active components that then catalyze the next reaction in the cascade, ultimately resulting in
cross-linked fibrin.12
The serine proteases are a family of enzymes that cut certain peptide bonds in
other proteins. This activity depends on a set of amino acid residues in the active site of the
enzyme - one of which is always a serine. In mammals, serine proteases perform many
important functions, especially in digestion, blood clotting, and the complement system. Three
protein-digesting enzymes secreted by the pancreas are serine proteases (chymotrypsin,
trypsin, elastase). Several activated clotting factors are serine proteases, including (Factors 10
(X), 11 (XI), and 12 (XII), Thrombin, Plasmin, which are involved in coagulation cascade.13
Serine proteases are highly conserved modular proteins that have evolved to
participate in biochemical reactions in blood coagulation. Hence the present study is carried
out to produce serine protease and to screen its coagulant activity.
6.2 NEED FOR THE STUDY:
Serine proteases or serine endopeptidases (newer name) are a class of peptidases
(enzymes that cleave peptide bonds in proteins) that are characterised by the presence of a
serine residue in the active site of the enzyme. Serine proteases are grouped into clans that
share structural homology and then further sub grouped into families that share close sequence
homology. The major clans found in humans include the chymotrypsin-like, the subtilisin-like,
the alpha/beta hydrolase, and signal peptidase clans.
Proteases constitute 60 – 65% of the global industrial enzyme market most of which
are serine & metallo-proteases. Serine proteases play diverse roles in human health, from nonspecific digestion to highly regulated functions like embryonic development, immune
response and blood coagulation. Tissue plasminogen activator (TPA) is a serine protease
occurring in animals including humans. Human-identical TPA (produced industrially by
genetically recombinant microorganisms) has an established medical use in the treatment of
ischemic stroke by its proteolytic activity it enables the action of another enzyme (plasmin),
which breaks down the protein (fibrin) of blood clots. Immunity, and inflammation, as well as
contributing to digestive enzymes in both prokaryotes and eukaryotes. Disorders of
coagulation can lead to an increased risk of bleeding (hemorrhage) or obstructive clotting
(thrombos) leading to serious consequences.
Currently a large proportion of the commercially available proteases are derived from
Bacillus Strains. The reason for this is their high pH & temperature stability. Proteases which
have a serine centre, exhibiting a wide range of PH 6 – 13 & having wide industrial
applications (Anders Tunlid1 et al, 1994). In the present study optimization of the production
of serine protease from Bacillus licheniformis organisms and its characterization to fulfill the
present industrial needs is to be conducted and coagulant activities of serine proteases is
screened.
6.3 REVIEW OF LITERATURE :
1. Saurabh, S., et.al, 9 The growth and protease production by Bacillus sp. (SBP-29) was
examined for poultry processing industries. The maximum protease activity was 3028 U/mL
using 1.5% (w/v) of soybean meal as substrate. Soybean meal is an inexpensive and readily
available, thus it can be used as the cost effective crude material for the production of an
extracellular protease. Inorganic nitrogen sources proved to be less favorable, for protease
production as strong catabolic repression was observed with ammonium ions. A maximum of
3208 U/mL of protease was produced in 18 h in a 10L bioreactor. The enzyme has
temperature and pH optima of 60°C and 9.5 respectively. However, the temperature stability
range is from 20-90 °C and pH stability range is from 6.0–12.0. The protease was completely
inhibited by phenylmethylsulfonyl fluoride (PMSF) and diodopropyl fluorophosphate (DFP),
with little increase (10-15%) in the production of upon addition of Ca++ and Mg++.
2. Saleh A. et.al, 8 The molecular weights of F. gigantica PIc and PII were 60,000 and 25,000,
respectively. F. gigantica PIc and PII had pH optima at 7.5 and 5.5 and KM of 2 and 5 mg
azocasein/mL, respectively. For amidolytic substrates, PIc had KM of 0.3 mM BAPNA/mL
and 0.5 mM BTPNA/mL at pH 8.0 and PII had KM of 0.6 mM BAPNA/mL at pH 6.5 with
reducing agent. F. gigantica PIc and PII had the same optimum temperature at 50 -C and were
stable up to 40 -C. All examined metal cations tested had inhibitory effects toward the two
enzymes. From substrate specificity and protease inhibitor studies, PIc and PII could be
designated as serine PIc and cysteine PII, respectively. D 2005 Elsevier Inc. All rights
reserved.
3. Jinkui Yang et.al,
5
An extracellular protease (PrC) was purified from an isolate of
Clonostachys rosea (syn. Gliocladium roseum) to apparent homogeneity. The protease had a
molecular mass of 33 kDa estimated by SDS-PAGE. The optimum activity of PrC was at pH
9–10 and 60 8C (over 10 min). The purified protease could degrade a broad range of
substrates including casein, gelatin and nematode cuticle. 80±5% of nematodes (Panagrellus
redivivus) were immobilized and degraded after treating with PrC for 48 h. The protease was
highly sensitive to PMSF (phenylmethyl sulfony fluoride) (5 mM) indicating it belonged to
the serine protease family. The N-terminal amino acid residues of PrC are ATQSNAPWGL,
which share a high degree of similarity with other cuticle-degrading proteases from
nematophagous and entomopathogenic fungi suggesting PrC play a role in infection process.
4. Usama F. Ali et.al,
7
Aspergillus terreus a local wild fungal isolate was tested for the
production serine alkaline protease (E.C. 3.4.1.14) when grown on whey amended with some
agro-industrial by-products. The addition of 30 % whey increased the activity to 2.25 units
/ml, the addition of 40% cane molass yield 3.40 units/ml, the addition of 30% beet molass
give 2.84 units/ml, the addition of 1% glutofeed give 2.88 units/ml while, the addition of 0.5%
of ragee elkon give 2.94 units/ml. The optimum condition for enzyme production was 40 ºC,
six days of incubation and pH 7.0. The enzyme was pH stable at a wide range of pH and also
thermostable at temperatures 40-90 ºC. The enzyme was found to compatible with certain
local detergents and suitable for detergent applications.
5. Charles et.al,
6
Aspergillus nidulans is a highly potent fungus used in the production of
alkaline protease. Extracellular alkaline protease was purified from A. nidulans in a two-step
procedure involving ammonium sulphate precipitation and Sephadex G-100 column
chromatography. The molecular mass of the enzyme was determined to be 42 kDa by SDSPAGE. The enzyme activity was also analyzed by zymogram with gelatin. The enzyme was
more stable over a wide range of pH (6–10) and the temperatures up to 50 °C. It showed
optimum enzyme activity at pH 8.0 and a temperature of 35 °C. The protease enzyme was
completely inhibited by the serine protease inhibitor of phenylmethylsulfonyl fluoride
(PMSF). The crystallization of the purified enzyme was performed by hanging drop vapour
diffusion method using PEG 6000 as the precipitant. The micro crystals occurred in 40% of
PEG 6000.
6. Jinkui Yang, et.al 5 worked on the gene encoding a cuticle-degrading serine protease was
cloned from three isolates of Lecanicillium psalliotae (syn. Verticillium psalliotae) by 3¢ and
5¢ RACE (rapid amplification of cDNA ends) method. The gene encodes for 382 amino acids
and the protein shares conserved motifs with subtilisin N and peptidase S8. Comparison of
translated cDNA sequences of three isolates revealed one amino acid polymorphism at
position 230. The deduced protease sequence shared high degree of similarities to other
cuticle-degrading proteases from other nematophagous fungi.
6.4 OBJECTIVES OF THE STUDY:
1. Procuring the Bacillus licheniformis from MTCC.
2. Sandardisation of process parameters (Time ,Temperature, pH, Carbon source, Metal ion,
Nitrogen source) for enhanced productivity.
3. Characterisation of serine protease.
4. Evaluation of coagulation activity.
ENCLOSURE-II
7.0 MATERIALS AND METHODS:
1. Procuring the Bacillus licheniformis from MTCC and screening for protease
production3.
Procuring and the organism was inoculated in skim milk agar and checked for the
protease production.
2. Standardisation of process parameters (temperature, pH, carbon source, metal ion,
nitrogen source) for enhanced productivity.
A modified medium from saurabh9, S et al 2007 was used for the production media and
standardised for the different parameters.
3. Characterization of serine protease1,4
Characterization:
a) SDS-PAGE
b) Native PAGE
c) Effect of temperature on enzyme activity
d) Effect on pH on enzyme activity
e) Effect of substrate concentration
f) Effect of activators
g) Effect of inhibitors
h) Thermal stability of the enzyme.
4. Screening for coagulant activity:10
a. Prothrombin Time (PT):
An aliquot of 0.1 ml of citrated plasma is incubated for 1 min at 37 °C. Then 0.2 ml of
human thromboplastin (Thromborel®, Behring Werke, Marburg) is added and the coagulometer (Schnittger + Gross coagulometer, Amelung, Brake) isstarted. The time to
clot formation is determined. The PT measures effects on the exogenous pathway of
coagulation.
b.
Thrombin Time (TT):
To 0.1 ml of citrated plasma 0.1 ml of diethyl barbiturate-citrate buffer, pH 7.6 (Behring Werke Marburg) is added and the mixture is incubated for 1 min at 37 °C. Then
0.1 ml of bovinetest- thrombin (30 IU/ml, Behring Werke Marburg) is added and the
coagulometer is started. The time to clot formation is determined. The TT measures effects on fibrin formation.
c. Activated Partial Thromboplastin Time (APTT).
To 0.1 ml of citrated plasma 0.1 ml of human placenta lipid extract (Pathrombin®,
Behring Werke, Marburg) is added and the mixture is incubated for 2 min at 37 °C.
The coagulation process is initiated by the addition of 0.1 ml 25 mM calcium chloride
when the coagulometer is started and the time to clot formation is determined. The
APTT measures effects on the endogenous pathway of coagulation.
7.1 SOURCE OF DATA:
1. Library, Bharathi College of Pharmacy.
2. E-library, Bharathi College of Pharmacy.
3. Library, RGUHS, Bangalore.
7.2 METHOD OF COLLECTION OF DATA:
The preliminary data required for the experimental study were obtained from
1. Internet.
2. Scientific Abstracts.
3. Scientific Journals.
4. Relevant Books on Enzymology.
5. Relevant Books on Biotechnology.
7.3 DOES THE STUDY REQUIRE ANY INVESTIGATION OR INTER VENTIONS
TO BE CONDUCTED ON PATIENTS OR OTHER HUMANS OR ANIMALS?
-NO-
7.4 HAS ETHICAL CLEARENCE BEEN OBTAINED FROM YOUR INSTITUTION
IN CASE OF 7.3?
- NOT APPLICABLE-
ENCLOSURE-III
8.0
REFERENCES:
1. Anders Tunlid, Stefan Rosen, Bo Eke, Lars Rask. Purification and characterization of an
extracellular serine protease from the nematode-trapping fungus Arthrobotrys oligospora,
Microbiology 1994;140:1687-95.
2. Ahmed Al Jabr , Mahmoud Abo-El-Saad . A Putative serine protease from larval midgut
of red palm weevil Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae):
Partial purification and biochemical characterization. Am J of Envron Sci. 2008;4(6):595601.
3. Asokan S, Jayanthi. Alkaline protease production by Bacillus licheniformis and Bacillus
coagulans. J of Cell and Tissue Res. March 5, 2010;10(1):2119-23.
4. Bayoumi R A, Louboudy S S, Sidkey N M, Abd-El-Rahman M A. Production,
purification, and characterization of thermo stable alkaline protease under solid state
fermentation conditions for application in bio-detergent technology. Egy J of Biotechnol.
2007;25(1):111-29.
5. Jinkui Yang, Xiaowei Huang, Baoyu Tian, Hui Sun, Junxin Duan, Wenping Wu &Keqin
Zhang. Characterization of an extracellular serine protease gene from the nematophagous
fungus Lecanicillium psalliotae. 2005; 27 April.
6. Charles P, Devanathan.V, Periasamy Anbu, Ponnuswamy M.N, Kalaichelvan P.T, ByungKi Hur. Purification, characterization and crystallization of an extracellular alkaline
protease from Aspergillus nidulans HA-10. J of Basic Microbiol 2008;48: 347–52.
7. Usama Ali F. Utilization of whey amended with some agro-industrial by-products for the
improvement of protease production by Aspergillus terreus and its compatibility with
commercial detergents. Res J of Agri and Biol Sci. 2008;4(6):886-91.
8. Saleh Mohamed A, Afaf S, Fahmy, Tarek Mohamed M, Soha Hamdy M. Proteases in
egg, miracidium and adult of Fasciola gigantica. Characterization of serine and cysteine,
Proteases from adult. Comparative Biochemistry and Physiology, Part B. 2005;142:192–
200.
9. Saurabh S, Jasmine I, Pritesh G, Rajendra Kumar S. Enhanced productivity of serine
alkaline protease by Bacillus sp. using soybean as substrate. Malays J of Microbiol.
2007;3(1):1-6.
10. Vogel HG. Drug Discovery and Evaluation: Pharmacological Assays. 3rd ed. Vol 1.
London:Springer; 2002. ISBN 3-540-42396-6:277-78.
11. Bennett T. P, Frieden E. Modern Topics in Biochemistry. Macmillan, London 1969;43-45.
12. http://www.wikipedia.org/wiki/coagulation.
13. http://www.lassesen.com/cfids/coagulation.htm/.