NATTOKINASE

By

PRANAV BHASKAR

CONTENTS

1. INTRODUCTION

2. METHODS

3. RESULTS AND DISCUSSIONS

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.

Biological enzymes are enzymes which regulate endogenous chemical

processes and have been called "the fountain of life" -- because without them,

life could not exist. These enzymes speed and regulate all chemical reactions

in the body in an orchestration of intelligence and control. Enzymes are made

in the body from proteins and are provided by the ingestion of enzyme rich

foods. During times of stress, sickness or reduced nutrient intake, the body

can fall behind in the demand for the constant upkeep and creation of

enzymes. Luckily the body has evolved to derive many of its enzymes from

food, which helps to reduce the burden of the high enzyme production needs.

Unfortunately, however, the enzyme content of foods has significantly

decreased over the years due to processing, soil depletion, refining and

preservation techniques of the food industry and a decreased consumption of

fermented foods and fresh foods, which are high in enzyme content. Enzymes

are an essential component of the diet -- like vitamins, minerals,

phytonutrients, fat, protein, carbohydrates, etc. -- and without them, a

deficiency state does occur.

HOW ENZYMES WORK?

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.

An enzyme-catalyzed reaction takes a different 'route'. The enzyme and

substrate form a reaction intermediate. Its formation has lower activation

energy than the reaction between reactants without a catalyst.

Route A reactant 1 + reactant 2 product

Route B reactant 1 + enzyme intermediate

intermediate + reactant 2 product + enzyme

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.

However, based on catalyzed reactions, the nomenclature committee of

the International Union of Biochemistry and Molecular Biology (IUBMB)

recommended the following classification.

1. Oxido-reductases catalyze a variety of oxidation-reduction reactions.

Common names include dehydrogenase, oxidase, reductase and catalase.

2. Transferases catalyze transfers of groups (acetyl, methyl, phosphate,

etc.). Common names include acetyl-transferase, methylase, protein kinase

and polymerase. The first three subclasses play major roles in the regulation

of cellular processes. Their chemical reactions are shown in Figure 2-E-1.

The polymerase is essential for the synthesis of DNA and RNA.

3. Hydrolases catalyze hydrolysis reactions where a molecule is split into

two or more smaller molecules by the addition of water. Common examples

are:

Proteases split protein molecules; e.g., HIV protease and caspase. HIV

protease is essential for HIV replication. Caspase plays a major role in

apoptosis. Nucleases split nucleic acids (DNA and RNA). Based on the

substrate type, they are divided into RNase and DNase. RNase catalyzes the

hydrolysis of RNA and DNase acts on DNA. They may also be divided

into exo-nuclease and endo-nuclease. The exo-nuclease progressively

splits off single nucleotides from one end of DNA or RNA. The endo-

nuclease splits DNA or RNA at internal sites. Phosphatase catalyzes

dephosphorylation (removal of phosphate groups). Example: calcineurin.

The immune-suppressive drugs FK506 and Cyclosporin A are the inhibitors

of calcineurin.

4. Lyases catalyze the cleavage of C-C, C-O, C-S and C-N bonds by means

other than hydrolysis or oxidation. Common names include decarboxylase

and aldolase.

5. Isomerases catalyze atomic rearrangements within a molecule.

Examples include rotamase, protein disulfide isomerase (PDI), epimerase

and racemase.

6. Ligases catalyze the reaction which joins two molecules. Examples

include peptide synthase, aminoacyl-tRNA synthetase, DNA ligase and RNA

ligase. The IUBMB committee also defines subclasses and sub-subclasses.

Each enzyme is assigned an EC (Enzyme Commission) number. For

example, the EC number of catalase is EC1.11.1.6. The first digit indicates

that the enzyme belongs to oxido-reductase (class 1). Subsequent digits

represent subclasses and sub-subclasses.

FACTORS AFFECTING CATALYTIC ACTIVITY OF ENZYMES

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.

Above this temperature the enzyme structure begins to break down

(denature) since at higher temperatures intra- and intermolecular bonds are

broken as the enzyme molecules gain even more kinetic energy.

2. pH

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.

3. Concentration of enzyme and substrate

The rate of an enzyme-catalyzed reaction depends on the concentrations of

enzyme and substrate. If the concentration of enzyme is increased the rate of

reaction increases. For a given enzyme concentration, the rate of reaction

increases with increasing substrate concentration up to a point, above which

any further increase in substrate concentration produces no significant

change in reaction rate. This is because the active sites of the enzyme

molecules at any given moment are virtually saturated with substrate. The

enzyme - substrate complex has to dissociate before the active sites are free to

accommodate more substrate. Provided that the substrate concentration is

high and that temperature and pH are kept constant, the rate of reaction is

proportional to the enzyme concentration.

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:

supports normal blood pressure

prevents blood clots from forming

dissolves existing blood clots

dissolves fibrin

enhances the body's production of plasmin and other clot-dissolving agents, including urokinase

THE DISCOVERY OF NATTOKINASE

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.

Doctor Hiroyuki Sumi had long researched thrombolytic enzymes searching

for a natural agent that could successfully dissolve thrombus associated with

cardiac and cerebral infarction (blood clots associated with heart attacks and

stroke). Sumi discovered nattokinase in 1980 while working as a researcher

and majoring in physiological chemistry at University of Chicago Medical

School. After testing over 173 natural foods as potential thrombolytic agents,

Sumi found what he was looking for when Natto was dropped onto artificial

thrombus (fibrin) in a Petri dish and allowed it to stand at 37oC

(approximately body temperature). The thrombus around the natto dissolved

gradually and had completely dissolved within 18 hours. Sumi named the

newly discovered enzyme "nattokinase", which means "enzyme in natto".

Sumi commented that nattokinase showed "a potency matched by no other

enzyme."

THE MECHANISM BEHIND THROMBUS

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.

Thrombolytic enzymes are normally generated in the endothelial cells of the

blood vessels. As the body ages, production of these enzymes begins to

decline, making blood more prone to coagulation. This mechanism can lead to

cardio-vascular disease, stroke, angina, venous stasis, thrombosis, emboli,

atherosclerosis, fibromyalgia (chronic fatigue), claudication, retinal pathology,

hemorrhoid, varicose veins, soft tissue rheumatisms, muscle spasm, poor

healing, chronic inflammation and pain, peripheral vascular disease,

hypertension, tissue oxygen deprivation, infertility, and other gynecology

conditions (e.g. endometriosis, uterine fibroids).

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).

NATTOKINASE'S (NK) EFFECT ON FIBRIN/BLOOD CLOTS

Fibrin is a protein that when activated forms fibrinogen, which is responsible

for blood clotting. This is an important and protective mechanism that

protects the body from excessive bleeding. However, in many instances, this

process becomes over-activated or becomes "stuck" in high gear. This

irregulation of clotting has been implicated in a variety of serious health

conditions, namely, cardiovascular disease. The magnificent thing about

Nattokinase is that it appears to have many, if not most, of the benefits of

pharmaceutical agents designed to regulate blood clotting (e.g., warfarin,

heparin, t-PA, urokinase, etc.) without any of the side effects of these

medications. Furthermore, while these medications have to be injected and

only provide a very brief time of benefit (a few hours), Nattokinase is effective

when taken orally and its benefits linger many times longer. Standard doses of

Nattokinase vary from 250-1,000 mg and positive effects can be seen with as

little as 50 mg.

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.

POTENT THROMBOLYTIC ACTIVITY OF NATTOKINASE

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.

THE PROLONGED ACTION OF NATTOKINASE

Nattokinase produces a prolonged action (unlike anti – thrombin drugs) in

two ways: it prevents coagulation of blood and it dissolves existing thrombus.

Both the efficacy and the prolonged action of NK can be determined by

measuring levels of EFA (euglobulin fibrinolytic activity) and FDP (fibrin

degradation products), which both become elevated as fibrin is being

dissolved. By measuring EFA & FDP levels, activity of NK has been determined

to last from 8 to 12 hours. An additional parameter for confirming the action

of NK following oral administration is a rise in blood levels of TPA antigen

(tissue plasminogen activator), which indicates a release of TPA from the

endothelial cells and/or the liver.

METHODS

1. SERIAL DILUTION

1 gm of soil was primarily dissolved in 100ml of distilled water and then

serially diluted simultaneously to the dilutions of order of 10-7.

2. INOCULATION OF CULTURE MEDIA

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

Gram’s staining is an empirical method of differentiating bacterial

species into two large groups (Gram +ve and Gram -ve) based on the

chemical and physical properties of their cell walls.

Not all bacteria cam be definitively classified by this technique, thus

forming Gram-variable and Gram-indeterminant groups.

This method was invented by Hans Christian Gram in 1884 to

discriminate between two types of bacteria with similar clinical

symptoms: Streptococcus pneumoniae and Klebsiella pneumonia

bacteria. Exception is Archaea, since these yield widely varying

responses that do not follow their phylogenetic groups.

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.

5. MAINTENANCE OF PURE CULTURE

The colonies, thus, obtained from the sub-culture were maintained by

streaking continuously on the nutrient agar slants.

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

Tryptophan, an essential amino acid, is oxidized by some bacteria by the

enzyme tryptophanse resulting in the formation of indole, pyruvic acid

and ammonia. The indole test is performed by inoculating a bacteria

into tryptone broths the indole produced during the reaction is detected

by adding Kovac’s reagent (di-methylamino benzaldehyde) which

produces a cherry red reagent layer as illustrated.

Tryptone -----------------→ Indole + Pyruvic acid + NH3

Indole + Kovac’s reagent-------------→ Rosindole (cherry red) + H2O

This exercise deals with the determination of Indole Production from

microbial catabolism of Tryptophan.

Methyl – Red and Voges – Proskauer 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

Citrate test is used to differentiate among Enteric bacteria on the basis

of their ability to utilize citrate as sole carbon source. The utilization of

citrate depends on the presence of an enzyme Citrase; produced by an

organism, that break down the citrate to Oxalo-acetate and acetic acid.

These products are later converted to Pyruvic acid and CO2

enzymatically.

The citrate test is performed by inoculating the micro-organism into an

organic synthetic medium; Simmon’s Citrate Agar where sodium citrate

is the only source of carbon and energy. Bromophenol blue is used as

indicator. When the citric acid is metabolized the CO2 generated

combines with sodium and water to form sodium carbonate, which

changes the colour of the indicator from green to blue and this gives a

positive test.

Bromophenol blue is green when acidic (pH 6.8 and below) and blue

when alkaline (pH 7.6 and higher).

B. Catalase Test

During aerobic respiration in the presence of oxygen microorganism

produce Hydrogen peroxide which is lethal to the cell. The enzyme

Catalase present in some microorganism breaks down hydrogen

peroxide to water and oxygen, and helps them in their survival. Catalase

test is performed by adding hydrogen peroxide to trypticase soy agar

slant culture. Release of free oxygen gas bubbles is a positive catalase

test.

C. Triple Sugar Iron Agar 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.

Production of gas from the fermentation of a sugar by an organism is

indicated by the appearance of bubbles or splitting in the butt or

pushing up of the entire slant from the bottom of the tube. Hydrogen

sulphide production by an organism is indicated by the reduction of the

ferrous sulphate of the medium to the ferric sulphide, which is

manifested as a black precipitate. The enteric bacilli which produce

hydrogen sulphide also ferment glucose but the large amount of black

precipitate can mask the yellow or acid contains in butt.

D. Gelatin Test

Proteins are organic molecule composed of amino acids in other words

protein contain carbon, hydrogen, oxygen and nitrogen through some

protein contain sulphur too. Amino acids are linked together by peptide

bond to form a small chain (a peptide) or large molecule (polypeptides)

of protein. Gelatin is a protein produced by hydrolysis of collagen, a

major components of connective tissue tendon in human and animals. It

dissolves in warm water (50oC) and exists as a liquid above 25oC and

solidifies (gel) when cooled below 25oC. Large protein molecules are

hydrolyzed by exo-enzyme known as gelatinase which acts to hydrolyze

this protein to amino acids.

Hydrolysis of gelatin in the laboratory can be demonstrated by growing

microbes in nutrient gelatin. Once the degradation of gelatin occurs in

the medium by an exo-enzyme, it can be detected by observing

liquification or testing with a protein precipitating material (i.e.,

flooding the gelatin agar medium with the mercuric chloride) solution

and observing the plates for cleaning around the line of growth, because

gelatin is also precipitated by chemicals that coagulate proteins which

the end product of degradation (i.e., amino acid) are not precipitated by

the same chemicals. This exercise deals with testing of gelatin

production by three microbes Bacillus subtilis, E. coli and Protease

vulgaris by 2 methods – stab inoculation of nutrient gelatin tubes to see

liquification of gelatin and incubation of gelatin agar plates to see the

formation of clear zones around the line of growth when flooded with

mercuric chloride.

E. Urease Test

Urea is a major organic waste product of protein digestion in most

vertebrate and is excreted in the urine. Some micro- organisms have the

ability to produce the enzyme urease. The urease is a hydrolytic enzyme

which attacks the carbon and nitrogen bond amide compounds (e.g.,

urea) with the liberation of ammonia.

Urea + water -------------→2NH3 + CO2

It is a useful diagnostic test for identify bacteria, especially to

distinguish members of the genus protease from the gram –ve

pathogens.

Urease Test is perfomed by growing the test organism on urea broth or

agar medium containing the ph indicator phenol red (pH 6.8). During

incubation microorganisms possessing urease will produce ammonia

that raises the ph becomes higher, the phenol red changes from yellow

colour (pH 6.8) to a red or deep pink colour. Failure of the development

of a deep pink colour due to no ammonia production is evidence of a

lack of urease production by the micro-organism.

F. Starch Hydrolysis Test

Amylase is an exo-enzyme that hydrolyzes starch polysaccharide into

maltose, a disaccharide and some monosaccharide such as glucose.

Starch is a complex carbohydrate composed of two constituents:

Amylose, a straight chain polymer of 200-300 glucose units and Amyl

pectin, a large branched polymer with phosphate groups Amylase

production is known in some bacteria while well known in fungi.

Amylase commercially produced from various Aspergilli is used in the

initial steps of several food fermentation processes to convert starch to

fermentable sugar. The ability to degrade starch is used as a criterion

for the determination of amylase production by a microbe. In a lab it is

tested by performing the starch test to determine the presence or

absence of starch in the medium by using Iodine Solution as an

indicator. Starch in the presence of iodine produces dark blue

colouration of the medium, and a yellow zone around the colony in an

otherwise blue medium indicates amylolytic activity. This exercise deals

with testing the hydrolysis of starch for the production of extracellular

amylase by three organisms viz. Bacillus subtilis, E. coli and Aspergillus

niger by inoculating these on starch agar medium.

G. Nitrate Reduction Test

Among bacteria there exists a variation in energy in metabolism. A

number of bacteria are capable of respiring under completely anaerobic

condition by utilizing Nitrate sulfate or carbonates as a terminal

inorganic electron acceptor. Reduction of nitrate takes place in the

presence of a stable electron donor to nitrate.

H. Motility Test

SIM medium is a semi-solid medium used for the determination of

sulphide production, indole formation and motility of enteric bacteria.

SIM medium contains source of organic sulphur (usually peptone) and

also sodium thio-sulphate. Since some bacteria may form this from one

source but not the other sulphur production usually occurs best under

anerobic or semi-aerobic condition. Therefore the medium should be

inoculated by stabbing the agar. SIM agar tubes can also be used for

motility test. Non-motile organism grows only along the line of

inoculation. Motile cultures show different growth or turbidity away

from the line of inoculation.

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.

During sporulation a vegetative cell gives rise to a new intercellular

structure termed as endospore that is surrounded by impermeable

layers called spore coats. Complete transformation of a vegetative cell

into a most spore forming species. An endospore develops into a

characteristic position within a cell that is central, sub-terminal or

terminal. Once an endospore is formed in a cell, the cell wall

disintegrate releasing the endospore that later becomes an independent

spore. Endospore can remain dormant for long period of time. Once

record describe the isolation of viable spores from a 3,000 years old

archeological specimen. However a free spore may return to its

vegetative or growing state with return of favourable conditions.

Endospores are formed by members of 7 genera, e.g., Bacillus

clostridium, Coxiella, Desulfotomaculum, Spoeolactobacillus,

Sporomusa, Thermoactinomyces. These include non-pathogenic soil

inhabitants (Bacillus and Clostridium) and pathogenic (Clostridium

tetanii and Bacillus anthracis).

The spores are differentially stained by using special procedures that

help dyes penetrate the spore wall. An aqueous primary stain

(malachite green) is applied and steam to enhance penetration of the

impermeable spore coats. Once stained, the endospores do not readily

decolorise and appear green within red cells.

J. Mannitol Fermentation

Fermentative degradation of various carbohydrates such as glucose,

sucrose, cellulose by microbes, under anaerobic condition is carried out

in a fermentation tube. A fermentation tube is a culture tube that

contains a Durham tube (i.e., a small tube placed in an inverted position

in the culture tube) for the detection of gas production as an end

product of metabolism. The fermentation broth; a specific

carbohydrates and a pH indicator (Phenol red) which is red at a neutral

pH (7.0) and turns yellow at or below a pH of 6.8 due to production of

an organic acid.

7. SCREENING OF THE ORGANISM PRODUCING NATTOKINASE

Casein Hydrolysis Test

Casein is the major protein found in milk. It is a macromolecule

composed of amino acids linked together by peptide bonds, CO-NH.

Some micro organisms have the ability to degrade the protein casein by

producing the proteolytic exoenzyme, called protease which breaks the

peptide bond CO-NH by introducing water into the molecule, liberating

smaller chains of amino acid called peptide, which is later broken down

into free amino acid. Casein hydrolysis can be demonstrated by

supplementing nutrient medium with skimmed milk. The medium is

opaque due to casein in colloidal suspension. Formation of a clear zone

adjacent to the bacterial growth, after inoculation and incubation of

agar plate culture, is an evidence of casein hydrolysis test.

Procedure:

Skim milk agar plates were prepared.

A single line streak of different colonies on different nutrient plates

were done with the help of inoculation loop.

The inoculated plates were incubated at 37°C for 24-48 hours.

8. MASS PRODUCTION OF ENZYME

Cultivation was carried in 250 ml conical flask containing 50 ml of

production medium. The flask was incubated for 48 hors on rotatory

shaker at 140 rpm at 37°C. After 48 hours of incubation turbidity in the

flask was observed which indicates that bacterial growth was present in

medium.

DOWNSTREAM PROCESSING

Downstream processing refers to the recovery and purification of

biosynthetic products particularly pharmaceuticals from natural

sources such as animal or plant source or fermentation broth, including

recycling of salvageable components and the proper treatment and

disposal of waste. It is an essential step in the manufacture of

pharmaceuticals such as antibiotics, hormones (e.g., insulin and Human

Growth hormone), antibodies (e.g., infliximab and abciximab) and

vaccines; antibodies and enzymes used in diagnostics; industrial

enzymes and natural fragrance and flavoured compound.

9. EXTRACTION OF ENZYMES

Since the enzyme released was extracellular, hence centrifugation is the

basic technique used to remove all cell debris from the enzyme released.

Therefore, after fermentation, the seed cultures were transferred into

centrifuge tubes and were centrifuged at 5000rpm for 15 minutes at

4°C. The supernatant thus obtained was collected and was used as crude

enzyme.

10. PURIFICATION OF ENZYME

A. Ammonium Sulfate Precipitation

The solubility of proteins is markedly affected by the ionic strength of

the medium. As the ionic strength is increased, protein solubility at first

increased. This is referred to as ‘salting in’. However, beyond a certain

point the solubility, begins to decrease and this is known as ‘salting out’.

At low ionic strength the activity coefficients of the ionizable groups of

the proteins are decreased so that their effective concentration is

decreased. This is because the ionizable groups become surrounded by

counter ions which prevent interactions between the ionizable groups.

These protein – protein interactions are decreased and the solubility is

increased.

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.

Because of differences in structure and amino acid sequence, proteins

differ in their salting in and salting out behaviour. This forms the basis

for the fractional precipitation of proteins by means of salt.

Ammonium sulfate is a particularly useful salt for the fractional

precipitation of proteins. It is available in highly purified form, has great

solubility allowing for significant changes in the ionic strength and is

inexpensive. Changes in the ammonium sulfate concentration of the

solution can be brought about either by adding solid substance or by

adding a solution of known saturation, generally, a fully saturated

solution (100%).

B. Dialysis

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

First, the concentrated protein solution is placed in dialysis bag with

small holes which allow water and salt to pass out of the bag while

protein is retained. Next the dialysis bag is placed in a large volume of

buffer and stirred for many hrs (16-24hrs), which allows the solution

inside the bag to equilibrate with the solution outside th4e bag with

respect to salt concentration. When the process of equilibration is

repeated several times (replacing the external solution with low salt

solution each time), the protein solution in the bag will reach a low salt

concentration:

After ammonium sulphate precipitation, solution contains a mixture of

buffer as well as excess salt. So we use the buffer we want for the next

step in the purification, which is ion exchange chromatography, as the

external solution during dialysis. After the three step dialysis process

where the protein solution is dialyzed against the starting buffer for the

ion exchange chromatography step, not only will the salt be removed

but the protein will now be in buffer needed for the next step and ready

to go. Sometimes, proteins will precipitate during the dialysis process

and you will need to centrifuge the solution after dialysis to remove any

particles which would interfere with the next step such as ion-exchange

chromatography where particles would clog the column and prevent the

chromatography step from working.

C. ION EXCHANGE CHROMATOGRAPHY

Ion – exchange chromatography separate molecules on the basis of

differences between the overall charges of the proteins. The affinity

with which a particular protein bind to a given ion exchanger depend on

the identities and the concentration of the other ions in solution

because of the competition among these various ions for the binding

sites on the ion exchanger.

A small volume of protein solution obtained after dialysis is applied to

the top of a column in which the ion exchanger has been packed and the

column is washed with the buffer solution. The protein mixture is bound

to the top most portion of the ion exchanger in the chromatography

column. The greater the binding affinity of a protein for the ion

exchanger, the more it will be retarded. Thus, proteins that bind tightly

to the ion exchanger can be eluted by changing the elution buffer to one

with a higher salt concentration (elution buffers of 5mM to 20mM was

used).

11. ESTIMATION OF THE CONCENTRATION OF NATTOKINASE BY

LOWRY’S METHOD

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.

Protein------- cuprous ion--------- FCR------Blue colored complex (Intensity of

blue colour α amount of protein in the sample).

Procedure:

With BSA (Bovine Serum Albumin)

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.

d. To all the test tubes, 5ml of solution C was added.

e. The test tubes were then kept at room temperature for 10mins.

f. 0.5ml of FC reagent was added.

g. The test tubes were kept in the dark for 30mins for the reaction to

proceed.

h. Then the O.D reading was taken at 660nm.

With Tyrosine:

Similar procedure was followed with tyrosine taken as the standard

protein working solution (conc. = 10 mg/10 ml)

12. ASSAY OF ENZYME (casein as a substrate)

Casein + water -----------> Amino acids.

The enzyme assay was done by the method of colorimetry.

Procedure:

1. The spectrophotometer was set for absorbance at 660nm using

the blank.

2. Test tubes were taken and labeled properly.

3. 0.5 ml of reagent was pipette out in each test tube.

4. 0.1 ml of enzyme solution was added in each tube except blank.

5. It was kept for 10 minutes incubation at 37°C.

6. Now 5ml of reagent C was added in each tube.

7. Volume of blank was made up by adding 0.1 ml of distilled water.

8. Each tube was mixed by swirling and kept for incubation.

9. Now each tubes solution was filtered using Whatman filter paper.

10. 2 ml of filtrate was used further in which 5ml of reagent E was

added.

11. FC reagents was further added in the ratio or 1:2 and kept for

30mins incubation in dark.

12. Finally the OD was taken.

13. SDS-PAGE (Sodium Dodecyl Sulphate Poly Acrylamide Gel

Electrophoresis)

A simple, effective and very high resolution method to fractionate and

analyze protein mixtures is the sodium dodecyl sulfate (SDS)

polyacrylamide gel electrophoresis (PAGE). Electrophoresis is a critical

tool used to separate complex mixtures of proteins, to purify proteins,

to elucidate homogeneity of proteins samples, but also as depicted in

our investigation, to investigate the subunit compositions of proteins.

SDS-PAGE separates proteins on the basis of their molecular size. This is

obtainable by allowing the SDS-covered proteins to migrate through the

pores of a polyacrylamide gel matrix, which consists of abundant pores

(vary in size and number with % acrylamide used).

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.

Through the use of a supporting medium called polyacrylamide, with

the application of an electric field through this medium, the SDS

negatively charged protein complexes in a protein mixture can be

separated. Polyacrylamide is a synthetic polymer, which is formed by

the polymerization of acrylamide monomer with additional bi-

functional cross-linking agents (aided by a catalyst). This polymerized

polyacrylamide matrix is a three-dimensional network of pores whose

size is determined by the percentage degree of acrylamide monomer

and cross-linker concentration utilized in the mixture (pore size

decreases with higher acrylamide gel concentrations) and is often

referred to as a separating or running gel. The pores within the

polyacrylamide gel are comparable in molecular size to the size of

protein molecules. Upon electrophoresis, which applies an electric field

through the pores in the gel matrix, the proteins are sieved through the

pores of the gel with the larger proteins having a slower migration rate

than the smaller proteins. The negative charges flow from the negative

cathode terminal into the upper buffer chamber, through the gel, and

into the lower buffer chamber, which is connected to the positive

terminal. Therefore, the negatively charged SDS coated proteins migrate

towards the anode. The combination of gel pore size and protein charge,

size and shape determines the migration ability of the protein.

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.

(2) Separating Gel mixture (5ml for a chamber) was prepared.

(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.

(6) The samples were prepared for electrophoresis. The sample

solutions were taken using a micropipette and carefully poured into the

wells through the 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.

14. ENZYME KINETICS

Effect of pH

The rate of almost all enzyme catalyzed reactions exhibits a

significant dependence on hydrogen ion concentration. Most intra-

cellular enzymes exhibits optical activity at pH values between 3 to 8.

The relationship of activity to hydrogen ion concentration reflects the

balance between enzyme denaturation at high or low pH and effects

on the charged state of the enzyme, the substrate, or both. For

enzymes whose mechanism involves acid-base catalysis, the residues

involved must be in the appropriate state of protonation for the

reaction to proceed.

Effect of Temperature

Raising the temperature increases the rate of both uncatalysed and

enzyme catalyzed reactions by increasing the kinetic energy and the

collision frequency of the reacting molecule. However, heat energy

can also increase the kinetic energy of the enzyme to a point that

exceeds the energy barrier for disrupting the non-covalent

interaction that maintain the enzyme’s three dimensional structure.

The polypeptide chain, then begins to unfold, or denature, with an

accompanying rapid loss of catalytic activity. The temperature range

over which an enzyme maintains a stable, catalytically competent

conformation depends upon and typically moderately exceeds the

normal temperature of the cells in which it resides. Enzymes from

humans generally exhibit stability at temperature up to 45 – 55oC. By

contrast, enzymes from the thermophilic micro-organisms that reside

in volcanic hot springs or under-sea hydrothermal vents may be

stable up to or above 100oC.

Effect of Activator

Enzyme activators are molecules that bind to enzymes and increase

their activity. These molecules are often involved in the allosteric

regulation of enzymes in the control of metabolism.

Effect of Inhibitor

Enzymes inhibitors are molecules that bind to enzymes and decrease

their activity. The binding of an inhibitor can stop a substance from

entering the enzyme’s active site and/or hinder the enzyme from

catalyzing its reaction. Inhibitor binding is either reversible or

irreversible.

RESULTS AND DISCUSSIONS

On the basis of colonies obtained on mixed culture plate and after staining

them with gram stains, it was concluded that the obtained bacterial colonies

2,7and 8 have following morphological characteristics:

COLONY NAME 2 7 8

COLOUR Off – white Off – white Off – white

FORM Irregular Circular Circular

ELEVATION Flat Flat Flat

MARGIN Erose Entire Entire

SIZE Small Small Small

TRANSPARENCY Opaque Opaque Opaque

GRAM’S STAINING +ve +ve +ve

SHAPE Rod and chain Rod Rod

Fig. Mixed culture

Fig. The axenic culture of colonies 2, 7 and 8

Then screening of the sub-cultured bacterial colonies were done on Casein Agar plates and it was

observed that the colonies isolated were having proteolytic enzymes and had shown clear zone on

the screening media:

Fig. The screening media showing clear zone.

The sub-cultured bacterial colonies were then identified with the help of various bio-chemical

tests. The results were as follows:

BIO

CH

EM

ICA

L T

ES

TS

COLONIES 2 7 8

Indole Production -ve -ve -ve

MR +ve +ve +ve

VP -ve -ve -ve

Citrate +ve +ve +ve

Catalase +ve +ve +ve

TSI Ferment

glucose only

Ferment Lac

+ glu

Ferment Lac

+ glu

Gelatin +ve -ve +ve

Urease -ve -ve -ve

Starch Hydrolysis +ve -ve -ve

Nitrate Reduction +ve -ve +ve

Motility Motile Motile Motile

Endospore Staining Numerous

oval

endospores

More

endospores

than veg. cells

More

endospores

than veg. cells

Mannitol

Fermentation +ve +ve -ve

On the basis of the results of the biochemical tests colony 2 and 7 were

recognized as Bacillus subtilis, our subject colony.

Fig. Indole production test, VP and Citrate test

Fig. Catalase test, TSI and Gelatin test

Fig. Starch Hydrolysis test for colony 2, 7 and 8

Fig. Nitrate reduction, motility and mannitol fermentation tests

Fig. stained endospore of Bacillus subtilis

After the identification of the desired bacterium, the extraction of enzyme

was preceded with colony 2 and 7 along with the bacterium Pseudomonas

aeruginosa, provided by the laboratory. Seed culture was performed with

each of the above mentioned colonies and the enzyme was extracted. The

enzyme thus obtained was crude and needs to be purified. We then purified

the enzyme by the process of ammonium sulfate precipitation followed by

dialysis and ion-exchange chromatography.

The concentration of the purified enzyme was then estimated by the

comparison with BSA standard and Tyrosine standard, after each step of

purification.

Sl. No.

Vol. of

BSA

(in

ml)

Vol. of

D/W

(in ml)

Solution C

(in ml)

I

n

c

u

b

a

ti

o

n

a

t

R

T

f

o

r

1

0

m

i

n

s

FC Reagent

(in ml)

I

n

c

u

b

a

ti

o

n

i

n

d

a

r

k

f

o

r

3

0

m

i

n

s

Concentration

Of protein (in

µg/ml)

OD at

660nm

Blank 0.0 1.0 5 0.5 0 0.0

1 0.2 0.8 5 0.5 40 0.056

2 0.4 0.6 5 0.5 80 0.096

3 0.6 0.4 5 0.5 120 0.142

4 0.8 0.2 5 0.5 160 0.235

5 1.0 0.0 5 0.5 200 0.277

Sl. No.

Vol. of

Test

(in

ml)

Vol. of

D/W

(in ml)

Solution C

(in ml)

FC Reagent

(in ml)

Concentration

Of protein (in

µg/ml)

OD at

660nm

2C 0.1 0.9 5 0.5 440 0.643

2A 0.1 0.9 5 0.5 152 0.212

7C 0.1 0.9 5 0.5 304 0.564

7A 0.1 0.9 5 0.5 96 0.948

PC 0.1 0.9 5 0.5 296 0.435

PA 0.1 0.9 5 0.5 84 0.120

*C stands for the crude enzyme sample and A stands for the ammonium precipitated sample

#2, 7 and P stands for colony 2, 7 and Pseudomonas aeruginosa

Table. Estimation of protein by Lowry’s Method

Sl. No.

Vol. of

Tyros

ine

(in

ml)

Vol. of

D/W

(in ml)

Solution C

(in ml)

I

n

c

u

b

a

ti

o

n

a

t

R

T

f

o

r

1

5

m

i

n

s

FC Reagent

(in ml)

I

n

c

u

b

a

ti

o

n

i

n

d

a

r

k

f

o

r

3

0

m

i

n

s

Concentration

Of protein (in

µg/ml)

OD at

660nm

Blank 0.0 1.0 5 0.5 0 0.0

1 0.2 0.8 5 0.5 200 0.225

2 0.4 0.6 5 0.5 400 0.414

3 0.6 0.4 5 0.5 600 0.571

4 0.8 0.2 5 0.5 800 0.597

5 1.0 0.0 5 0.5 1000 0.750

Sl. No.

Vol. of

Test

(in

ml)

Vol. of

D/W

(in ml)

Solution C

(in ml)

FC Reagent

(in ml)

Concentration

Of protein (in

µg/ml)

OD at

660nm

2C 0.1 0.9 5 0.5 650 0.489

2A 0.1 0.9 5 0.5 520 0.390

7C 0.1 0.9 5 0.5 730 0.545

7A 0.1 0.9 5 0.5 480 0.374

PC 0.1 0.9 5 0.5 880 0.569

PA 0.1 0.9 5 0.5 340 0.362

Table. Estimation by Tyrosine standard

After estimation of the concentration of the enzyme in the sample was then

assayed on the substrate (casein solution).

S.N

Vol. of 1

%

Casein

solution

(in ml)

Incu

bat

ion

at

37

°C f

or

10

min

s.

Enzy

me

solut

ion

(in

ml)

Swir

lin

g a

nd

in

cub

ati

on

at

37

°C f

or

10

min

s.

TC

A

Re

g C

(in

ml

)

D/

W

(in

ml)

Swir

lin

g a

nd

in

cub

ati

on

at

37

°C f

or

30

min

s.

Fil

trat

ion

of

each

sa

mp

le u

sin

g W

hat

ma

nn

fil

ter

pap

er Filtrate

(in ml)

Solut

ion C

(in

ml)

FC

Re

age

nt

(in

ml)

O.D at

660

nm

Conce

ntratio

n (in

µg/ml

)

Blank 0.5 - 5 0.1 2 5 0.5 0.0 0.0

2C 0.5 0.1 5 - 2 5 0.5 0.256 340

2A 0.5 0.1 5 - 2 5 0.5 0.09 120

7C 0.5 0.1 5 - 2 5 0.5 0.175 230

7A 0.5 0.1 5 - 2 5 0.5 0.055 70

PC 0.5 0.1 5 - 2 5 0.5 0.658 870

PA 0.5 0.1 5 - 2 5 0.5 0.268 350

Table. Enzyme assay on casein solution

The effect of temperature, pH, activator, inhibitor and substrate concentration

on enzyme activity was studied and the following observations were

tabulated:

Colony pH

Temperature

(in ˚C)

Activator Inhibitor

Substrate

concentration

(in %age)

2 4.0 37 CaCl2 HgCl2 2.5

7 4.0 27 FeCl3 HgCl2 2.5

P 7.0 4 MgSO4 HgCl2 2.5

Table. Characterisation of enzyme

The activity of the enzyme sample obtained from the three colonies was found

to be maximal at the above mentioned parameters. Anomaly has been found

in the effect of pH, temperature and activator, however, all the three samples

show similarity in activity for inhibitor and %age concentration of the

substrate provided.

CALCULATIONS: ENZYME ACTIVITY

COLONY

VOL. OF

ENZYME

USED

(ml)

CRUDE

AFTER

AMMONIUM

SULFATE

PRECIPITATION

AFTER ION-

EXCHANGE

CHROMATOGRAPHY

OD (A) CONC.

(µg/ml) OD (A)

CONC.

(µg/ml) OD (A)

CONC.

(µg/ml)

2 0.1 0.489 650 0.390 520 0.206 270

7 0.1 0.545 730 0.374 480 0.175 240

P 0.1 0.569 880 0.362 340 0.710 950

Table. Concentration of enzyme after each purification step

The respective activity of the enzyme samples were calculated by using

the formula as follows:

Enzyme activity = concentration/mol. Wt. of tyrosine

Enzyme activity is given by micromoles/min.

COLONY

VOL. OF

ENZYME

USED

(ml)

CRUDE

AFTER AMMONIUM

SULFATE

PRECIPITATION

AFTER ION-

EXCHANGE

CHROMATOGRAPHY

OD

(A)

ACTIVITY

(µmol/min)

OD

(A)

ACTIVITY

(µmol/min)

OD

(A)

ACTIVITY

(µmol/min)

2 0.1 0.489 3.587 0.390 2.869 0.206 1.490

7 0.1 0.545 4.028 0.374 2.649 0.175 1.324

P 0.1 0.569 4.856 0.362 1.876 0.710 5.243

Table. Activity of enzyme after each purification step

The specific activity of the enzyme samples were calculated by using the

formula as follows:

Specific activity = units per ml of enzyme / units per mg of enzyme

Units per ml of enzyme = micromole of tyrosine eq. released into

volume of assay / vol. of enzyme used X

incubation time X vol. used in colorimetric

determination

COLONY CRUDE

AFTER

AMMONIUM

SULFATE

PRECIPITATION

AFTER ION-

EXCHANGE

CHROMATOGRAPHY

2 10.326 10.340 10.348

7 10.345 10.345 10.347

P 10.346 10.344 10.347

Table. Specific activity after each purification step

The enzyme samples obtained after each purification step of the three

colonies were subjected to SDS – PAGE to determine the molecular weight by

running them with marker. A mixture of egg albumin and 1% BSA was used as

marker that gave protein bands between 14 to 60 kD.

Fig. SDS – PAGE

WELL NO. SAMPLE MOL. WT.

(IN kD)

1 5mM P 55

2 15mM 7 52

3 5mM 2 60

4 Dialysed P

A 60

B 54

C 30

5 Dialysed 7 A 60

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.

Fig. Enzyme dissolving blood clots

Fig. Clots completely dissolved

APPENDIX

I. CULTURE MEDIA

a. Casein Agar (pH = 7.0 + 0.2)

Skim milk powder 1gm

Glucose 1gm

Peptone 5gm

Yeast Extract 2.5gm

Agar 10.5gm

Distilled Water 1000ml

b. Mannitol Fermentation Media (pH = 7.3)

Peptone 10gm

Mannitol 5gm

Sodium chloride 15gm

Phenol red 0.018gm

Distilled Water 1000ml

c. MRVP Broth (pH = 6.9)

Peptone 7gm

Dextrose/Glucose 5gm

Potassium phosphate 5gm

Distilled Water 1000ml

d. Nitrate Broth

Potassium nitrate 0.2gm

Peptone 5gm

Distilled Water 1000ml

e. Nutrient Agar Media (pH = 7.0 + 0.2)

Beef Extract 3gm

Peptone 5gm

Sodium chloride 5gm

Agar 20gm

Distilled Water 1000ml

f. Nutrient Gelatin (pH = 6.8)

Peptone 5gm

Beef extract 3gm

Gelatin 120gm

Distilled Water 1000ml

g. Production Media (pH = 7.0 to 7.2)

Glucose 10gm

Yeast extract 10gm

Dipotassium hydrogen phosphate 1gm

Magnesium sulfate 0.5gm

h. SIM Agar Media (pH = 7.3)

Peptone 30gm

Beef extract 3gm

Ferrous ammonium sulfate 0.2gm

Sodium thiosulfate 0.025gm

Agar 3gm

Distilled water 1000ml

i. Simmon’s Citrate Agar (pH = 6.9)

Ammonium dihydrogen phosphate 1gm

Dipotassium phosphate 1gm

Sodium chloride 5gm

Sodium citrate 2gm

Magnesium sulfate 0.2gm

Agar 15gm

Bromothymol blue 0.8gm

Distilled Water 1000ml

j. Starch Agar

Starch 20gm

Peptone 5gm

Beef extract 3gm

Agar 15gm

Distilled Water 1000ml

k. Triple Sugar Iron Agar (pH = 7.4)

Peptone 15gm

Protease peptone 5gm

Beef extract 3gm

Yeast extract 3gm

Lactose 10gm

Sucrose 10gm

Dextrose 1gm

Sodium chloride 5gm

Ferrous sulfate 0.2gm

Sodium thiosulfate 0.3gm

Phenol red 0.024gm

Agar 12gm

Distilled water 1000ml

l. Tryptone Broth (pH = 6.9)

Tryptone 1gm

Distilled water 100ml

m. Urea Agar Media (pH = 6.8)

Peptone 1gm

Sodium chloride 5gm

Potassium monohydrogen or

dihydrogen phosphate 2gm

Agar 2gm

Distilled water 1000ml

Glucose 1gm

Phenol red (0.2% solution) 6ml

Urea (20% aqueous in 100ml from 1000ml)

II. REAGENTS

a. Acrylamide bis-acrylamide solution

38.6% acrylamide + 1.4% bisacrylamide

b. Ammonium per sulfate solution

10% freshly prepared APS

c. BSA Standard

BSA (Stock) 10mg/40ml of distilled water

BSA (Working) 10ml Stock + 40 ml Distilled water

d. Destaining Solution

Glacial acetic acid 7.5%

Methanol 10%

e. FC Reagent

Distilled water and FC reagent were mixed in the ratio of 2:1

f. Nitrate Test Reagent

Sol. A: 8 gm sulphanilic acid + 1000ml acetic acid

Sol. B: 5gm α-naphalamine + 1000ml acetic acid

Equal volume of solution A and B were mixed, immediately before

use.

g. Resolving Gel

Distilled water 2.3ml

Acrylamide bis-acrylamide solution 2.3ml

10% SDS 0.05ml

10% APS 0.05ml

1.5M Tris (pH = 8.8) 1.3ml

TEMED 0.005ml

h. SDS (Sodium dodecyl sulfate)

10% freshly prepared SDS

i. Solution C

Sol. A: 2% Na2CO3 + 0.1N NaOH

Sol. B: 0.5% CuSO4 + 1% Potassium sodium tartarate

Sol. C: 50ml Sol. A + 1ml Sol. B

j. Stacking Gel

Distilled water 1.4ml

Acrylamide bis-acrylamide solution 0.33ml

10% SDS 0.02ml

10% APS 0.02ml

1.0M Tris (pH = 6.8) 0.25ml

TEMED 0.002ml

k. Staining Solution

Coomassie brilliant blue 0.25%

Iso-propanol 40%

l. TEMED

10μl TEMED + 1ml distilled water

m. Tyrosine Standard

Tyrosine (stock) 10mg/10ml of Distilled water

Tyrosine (working) 10ml stock + 40ml Distilled water

III. BUFFERS

a. Electrophoresis Buffer (10X)

SDS 0.1gm

Tris buffer 0.62gm

Glycine 1.47gm

b. Ellusion Buffer

Sodium phosphate – Potassium phosphate Buffer (pH = 7.0)

c. Gradient Buffer

5ml 5mM NaCl + 0.5ml 10mM Tris + 4.5ml D/W

10ml 5mM NaCl + 0.5ml 10mM Tris + 4.5ml D/W

15ml 5mM NaCl + 0.5ml 10mM Tris + 4.5ml D/W

20ml 5mM NaCl + 0.5ml 10mM Tris + 4.5ml D/W

d. Lower Tris (pH = 8.8)

1.5M tris buffer

e. Sodium phosphate – Potassium phosphate Buffer

Desired pH Vol. of KH2PO4

(in ml)

Vol. of Na2PO4

(in ml)

Final Volume

(in ml)

5.4 970 30 1000

6.0

7.0

8.0

880

389

55

120

611

945

1000

1000

1000

The two solutions must be used of equal molarity. 0.1M

KH2PO4 (anhyd.) solution can be prepared by dissolving

1.362gm in 100ml D/W. 0.1M Na2HPO4. 2H20 solution can be

prepared by dissolving 1.78gm in 100ml.

f. Tank Buffer

1X electrophoresis buffer

g. Upper Tris

1M tris buffer