Documente Academic
Documente Profesional
Documente Cultură
By
PRANAV BHASKAR
CONTENTS
1. INTRODUCTION
2. METHODS
4. APPENDIX
INTRODUCTION
Enzymes are the biomolecules that act as efficient catalysts and help complex
reactions occur everywhere in life. They speed up reactions by providing an
alternative reaction pathway of lower activation energy. Like all catalysts,
enzymes take part in the reaction - that is how they provide an alternative
reaction pathway. But they do not undergo permanent changes and so remain
unchanged at the end of the reaction. They can only alter the rate of reaction,
not the position of the equilibrium.
Most chemical catalysts catalyze a wide range of reactions. They are not
usually very selective. In contrast enzymes are usually highly selective,
catalyzing specific reactions only. This specificity is due to the shapes of the
enzyme molecules. Many enzymes consist of a protein and a non-protein
(called the cofactor). The proteins in enzymes are usually globular. The intra-
and intermolecular bonds that hold proteins in their secondary and tertiary
structures are disrupted by changes in temperature and pH. This affects
shapes and so the catalytic activity of an enzyme is pH and temperature
sensitive. Enzymes are widely used commercially, for example in the
detergent, food and brewing industries. Protease enzymes are used in
'biological' washing powders to speed up the breakdown of proteins in stains
like blood and egg. Pectinase is used to produce and clarify fruit juices.
For two molecules to react they must collide with one another. They must
collide in the right direction (orientation) and with sufficient energy.
Sufficient energy means that between them they have enough energy to
overcome the energy barrier to reaction. This is called the activation energy.
Enzymes have an active site. This is part of the molecule that has just the
right shape and functional groups to bind to one of the reacting molecules.
The reacting molecule that binds to the enzyme is called the substrate.
So the enzyme is used to form a reaction intermediate, but when this reacts
with another reactant the enzyme reforms.
CLASSIFICATION OF ENZYMES
There are three major groups of biological enzymes: (1) Food Enzymes, (2)
Digestive Enzymes and (3) Metabolic Enzymes. In the past, the therapeutic
use of enzymes has largely focused on the use of digestive enzymes. Digestive
enzymes can be directly beneficial because they assist in digestion, help
regulate immune responses in the intestinal tract, and relieve the body of its
relative requirement of digestive enzyme production, allowing for biological
energy and resources to be further allocated to the production of metabolic
enzymes, indirectly.
1. Temperature
As the temperature rises, reacting molecules have more and more kinetic
energy. This increases the chances of a successful collision and so the rate
increases. There is a certain temperature at which an enzyme's catalytic
activity is at its greatest. This optimal temperature is usually around human
body temperature (37.5 oC) for the enzymes in human cells.
Each enzyme works within quite a small pH range. There is a pH at which its
activity is greatest (the optimal pH). This is because changes in pH can make
and break intra- and intermolecular bonds, changing the shape of the enzyme
and, therefore, its effectiveness.
NATTOKINASE
Nattokinase, like plasmin, is a potent fibrinolytic enzyme extracted and highly
purified from a traditional Japanese food called Natto. Nattokinase is a serine
endo-peptidase with a molecular weight of 20,000 Daltons and a point of
ionization (pI) of 8.6. Natto is a fermented soybean derivative, a soy cheese
that has been a staple in the Japanese diet, for over 1000 years for its popular
taste and as a folk remedy for heart and vascular diseases. Natto is produced
by a fermentation process by adding Bacillus natto, a beneficial bacteria, to
boiled soybeans resulting in the production of the nattokinase enzyme.
Nattokinase enhances the body's natural ability to fight blood clots, and has an
advantage over blood thinners because it has a prolonged effect without side
effects.
Nattokinase:
dissolves fibrin
Blood has a sticky quality that helps it clot and stop the bleeding from
wounds. When a wound occurs, blood platelets rush to the wound site and
cause a series of reactions that produce strands of fibrin. These fibrin strands
form a thin, web-like structure that covers the wound and stops the bleeding.
Research has established that the fibrin strands are the main cause of sluggish
blood, so researchers next began looking for a substance that would act to
maintain healthy levels of fibrin. That breakthrough discovery was
Nattokinase, a natural, food-based supplement that supports healthier fibrin
levels so that blood flows at a faster rate, reducing blood pressure and
cholesterol levels.
Blood clots (or thrombi) form when strands of protein called fibrin
accumulate in a blood vessel. In the heart, blood clots cause blockage of blood
flow to muscle tissue. If blood flow is blocked, the oxygen supply to that tissue
is cut off and it eventually dies. This can result in angina and heart attacks.
Clots in chambers of the heart can mobilize to the brain. In the brain, blood
clots also block blood and oxygen from reaching necessary areas, which can
result in senility and/or stroke.
Since endothelial cells exist throughout the body, such as in the arteries, veins
and lymphatic system, poor production of thrombolytic enzymes can lead to
the development of thrombotic conditions virtually anywhere in the body.
It has recently been revealed that thrombotic clogging of the cerebral blood
vessels may be a cause of dementia. It has been estimated that sixty percent of
senile dementia patients in Japan is caused by thrombus. Thrombotic diseases
typically include cerebral hemorrhage, cerebral infarction, cardiac infarction
and angina pectoris, and also include diseases caused by blood vessels with
lowered flexibility, including senile dementia and diabetes (caused by
pancreatic dysfunction). Hemorrhoids are considered a local thrombotic
condition. If chronic diseases of the capillaries are also considered, then the
number of thrombus related conditions may be much higher. Cardiac
infarction patients may have an inherent imbalance in that their thrombolytic
enzymes are weaker than their coagulant enzymes. Nattokinase holds great
promise to support patients with such inherent weaknesses in a convenient
and consistent manner, without side effects. Nattokinase is capable of directly
and potently decomposing fibrin as well as activating pro-urokinase
(endogenous).
Fibrinolytic enzymes, which break down fibrin and thrombi, are normally
generated in the endothelial cells. As the body ages, production of these
enzymes begins to decline, making blood more prone to coagulation. Since
these cells exist throughout the body, such as in the arteries, veins and
lymphatic system, poor production of thrombolytic enzymes can lead to the
development of clotting-prone conditions virtually anywhere in the body. This
hyper-coagulability has been linked to a variety of conditions. Underlying
connective tissue weakness due to nutritional deficiencies and dysfunction of
the endothelium gives rise to inflammatory and repair mechanism. Once
initiated, this pro-inflammatory/pro-oxidative process is not only the
underlying process of atherosclerosis and vascular dysfunction, but also
causes a propensity to thrombi and thrombo-emboli. More than 50 important
substances that affect blood coagulation have been found in the blood and
tissues, some of which are pro-coagulants and some of which are
anticoagulants. In general, however, once damage has occurred to the blood
and blood vessels, the process of coagulation and clotting involve the
following: Damaged, weakened or traumatized blood vessel or blood vessel
wall, as initiated by nutritional deficiencies, trauma, and/or infection (can be
chronic or acute). Pro-thrombin Activator catalyzes the conversion of pro-
thrombin to thrombin. Thrombin acts as an enzyme to convert fibrinogen into
fibrin fibers. Fibrin fibers cause clotting. The final clot is composed of a
meshwork of fibrin fibers, running in all directions and entrapping blood cells,
platelets and plasma. Normally, the body has its own anti-coagulants, which
are able to keep balance between the pro-coagulants, allowing for repair and
healing, but not overshooting to cause pathological mechanisms. However,
chronic nutritional deficiencies, infection, cell senility, and/or trauma can
overwhelm the body's endogenous coagulation homeostasis, resulting in
thrombus and emboli. Although it is extremely important to treat the
underlying cause, such as replenishing the necessary nutritional factors to
allow for the formation and repair of healthy connective tissue and to support
proper endothelial function, often immediate and acute modulation of a
decompensated clotting system is needed. Until now, the only tools available
to target a decompensated clotting system were potent pharmaceutical agents
("clot busters") with known serious side effects. Now, however, an ideal
candidate appears to be Nattokinase, which can safely accomplish this task in
many instances.
The human body produces several types of enzymes for making thrombus,
but only one main enzyme for breaking it down and dissolving it - plasmin.
The properties of nattokinase closely resemble plasmin. Nattokinase enhances
the body's natural ability to fight blood clots in several different ways;
Because it so closely resembles plasmin, it dissolves fibrin directly. In
addition, it also enhances the body's production of both plasmin and other
clot-dissolving agents, including urokinase (endogenous). In some ways,
nattokinase is actually superior to conventional clot-dissolving drugs. T-PAs
(tissue plasminogen activators) like urokinase (the drug), are only effective
when taken intravenously and often fail simply because a stroke or heart
attack victim's arteries have hardened beyond the point where they can be
treated by any other clot-dissolving agent. Nattokinase, however, can help
prevent that hardening with an oral dose of as little as 100 mg a day.
Nutrient agar plates was prepared and with the help of L-rod, different
dilution samples (10-3, 10-4,10-5 and 10-6) were inoculated and left for
incubation at 37oC for overnight.
3. GRAM’S – STAINING
The heat fixed smears of the bacterial colonies is stained with crystal
violet. The primary stain, i.e., Crystal violet (CV) dissociates in the
aqueous solution into CV+ and Cl- ions. These ions penetrate through the
cell wall and cell membrane of Gram +ve and Gram –ve cells. CV+
interacts with negatively charged components of bacterial cells and
stains the cells purple.
I- or I3- interacts with CV+ and forms a large complex (CV-I) within the
outer and inner layers of the cell. When a decolorizing agent (95%
EtOH) is added, it interacts with the lipids of the cell membrane. A Gram
–ve cell loose its outer membrane, exposing the peptidoglycan layer. CV-
I complexes are washed from the gram –ve cells along with the outer
membrane. Whereas gram +ve cell becomes dehydrated after EtOH
treatment. The large CV-I complexes get trapped within the cell due to
the multilayered nature of its peptidoglycan layer.
After decolorisation, Gram +ve retains its purple colour while Gram –ve
loses its purple colour. Counterstain (positively charged Safranin or
Basic Fuchsin) is applied to give Gram –ve bacteria a pink or a red
colour.
4. SUB-CULTURING
With the help of the inoculation loop, the cells from the distinct colonies
grown on the mixed culture plates were taken and streaked (continuous
streak) on the freshly prepared agar plates and left for incubation at
37oC for overnight.
6. BIOCHEMICAL TESTS
A. IMViC Tests
The IMViC tests are a group of individual tests used in microbiology lab
testing to identify an organism in the coliform group. A coliform is a
gram negative, aerobic or facultative aerobic rod which produces gas
from lactose within 48 hours. The presence of some coliforms indicate
fecal contamination. These IMViC tests are useful for differentiating the
family Enterobacteriaceae, especially when used alongside the Urease
test. These four tests include:
Indole Production Test
The methyl red (MR) and Voges – Proskauer (VP) tests are used to
differentiate two major types of facultatively anaerobic enteric bacteria
that produce large amount of acid and those produce neutral product
acetoin as end product. Both these are performed on the same medium
MR-VP broth. Opposite results are usually obtained for the MR and VP
tests, i.e., MR +ve, VP –ve or MR –ve, VP +ve. In these tests, if an
organism produces large amount of organic acids, formic acid, acetic
acid, lactic acid and Succinic acid (end product) from glucose, the
medium will remain red (a +ve test) after the addition of methyl red, a
pH indicator (i.e., pH <4.4). In other organisms, methyl red will turn
yellow (a –ve test) due to the elevation of pH > 6.0 because of the
enzymatic conversion of the organic acid (produced during the glucose
fermentation) to non-acidic end products such as EtOH and acetoin
(acetyl methyl carbinol).
Citrate Test
Bromophenol blue is green when acidic (pH 6.8 and below) and blue
when alkaline (pH 7.6 and higher).
B. Catalase Test
TSI Agar medium is composed of three sugars; lactose sucrose and very
small amount of (1%) glucose, iron (ferrous sulphate) and phenol red as
indicator. The indicator is employed for the detection of fermentation of
sugars indicated by the change in colour of the medium due to the
production of organic acid, hydrogen sulphide. If an organism ferments
any of the three sugars or will become yellow due to the production of
acids as end product of fermentation. The enteric pathogens, however,
are capable of fermenting only glucose and medium turns yellow within
24 hours of incubation and in aerobic conditions of the slants the
reaction reverts and becomes alkaline showing again the red colour in
the slanted position of the tube while the anaerobic butt will remain
yellow (presence of acid) because the same organism is unable to cause
a reversion in the anaerobic condition present in the butt. Thus
Salmonella and Shigella shows a yellow butt and red slant, after 24-48
hours of incubation, indicating glucose fermentation only. No change in
the medium indicates that none of the sugar has been fermented.
D. Gelatin Test
E. Urease Test
H. Motility Test
I. Endospore Staining
Some bacteria are capable of changing into dormant structure that are
metabolically inactive and does not grow on reproduce. Since these
structures are formed inside the cells hence called endospore. The
German botanist Ferdinard Cohn (1828-98) discovered the existence of
endospore in bacteria. These are remarkably resistant to heat, radiation
chemicals and reagent that are typically lethal to the organism. The heat
resistant of spores has been linked to their high content of calcium and
dipicolinic acid. A single bacterium forms a single spore by a process
called sporulation. Sporulation takes place either by depletion of an
essential nutrient or during unfavourable environmental condition.
J. Mannitol Fermentation
Procedure:
DOWNSTREAM PROCESSING
9. EXTRACTION OF ENZYMES
At high ionic strengths much water becomes bound by the added ions
that not enough remains to properly hydrate the proteins. As a result,
protein – protein interactions exceed protein – water interactions and
the solubility decreases.
After a protein has been ammonium sulphate precipitate and taken back
up in buffer at a much greater protein concentration than before
precipitation, the solution will contain a lot of residual ammonium
sulphate which was bound to the protein. One way to remove this
excess salt is to dialyses the protein against a buffer low in salt
concentration
Protein reacts with FCR to give a blue color complex .The color so
formed is due to the reaction of the alkaline copper with the protein and
the reduction of phosphomolybdate and phosphotungstate components
in the FCR by the aromatic amino acids such as tyrosine and tryptophan
in proteins.The above reduced components combine with copper of the
copper sulphate and give blue colored complex which is read at 660nm.
Procedure:
a. Six test tubes were taken and filled with 0, 0.2, 0.4, 0.6, 0.8 and 1ml of
protein working solution
b. 0.2ml each of the crude protein sample was taken in four different
test tubes as ‘test’.
c. The volume was made up to 1ml in all the test tube by using distilled
water.
e. The test tubes were then kept at room temperature for 10mins.
g. The test tubes were kept in the dark for 30mins for the reaction to
proceed.
With Tyrosine:
Procedure:
9. Now each tubes solution was filtered using Whatman filter paper.
11. FC reagents was further added in the ratio or 1:2 and kept for
30mins incubation in dark.
SDS is an anionic detergent, which binds and reacts with the proteins in
the solution, and destroys the tertiary structure of the protein, leading
to partial unfolding of the polypeptide chain. In addition, SDS binds to
both the hydrophilic and hydrophobic regions of the polypeptide chain,
giving the protein an excessively net negative charge, which
diminuishes any intrinsic amino acid charge. The protein also adopts a
cylindrical shape, which is coated along its' entire surface with
negatively charged sulfonate ions. In addition, beta-mercaptoethanol
may be used to reduce disulfide bonds, which forms mixed disulfides
with cystein side chains. However, the SDS coated proteins are now
denatured and biologically non-functionable. A general rule when using
SDS is that the amount of SDS bound per gram of protein is found to be
constant at a SDS: protein ratio of about 1.4 gram SDS per gram protein.
Moreover, this ratio is achieved under reducing conditions, but is
altered with carbohydrate-containing proteins, such as
immunoglobulin, which are known to bind less SDS than other similar
sized proteins. Therefore, an inverse relationship develops between the
mobility versus the proteins logarithm mass. A calibration curve with a
set of standard proteins of known mass can be projected and then used
to determine the molecular mass weights of unknown proteins through
a method of comparison.
Procedure:
(1) The glass plates and spacers were thoroughly cleaned and dried,
then assembled with the help of Bulldog clips. Silicon was applied
around the edges of the spacers to hold them in place and seal the
chamber between the glass plates.
(3) The gel solution was poured into the chamber between the glass
plates and kept for 30 - 60 min.
(4) Stacking Gel mixture (2 ml) was prepared and poured. Then the
comb was placed into the Stacking Gel.
(5) After the stacking gel has polymerized the comb was removed
without distorting the shapes of the wells. The gel was installed into the
electrophoresis apparatus. The tank was filled with electrode buffer.
(7) D.C. current was applied and allowed the electrophoresis unit to run
for about 3 hrs.
(8) After 3 hrs the gel was removed from the plates and immersed into
the Staining Solution for overnight with uniform shaking. The protein
absorbs the Coomassie Brilliant Blue.
(9) The gel was transformed into a suitable container with at least 100
ml of destaining solution and shaked gently continuously. The unbound
dye was removed. The destaining solution was changed frequently
particularly during initial period, until the background of the gel was
colorless source.
Effect of pH
Effect of Temperature
Effect of Activator
Effect of Inhibitor
COLONY NAME 2 7 8
The sub-cultured bacterial colonies were then identified with the help of various bio-chemical
tests. The results were as follows:
COLONIES 2 7 8
Mannitol
+ve +ve -ve
Fermentation
On the basis of the results of the biochemical tests colony 2 and 7 were
recognized as Bacillus subtilis, our subject colony.
Vol. of I Concentration
Vol. of FC Reagent
BSA Solution C I n OD at
Sl. No. D/W
(in (in ml) n c Of protein (in 660nm
(in ml) (in ml)
ml) c u µg/ml)
u b
Blank 0.0 1.0 5 b 0.5 a 0 0.0
a ti
1 0.2 0.8 5 0.5 40 0.056
ti o
2 0.4 0.6 5 o 0.5 n 80 0.096
n
3 0.6 0.4 5 0.5 120 0.142
i
4 0.8 0.2 5 0.5 160 0.235
a n
5 1.0 0.0 5 t 0.5 200 0.277
Vol. of Concentration
Vol. of FC Reagent d
Test Solution C OD at
Sl. No. D/W R a
(in (in ml) Of protein (in 660nm
(in ml) T (in ml) r
ml) µg/ml)
k
2C 0.1 0.9 5 0.5 440 0.643
f
2A 0.1 0.9 5 0.5 152 0.212
o f
7C 0.1 0.9 5 r 0.5 o 304 0.564
r
7A 0.1 0.9 5 0.5 96 0.948
1
PC 0.1 0.9 5 0.5 296 0.435
0 3
m 0
i m
PA 0.1 0.9 5 n 0.5 i 84 0.120
s n
s
*C stands for the crude enzyme sample and A stands for the ammonium precipitated
sample
Temperature Substrate
Colony pH Activator Inhibitor concentration
(in ˚C) (in %age)
MOL. WT.
WELL NO. SAMPLE
(IN kD)
1 5mM P 55
2 15mM 7 52
3 5mM 2 60
A 60
4 Dialysed P B 54
C 30
A 60
5 Dialysed 7
B 45
6 Dialysed 2 60
7 Marker 60
The target enzyme nattokinase, is believed to possess fibrinolytic properties,
i.e., it functions as ‘clot bursters’. When applied on clotted blood, the enzyme
successfully dissolved the thrombus.
Glucose 1gm
Peptone 5gm
Agar 10.5gm
Peptone 10gm
Mannitol 5gm
Peptone 7gm
Dextrose/Glucose 5gm
d. Nitrate Broth
Peptone 5gm
Agar 20gm
Peptone 5gm
Gelatin 120gm
Glucose 10gm
Peptone 30gm
Agar 3gm
Agar 15gm
j. Starch Agar
Starch 20gm
Peptone 5gm
Agar 15gm
Peptone 15gm
Lactose 10gm
Sucrose 10gm
Dextrose 1gm
Agar 12gm
Tryptone 1gm
Peptone 1gm
Potassium monohydrogen or
Agar 2gm
Glucose 1gm
II. REAGENTS
a. Acrylamide bis-acrylamide solution
c. BSA Standard
d. Destaining Solution
Methanol 10%
e. FC Reagent
g. Resolving Gel
TEMED 0.005ml
i. Solution C
j. Stacking Gel
TEMED 0.002ml
k. Staining Solution
Iso-propanol 40%
l. TEMED
III. BUFFERS
a. Electrophoresis Buffer (10X)
SDS 0.1gm
Glycine 1.47gm
b. Ellusion Buffer
c. Gradient Buffer
f. Tank Buffer
1X electrophoresis buffer
g. Upper Tris
1M tris buffer