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 intraand 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 Route B

reactant 1 + reactant 2 reactant 1 + enzyme

product intermediate product + enzyme

intermediate + reactant 2

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 endonuclease 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 prothrombin 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. CVI 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 CO 2 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 CO 2 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. A. PURIFICATION OF ENZYME 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 bifunctional 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 intracellular 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 COLOUR FORM ELEVATION MARGIN SIZE TRANSPARENCY GRAM’S STAINING SHAPE 2 Off – white Irregular Flat Erose Small Opaque +ve Rod and chain 7 Off – white Circular Flat Entire Small Opaque +ve Rod 8 Off – white Circular Flat Entire Small Opaque +ve 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:

COLONIES
Indole Production MR VP

2
-ve +ve -ve +ve +ve
Ferment glucose only

7
-ve +ve -ve +ve +ve
Ferment Lac + glu

8
-ve +ve -ve +ve +ve
Ferment Lac + glu

BIOCHEMICAL TESTS

Citrate Catalase TSI Gelatin Urease Starch Hydrolysis Nitrate Reduction Motility Endospore Staining Mannitol Fermentation

+ve -ve +ve +ve Motile
Numerous oval endospores

-ve -ve -ve -ve Motile
More endospores than veg. cells

+ve -ve -ve +ve Motile
More endospores than veg. cells

+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.
Vol. of BSA (in ml) 0.0 0.2 0.4 0.6 0.8 1.0 Vol. of Test (in ml) 0.1 0.1 0.1 0.1 0.1 Vol. of D/W (in ml) 1.0 0.8 0.6 0.4 0.2 0.0 Vol. of D/W (in ml) 0.9 0.9 0.9 0.9 0.9 Solution C (in ml) I n c u b a ti o n FC Reagent (in ml) 0.5 0.5 0.5 0.5 a t 0.5 0.5 FC Reagent R T (in ml) 0.5 f o r 0.5 0.5 0.5 1 0 m i n s 0.5 3 0 m i n s f o r d a r k i n I n c u b a ti o n Concentration Of protein (in µg/ml) 0 40 80 120 160 200 Concentration Of protein (in µg/ml) 440 152 304 96 296 OD at 660nm OD at 660nm

Sl. No.

Blank 1 2 3 4 5

5 5 5 5 5 5

0.0 0.056 0.096 0.142 0.235 0.277

Sl. No.

Solution C (in ml)

2C 2A 7C 7A PC

5 5 5 5 5

0.643 0.212 0.564 0.948 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) 0.0 0.2 0.4 0.6 0.8 1.0 Vol. of Test (in ml) 0.1 0.1 0.1 0.1 0.1

Vol. of D/W (in ml)

Solution C (in ml)

Blank 1 2 3 4 5

1.0 0.8 0.6 0.4 0.2 0.0 Vol. of D/W (in ml) 0.9 0.9 0.9 0.9 0.9

5 5 5 5 5 5

I n c u b a ti o n

FC Reagent (in ml)

0.5 0.5 0.5 0.5

I n c u b a ti o n

Concentration Of protein (in µg/ml) 0 200 400 OD at 660nm

0.0 0.225 0.414 0.571 0.597 0.750

a t

0.5 0.5

i n

600 800 1000

Sl. No.

Solution C (in ml)

R T

FC Reagent (in ml) 0.5 0.5 0.5

d a r k

Concentration Of protein (in µg/ml) 650 OD at 660nm

2C 2A 7C 7A PC

5 5 5 5 5

f o r

0.489 0.390 0.545 0.374 0.569

f o r

520 730 480

PA

0.1

0.9

5

1 5 m i n s

0.5 0.5 3 0 m i n s

880

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).
Vol. of 1 % Casein solution (in ml)
Incubation at 37°C for 10 mins.

S.N

Blank 2C 2A 7C 7A PC PA

0.5 0.5 0.5 0.5 0.5 0.5 0.5

0.1 0.1 0.1 0.1 0.1 0.1

5 5 5 5 5 5 5

0.1 -

Filtration of each sample using Whatmann filter paper

Swirling and incubation at 37°C for 10 mins.

Swirling and incubation at 37°C for 30 mins.

Enzy me solut ion (in ml)

TC A Re gC (in ml )

D/ W (in ml)

Filtrate (in ml)

Solut ion C (in ml)

FC Re age nt (in ml) 0.5 0.5 0.5 0.5 0.5 0.5 0.5

O.D at 660 nm

Conce ntratio n (in µg/ml )

2 2 2 2 2 2 2

5 5 5 5 5 5 5

0.0 0.256 0.09 0.175 0.055 0.658 0.268

0.0 340 120 230 70 870 350

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:
Temperature Colony pH (in ˚C) 2 7 P 4.0 4.0 7.0 37 27 4 CaCl2 FeCl3 MgSO4 HgCl2 HgCl2 HgCl2 Activator Inhibitor Substrate concentration (in %age) 2.5 2.5 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 OD (A) 0.390 0.374 0.362 CONC. (µg/ml) 520 480 340 AFTER IONEXCHANGE CHROMATOGRAPHY CONC. (µg/ml) 270 240 950

OD (A) 2 7 P 0.1 0.1 0.1 0.489 0.545 0.569

CONC. (µg/ml) 650 730 880

OD (A) 0.206 0.175 0.710

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) OD (A) 2 7 P 0.1 0.1 0.1 0.489 0.545 0.569 CRUDE AFTER AMMONIUM SULFATE PRECIPITATION OD (A) 0.390 0.374 0.362 ACTIVITY (µmol/min) 2.869 2.649 1.876 AFTER IONEXCHANGE CHROMATOGRAPHY OD (A) 0.206 0.175 0.710 ACTIVITY (µmol/min) 1.490 1.324 5.243

ACTIVITY (µmol/min) 3.587 4.028 4.856

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 10.340 10.345 10.344 AFTER IONEXCHANGE CHROMATOGRAPHY 10.348 10.347 10.347

2 7 P

10.326 10.345 10.346

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. 1 2 3 SAMPLE 5mM P 15mM 7 5mM 2 A 4 Dialysed P B C A 5 6 7 Dialysed 7 B Dialysed 2 Marker 60 60 45 MOL. WT. (IN kD) 55 52 60 60 54 30 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 Glucose Peptone Yeast Extract Agar Distilled Water 1gm 1gm 5gm 2.5gm 10.5gm 1000ml

b. Mannitol Fermentation Media (pH = 7.3) Peptone Mannitol Sodium chloride Phenol red Distilled Water c. MRVP Broth (pH = 6.9) Peptone Dextrose/Glucose Potassium phosphate Distilled Water d. Nitrate Broth Potassium nitrate 0.2gm 7gm 5gm 5gm 1000ml 10gm 5gm 15gm 0.018gm 1000ml

Peptone Distilled Water

5gm 1000ml

e. Nutrient Agar Media (pH = 7.0 + 0.2) Beef Extract Peptone Sodium chloride Agar Distilled Water f. Nutrient Gelatin (pH = 6.8) Peptone Beef extract Gelatin Distilled Water 5gm 3gm 120gm 1000ml 3gm 5gm 5gm 20gm 1000ml

g. Production Media (pH = 7.0 to 7.2) Glucose Yeast extract 10gm 10gm

Dipotassium hydrogen phosphate 1gm Magnesium sulfate h. SIM Agar Media (pH = 7.3) Peptone Beef extract Ferrous ammonium sulfate 30gm 3gm 0.2gm 0.5gm

Sodium thiosulfate Agar Distilled water

0.025gm 3gm 1000ml

i. Simmon’s Citrate Agar (pH = 6.9) Ammonium dihydrogen phosphate 1gm Dipotassium phosphate Sodium chloride Sodium citrate Magnesium sulfate Agar Bromothymol blue Distilled Water j. Starch Agar Starch Peptone Beef extract Agar Distilled Water k. Triple Sugar Iron Agar (pH = 7.4) Peptone Protease peptone Beef extract 15gm 5gm 3gm 20gm 5gm 3gm 15gm 1000ml 1gm 5gm 2gm 0.2gm 15gm 0.8gm 1000ml

Yeast extract Lactose Sucrose Dextrose Sodium chloride Ferrous sulfate Sodium thiosulfate Phenol red Agar Distilled water l. Tryptone Broth (pH = 6.9) Tryptone Distilled water m. Urea Agar Media (pH = 6.8) Peptone Sodium chloride Potassium monohydrogen or dihydrogen phosphate Agar Distilled water Glucose Phenol red (0.2% solution)

3gm 10gm 10gm 1gm 5gm 0.2gm 0.3gm 0.024gm 12gm 1000ml

1gm 100ml

1gm 5gm

2gm 2gm 1000ml 1gm 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) BSA (Working) d. Destaining Solution Glacial acetic acid Methanol 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 Acrylamide bis-acrylamide solution 2.3ml 2.3ml 7.5% 10% 10mg/40ml of distilled water 10ml Stock + 40 ml Distilled water

10% SDS 10% APS 1.5M Tris (pH = 8.8) TEMED h. SDS (Sodium dodecyl sulfate) 10% freshly prepared SDS i. Solution C Sol. A: 2% Na2CO3 + 0.1N NaOH

0.05ml 0.05ml 1.3ml 0.005ml

Sol. B: 0.5% CuSO4 + 1% Potassium sodium tartarate Sol. C: 50ml Sol. A + 1ml Sol. B j. Stacking Gel Distilled water Acrylamide bis-acrylamide solution 10% SDS 10% APS 1.0M Tris (pH = 6.8) TEMED k. Staining Solution Coomassie brilliant blue Iso-propanol l. TEMED 10μl TEMED + 1ml distilled water 0.25% 40% 1.4ml 0.33ml 0.02ml 0.02ml 0.25ml 0.002ml

m. Tyrosine Standard Tyrosine (stock) Tyrosine (working) 10mg/10ml of Distilled water 10ml stock + 40ml Distilled water

III. BUFFERS
a. Electrophoresis Buffer (10X) SDS Tris buffer Glycine 0.1gm 0.62gm 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) 5.4 970 Vol. of Na2PO4 (in ml) 30 Final Volume (in ml) 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