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Mixture of protein digesting enzymes known as proteolytic enzymes or proteases include several other substances as welleither of two proteases extracted from plant family bromeliaceae i.e., Stem bromelain - EC 3.4.22.32 Fruit bromelain - EC 3.4.22.33May also refer to a combination of those enzymes along with other compounds produced in an extractReferred to as sulfhydryl proteases since a free sulfhydral group of a cysteine side chain is essentialThe other substances typically include peroxidase, acid phosphatase, protease inhibitors, and calcium

WHAT ACTUALLY IS BROMELAIN??

temperature 40-60 COptimal temperature 50-60 CDeactivation temperature above65 C approx.Effective pH 4.0-8.0Optimal pH 4.5-5.5Molecular weight 28.4 kD

COILS HELIXES AND HELICAL TURNS The term "bromelain" can refer to either of two protease enzymes extracted from the plant family Bromeliaceae, or it can refer to a combination of those enzymes along with other compounds produced in an extract.

1PEEK IN TO THE PASTFirst isolation Vicente Marcano in 1891 from fruit of pineapple.In 1892, Chittenden, Joslin and Meara investigated the matter fully and named it BromelinLater, Bromelian was introduced and orignally applied to any protease from any member of family Bromeliaceae.In 1957 first introduced as therapeutic supplementPioneer research at Hawaii but recent in countries in Asia, Europe and Latin America. Germany has recently taken a great interest in bromelian research.13th most widely used herbal medicine in Germany.

2IT COMES FROMPineapple plant (Ananas sp.)Stem most common commercial sourceTraditionally as a medicinal plant among natives of South and Central America.Produced in Thailand, Taiwan, and other tropical parts of the world where pineapples are grown.Prepared from stem part of pineapple after harvesting the fruit.

Bromelain is present in all parts of the pineapple plant (Ananas sp.) However, the stem is the most common commercial source, presumably because it is readily available after the fruit has been harvested. Pineapples have had a long tradition as a medicinal plant among the natives of South and Central America.It is produced in Thailand, Taiwan, and other tropical parts of the world where pineapples are grown.

Bromelain is prepared from the stem part of the pineapple plant after harvesting the fruit. This stem part is peeled, crushed, and pressed to get the juice containing the soluble bromelain enzyme. Further processing includes purification and concentration of the enzyme

3ROLE OF THE STUDBromelain bloods fibrolytic activity and kininogen and bradykinin serum and tissue levels as well as reduce excretion of proinflammatory cytokines and chemokinesAlso effects prostaglandin synthesisInhibits fibrinogen synthesisDirectly degrades fibrin and fibrinogencleave at Lys-, Ala-, Tyr-, Gly- Is activated by cysteine, bisulfite salt, NaCN, H2S, Na2S, and benzoate.inhibited by Hg++, Ag+, Cu++, a-1-antitrypsin, estatin A&B, Iodoacetate, TLCK, TPCK

Bromelain, increases blood fibrinolytic activity as well as inhibiting fibrinogen synthesis; bromelain also directly degrades fibrin and fibrinogen. Kininogen and bradykinin serum and tissue levels are lowered by bromelain and it also affects prostaglandin synthesis, which gives it's antiinflammatory effects. Bromelain has been found to reduce the excretion of proinflammatory cytokines as well as chemokines in a study into its possible mechanism of action in ulcerative colitis, inflammatory bowel disease, Crohn's disease

4PAY BACK TIMEproduct name AnanaseVarious uses in Folk medicineExplored as a potential healing agent in alternative medicine. Work by blocking some proinflammatory metabolites when applied topicallyUsed for reducing swellingInvolved in the migration of neutrophils to the site of acute inflammation. Used for treating arthiritisWhen used in conjunction with trypsin and rutin is as effective as prescribed analgesics in the osteoarthiritis management.Meat tenderizing

Available in some countries as a product under the name 'Ananase', bromelain began its reputation for various uses in folk medicine and continues to be explored as a potential healing agent in alternative medicine. First introduced in medical research in 1957, bromelain may work by blocking some proinflammatory metabolites when applied topically. Bromelain may be used after surgery to reduce swelling. Preliminary research indicates that bromelain may affect migration of neutrophils to sites of acute inflammation. As a potential anti-inflammatory agent, it may be useful for treating arthritis, but has neither been confirmed in human studies for this use nor is it approved with a health claim for such an effect by the Food and Drug Administration or European Food Safety Authority. The Natural Medicines Comprehensive Database suggests that bromelain, when used in conjunction with trypsin and rutin is as effective as some prescription analgesics in the management of osteoarthritis.

5WHAT ELSE??Other effects include: Hay feverTreating a bowel condition that includes swelling and ulcer ulcerative colitisRemoving dead and damaged tissue after a burn debridementPreventing the collection of water in the lung pulmonary edemaRelaxing musclesImproving the absorption of antibioticsPreventing cancer Shortening laborHelp the body in reducing fatsSupplement may effect heart ratesystemic enzyme therapy

Bromelain may also be used for a variety of other effects that remain scientifically unconfirmed and not authorized by regulatory authorities like the Food and Drug Administration. Systemic enzyme therapy (consisting of combinations of proteolytic enzymes such as bromelain, trypsin, chymotrypsin, and papain) has been investigated in Europe to evaluate the efficacy of proteolytic enzymes in the treatment of breast, colorectal, and plasmacytoma cancer patients. In mice with experimental colitis, 6 months of dietary bromelain from pineapple stem or from fresh juice decreased the severity of colonic inflammation and reduced the number of cancerous lesions in the colon.

6DIASTASE Diastase are any one of a group of enzymes which catalyses the breakdown of starch into maltose.first enzyme discovered. It was extracted from malt solution in 1833 by Anselme Payen and Jean-Franois Persoz, chemists at a French sugar factory.The name "diastase" comes from the Greek (diastasis) (a parting, a separation)

because when beer mash is heated, the enzyme causes the starch in the barley seed to transform quickly into soluble sugars and hence the husk to separate from the rest of the seed.[4][5] 7ALPHA AMYLASEEC NUMBER: 3.2.1.1 is 1,4-a-D-Glucan glucanohydrolaseALTERNATIVE NAME : glucogenaseLocation: it is secreted in saliva and pancreas, found in humans and other animals food reserve of fungi/Acts on starch, glycogen and related polysaccharides and oligosaccharides in a random manner; reducing groups are liberated in the alpha-configuration.Causes hydrolyses alpha-bonds of large alpha-linked polysaccharides, such as starch and glycogen, yielding glucose and maltose.

STRUCTURE 679 amino acid residues with a molecular weight of 75112 residuesIt has 3 domains A B CDOMAIN A: These domains are generally found on all -amylase enzymes. The A domain constitutes the core structure, with a (/)8-barrel. DOMAIN B :consists of a sheet of four anti-parallel -strands with a pair of anti-parallel -strands. Long loops are observed between the -strands. Located within the B domain is the binding site for Ca2+-Na+-Ca2+. DOMAIN C consisting of eight -strands is assembled into a globular unit forming a Greek key motif. It also holds the third Ca2+ binding site in association with domain AACTIVE SITE:Positioned on the C-terminal side of the -strands of the (/)8-barrel in domain A is the active site. The catalytic residues involved for the BSTA active site are Asp234, Glu264, and Asp331

AMYLOSE IN STARCH

GLUCOSE RESIDUE CLEAVED BY AMYLASE

MECHANISM OF action of alpha amylose:Alpha amylose converts amylose or starch to maltose. Alpha-amylase hydrolyzes bonds between glucose repeats. Starch is a polysachride made up of 2 residues amylose and amylopectin. Alpha amylase is breaks down starch by hydrolysis to release maltoseThe reaction occurs in 3 steps:The simple glucose molecules have a bond between the 1 carbon in the ring and the 4 carbon in the ring. They are joined by an oxygen. AMYLOSEBelow is the building block of starch, glucose. Notice that when the single oxygen is left in the bond in the amylose, the two glucose molecules must have lost two hydrogen and an oxygen in the formation of this bond- or one H2O molecule. This chemical reaction is called dehydration, because it generates water.

Similarly, when this bond breaks to generate a free glucose or pairs of free maltose,there is the addition of a water molecule to make the OH at carbon 1 and carbon 4. This is called hydrolysis or Hydro (water) + Lysis (breaking). One of the disaccharides or monosaccharides gets an OH- from the water molecule and the other part gets an H+ from the water. Hydration is adding a water without splitting the molecule apart.10INDUSTRIAL APPLICATION:used in ethanol production to break starches in grains into fermentable sugars.detergents, especially dishwashing and starch-removing detergents.in textile weaving, starch is added for warping.-Amylase is used for the production of malt, as the enzyme is produced during the germination of cereal grainsChecking out pancerititis the amylase levels are measured in the pancertic cells.

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ENZYME: TRYPSIN*TYRPSINOGEN*HISTORY & SOURCE***1876, first named by Kuhne who described the proteolytic activity of this pancreatic enzyme.1931, Norothrop and Kunitz purified trypsin by crystallization.1974, three dimensional structure was determined

PancreasTRYPSINOGEN TRYPSIN

Bovine Pancreasexpresses two forms of trypsin:dominant cationic formminor anionic formThese protein sequences share 72% identity, while their coding regions share 78% identity.CONVERSION***TRYPSINOGEN TRYPSIN {Ph 9.3} {ph 10.5}Trypsinogen is activated by removal of a terminal hexapeptide to yield single-chain -trypsin. Limited autolysis produces other active forms having two or more peptide chains bound by disulfide bonds. Predominant forms are *-trypsin, having two peptide chains and *-, a single chainREACTION CATALYZED***Process catalyzed by trypsin

*Trypsin Proteolysis* {Trypsinisation}

Trypsin is considered as an endopeptidase* Cleavage occurs within the polypeptide chain rather than at the terminal amino acids located at the ends of polypeptides.TRYPSIN

SERINE PROTEASE

HYDROLYZE PROTEINSUSED FOR***Tissue dissociationMitochondria isolationin vitro studies of proteins Various hemagglutination proceduresDNA FingerprintingEnvironmental monitoringReduction of cell density in tissue cultureCleavaging fusion proteinsGenerating glycopeptides from purified glycoproteins

What is Alkaline Phosphatase?Alkaline phosphatase (EC 3.1.3.1)comprises a group of enzymes that catalyze the hydrolysis of phosphate esters in an alkaline environment, generating an organic radical and inorganic phosphate. This has many isoenzymes includingIntestinal (ALPI), Chromosome 2Placental (ALPP)Liver/bone/kidney (ALPL) Chromosome 1It belongs to Alpha and Beta class of proteins

Alkaline phosphatase is pertinent in recycling phosphate within living cells. Alkaline Phosphatases are a group of enzymes found primarily the liver (isoenzyme ALP-1) and bone (isoenzyme ALP-2). There are also small amounts produced by cells lining the intestines (isoenzyme ALP-3), the placenta, and the kidney (in the proximal convoluted tubules). The structure of alkaline phosphatase differs depending on where that particular enzyme originated. Alkaline phosphatase from bone looks different from alkaline phosphatase from the liver. These different structures are called isozymes

17Alkaline phosphatase is a glycoprotein mainly parallel beta sheets Core has 3 layers: a/b/a.In general, alkaline phosphatase is a dimer containing nearly identical subunits which each have two molecules of zinc and one molecule of magnesium ion. One molecule of zinc is tightly bound, giving the structure stability and the other is loosely bound which provides for the catalytic activity.STRUCTURE

General Mechanism

IIIIIIIV19Properties AND FUNCTIONThis enzyme was partially purified and studied by Kunitz (1960)It is a hydrolase enzyme found in bacteria and mammals Optimum pH: 8 9Activators: Mg2+Wide specificityInhibitors: acidic pH, chelators of the metal ions, urea and high levels of Zn2+The property of dephosphorylation allows for uses in molecular biology, in pasteurization and in nature by bacteria.It catalyses the following reactionA phosphate monoester + H(2)O an alcohol + phosphateElevated levels (five-times higher than normal) are found in the blood serum of people suffering from various bone and liver diseases. Irritable bowel syndrome, germ-cell tumors and liver infections will raise serum levels of Alkaline Phosphatase to a lesser extent.Alkaline phosphatase (ALP) is an enzyme normally present in raw milk and it is inactivated when thermal treatment conditions are slightly higher than those required for the destruction of the pathogenic bacteria. Hence, the chemical test carried out to determine alkaline phosphatase (ALP) concentration in pasteurized milk is used to verify if thermal treatment has been done correctly. Alkaline phosphatase is useful in the dairy industry as a marker for successful pasteurization in cow's milk. At the high temperatures of pasteurization, alkaline phosphatase is supposed to denature. To test this, experts sometimes add a para-nitrophenylphosphate substrate to the milk. If alkaline phosphatase is present, it will break down this phosphate molecule, causing the milk to change color to yellow. Properly pasteurized milk will not change cALP activity can be used to measure the effectiveness of pasteurization.

20One of the most important functions of alkaline phosphatase is as an indicator for disease.Alkaline Phosphatase TestThe blood serum level of Alkaline Phosphatase is used as a marker for disease. Elevated levels (five-times higher than normal) are found in the blood serum of people suffering from various bone and liver diseases. Irritable bowel syndrome, germ-cell tumors and liver infections will raise serum levels of Alkaline Phosphatase to a lesser extent.

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PEPSIN ClassificationEC number 3.4.23.3Member of the aspartate protease family First animal enzyme to be discoveredSecond to be crystallized

Discovery Theodor Schwann Northrop

22Structure:

Two aspartate molecules at the active site Three sulphide bridges23 PEPSINOGEN - primary structure has an additional 44 amino acids Released by chief cells in the stomach HCL causes activation Pepsinogen pepsin( autocatalysis in acidic env)

A tricky businessTARGETS: Amide bonds of aromatic amino acids like tryptophan, phenylalanine and tyrosine

Tryptophan Phenylalanine

24Temperature: 37C-42CpH: 1.5 2Stable until pH 8- can be reactivated upon re- acidificationActivity and Stability:Imbalance in pHInability to digest proteinDeficiency:25PAPAIN

Papaya Proteinase ICysteine protease hydrolaseEnzyme extraction

Family & structureSource: present inpapaya(Carica papaya) andmountain papaya(Vasconcellea cundinamarcensis).Cysteine protease(EC3.4.22.2)enzymeFamily: members found in baculovirus,eubacteria, yeast, and practically all protozoa, plants and mammals, lysosomal or secretedcontains 345 amino acid residues,and consists of a signal sequence (1-18), apropeptide(19-133) and the mature peptide (134-345). The amino acid numbers are based on the mature peptide. The protein is stabilised by threedisulfide bridges.

Mechanism of actionmechanism by which it breakspeptide bondsinvolves deprotonation of Cys-25 by His-1591. Deprotonation of thiol in cysteine by basic histidine2. Nucleophilic attack by deprotonated cysteine on substrate carbonyl atom

applicationsThe main function of the papain enzyme is to aid in digestion and to promote effective digestive health. This is done by breaking down all the protein in the body for easy digestion.The papain enzyme as a meat tenderizer has been used for many years. Since it is a proteolytic enzyme that tenderizes meat, it also acts as a clarifying agent in many food industry processes.It is used in treatment of stings that are administered by jellyfish, bees, wasps or insects by breaking down the toxin and the venom.It boosts the immune system and is seen to be beneficial in food allergies and tumors

Introduction (Cellulase)

Cellulaserefers to an entourage of enzymes produced chiefly by fungi, bacteria and protozoans that catalyze cellulolysis (i.e. thehydrolysisof cellulose). However, there are also cellulases produced by a few other types of organisms, such as sometermitesand the microbial intestinal symbionts of other termites.Several different kinds of cellulases are known, which differ structurally and mechanistically.Some species of fungi and bacteria are able to exhaustively digest crystalline cellulose in pure culture are said to have complete or true cellulases.

The majority of organisms that produce cellulases can only hydrolyze the cellulose in their diets to certain extent. they are known as incomplete cellulases.

These cellulases unable to digest cellulose exhaustively can still generate sufficient amount of glucose for their producers. Endogenous cellulases of termites belong to this category.

Complete vs. incomplete cellulases32Types of reactions/ Classification

General types of cellulases based on the type of reaction catalyzed:

Cleaves internal bonds at Endocellulase (EC 3.2.1.4) randomly amorphous sites that create new chain ends.Cellobiase (EC 3.2.1.21) orbeta-glucosidase hydrolyses the exocellulase product into individual monosaccharides.Cellulose phosphorylases depolymerize cellulose using phosphates instead of water.

Uses

Cellulase is used for commercial food processing incoffee. It performshydrolysisof cellulose during drying ofbeans.Furthermore, cellulases are widely used in textile industry and in laundry detergents. They have also been used in the pulp and paper industry for various purposes, and they are even used for pharmaceutical applications. Cellulase is used in the fermentation of biomass intobio fuels, although this process is relatively experimental at present. Cellulase is used as a treatment for phytobezoars, a form of cellulosebezoarsfound in the humanstomach.

34Succinyl coenzyme A synthetase

Succinyl coenzyme A synthetaseis an enzyme thatcatalyzesthe reversible reaction ofsuccinyl-CoAtosuccinate.Source

Bacteria e.g.E.coli

MammalsChemical ReactionSuccinyl CoA synthetase catalyzes the followingreversible reaction:

Succinyl CoA + Pi + NDP Succinate + CoA + NTP

Succinyl CoAsuccinate

MechanismThe enzyme facilitates coupling of the conversion of succinyl CoA to succinate with the formation of NTP from NDP and Pi. The first step involves displacement ofCoAfrom succinyl CoA by anucleophilicinorganic phosphate molecule to form succinyl phosphate. The enzyme then utilizes ahistidineresidue to remove the phosphate group from succinyl CoA and generate succinate. Finally, the phosphorylated histidine transfers the phosphate group to a nucleoside diphosphate, which generates the high-energy carrying nucleoside triphosphate. Mechanism

UsesSuccinyl-CoA synthetase plays a key role in

the citric acid cycle ketone metabolism heme synthesis

Urokinase sourcesSOURCE ORGANISM

Human urine.much lower concentrations in human plasma. Other organism may include rat, mouse, yeast etc.

SOURCE TISSUE

Ovaryproduced by kidney cells.produced by a variety of tumorcells and involved in the formation of tumormetastasis.Phagocytic cells

Urokinase is found in humanurineand in much lower concentrations in human plasma. In the body, urokinase is produced by kidney cells, and its presence in the urine promotes the dissolution of any bloodclotsin the urine-collecting system of the kidneys or thebladder. Urokinase is also produced by a variety oftumorcells, and it is thought to be involved in the formation of tumormetastases. Urokinase can be inhibited by plasmainhibitorproteins. The presence of cell receptors and inhibitors suggests that the regulation of urokinase function is complex.

Read more:http://www.answers.com/topic/urokinase#ixzz1qg5lkDNo41

MOLECULAR CHARACTERISTICS411-residue proteinthreedomains: serine protease domain, kringle domain and growth factor domain. synthesized as a prourokinase or single-chain urokinase form ; activated by proteolytic cleavage. The two resulting chains are kept together by disulphidebond.found in multiple molecular sizes. Low molecular weight (33-KDa) and high molecular weight (57-KDa). Urinary Urokinase contained predominantly the LMW formIt was found that urinary UK contained predominantly the LMW form. It was suggested that the HMW form is the native structure and that it is converted to the LMW form by cellular proteases. However, under certain conditions, HMW form was found to be more active when assayed with standard enzyme kinetic methods.42

REACTION CATALYSEDPLASMINOGEN + H20 PLASMINSpecific cleavage of Arg-Val bond in plasminogen to form plasmin.Reaction type: Hydrolysis of peptide bondhttp://www.cs.stedwards.edu/chem/Chemistry/CHEM44/serenil/metastasis.html

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USESused clinically for therapy of thrombotic disordersused in medicine to dissolve blood clots.employed in clinical medicine in the treatment ofacute myocardial infarction and arterial blood clots in the legs and arms.Used in peritoneal dialysis.

LuciferaseSourceLuciferaseis a generic term for the class of oxidativeenzymes used inbioluminescence62 kDa molecular weightpH optimum of 7.8A variety of organisms regulate their light production using different luciferasesbacteriaFirefliesJack-O-Lantern mushroomMetridia longa (marine copepod)Dinoflagellate, etc

Luciferase can be produced in the lab throughgenetic engineeringfor a number of purposes. Luciferasegenescan be synthesized and inserted into organisms or transfected into cells.Mice,silkworms, andpotatoesare just a few organisms that have already been engineered to produce the protein45

Chemical reactionThe chemical reaction catalyzed by firefly luciferase takes place in two steps:

luciferin+ATPluciferyl adenylate+PPiluciferyl adenylate + O2oxyluciferin+AMP+ light

Light is emitted because the reaction forms oxyluciferin in an electronicallyexcited state. The reaction releases a photon of light as oxyluciferin returns to theground state.46Mechanism

StructureThe protein structure of firefly luciferase consists of two compactdomain

The N-terminal domainThe C-terminal domain

USESgene report and detectionRNAi system researchinteraction between proteinscell analysisdetection of Microorganism

Asparaginase

EC 3.-.-.-Hydrolases. [16,495PDB entries]EC 3.5.-.-Acting on carbon-nitrogen bonds, other than peptide bonds. [1,218PDB entries]EC 3.5.1.-In linear amides. [444PDB entries]EC 3.5.1.1Asparaginase.[27PDB entries]50About proteinA tetrameric protein composed of four identical subunits, each subunit contains 326 amino acid residues. The two threonine residues present at the active site are required for activity.Molecular Weight:Monomer: 36.8 kDa Optimal pH:8.0- 8.6

ADVANTAGESTreatment of acute lymphoplastic leukemia.Aspariginase can be used as a food processing aid to reduce the formation of acrylamide, a suspected carcinogen, in starchy food products.

DISADVANTAGESanaphylaxispancreatitis coagulopathy

Mechanism of action as a food processing aidAspariginase can be used as a food processing aid to reduce the formation of acrylamide, a suspected carcinogen, in starchy food products. Acrylamide is a chemical compound that is formed in starchy foods when they are baked or fried. During heating theamino acidasparagine, naturally present in starchy foods, is converted into acrylamide in a process called theMaillard reaction. The reaction is responsible for giving baked or fried foods their brown color, crust and toasted flavor.By adding asparaginase before baking or frying the food, asparagine is converted into another common amino acid,aspartic acid, andammonium. As a result, asparagine cannot take part in the Maillard reaction, and therefore the formation of acrylamide is significantly reduced. Complete acrylamide removal is probably not possible due to other, minor asparagine-independent formation pathways[1].As a food processing aid, asparaginases can effectively reduce the level of acrylamide up to 90% in a range of starchy foods without changing the taste and appearance of the end product[4].[edit]Mechanism of action as a drugThe rationale behind asparaginase is that it takes advantage of the fact that ALL leukemic cells are unable to synthesize the non-essential amino acid asparagine, whereas normal cells are able to make their own asparagine; thus leukemic cells require high amount of asparagine. These leukemic cells depend on circulating asparagine. Asparaginase, however, catalyzes the conversion ofL-asparagine to aspartic acid and ammonia. This deprives the leukemic cell of circulating asparagine.The mainside effectis anallergicor hypersensitivity reaction;Asparaginase has also been associated withpancreatitis. Additionally, it can also be associated with a coagulopathyas it decreases protein synthesis, including synthesis of coagulation factors (eg progressive isolated decrease offibrinogen) and anticoagulant factor (generallyantithrombinIII; sometimesprotein C&Sas well), leading tobleedingorthrombotic eventssuch as stroke.

Anaphylaxisis defined as "a seriousallergic reactionthat is rapid in onset and may cause death".[1]It typically results in a number of symptoms including an itchy rash, throat swelling, and lowblood pressure. Common causes include insect bites, foods, and medications.Pancreatitisisinflammationof thepancreas. It occurs when pancreatic enzymes (especiallytrypsin) that digest food are activated in the pancreas instead of the small intestine. It may beacute beginning suddenly and lasting a few days, orchronic occurring over many years. It has multiple causes and symptoms.Coagulopathy(also calledclotting disorderandbleeding disorder) is a condition in which the bloods ability to clot is impaired. This condition can cause prolonged or excessive bleeding, which may occur spontaneously or following an injury or medical and dental procedures.

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POTENTIALfusion protein of asparaginase-TTP-CETPC could also be useful for the development of a vaccine against atherosclerosis.