WHAT ACTUALLY IS BROMELAIN…?? - PowerPoint PPT Presentation

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  1. WHAT ACTUALLY IS BROMELAIN…?? • Mixture of protein digesting enzymes known as proteolytic enzymes or proteases – include several other substances as well • either of two proteases extracted from plant family bromeliaceae i.e., Stem bromelain - EC Fruit bromelain - EC • May also refer to a combination of those enzymes along with other compounds produced in an extract • Referred to as sulfhydryl proteases since a free sulfhydral group of a cysteine side chain is essential • The other substances typically include peroxidase, acid phosphatase, protease inhibitors, and calcium β COILS α HELIXES AND HELICAL TURNS temperature 40-60 °C Optimal temperature 50-60 °C Deactivation temperature above65 °C approx. Effective pH 4.0-8.0 Optimal pH 4.5-5.5 Molecular weight 28.4 kD

  2. PEEK IN TO THE PAST… • First isolation  Vicente Marcano in 1891 from fruit of pineapple. • In 1892, Chittenden, Joslin and Meara investigated the matter fully and named it ‘Bromelin’ • Later, Bromelian was introduced and orignally applied to any protease from any member of family Bromeliaceae. • In 1957 first introduced as therapeutic supplement • Pioneer 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.

  3. IT COMES FROM… • Pineapple plant (Ananas sp.) • Stem  most common commercial source • Traditionally 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.

  4. ROLE OF THE STUD… • Bromelain bloods fibrolytic activity and kininogen and bradykinin serum and tissue levels as well as reduce excretion of proinflammatory cytokines and chemokines • Also effects prostaglandin synthesis • Inhibits fibrinogen synthesis • Directly degrades fibrin and fibrinogen • cleave 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

  5. PAY BACK TIME… • product name  ‘Ananase’ • Various uses in Folk medicine • Explored as a potential healing agent in alternative medicine. • Work by blocking some proinflammatory metabolites when applied topically • Used for reducing swelling • Involved in the migration of neutrophils to the site of acute inflammation. • Used for treating arthiritis • When used in conjunction with trypsin and rutin is as effective as prescribed analgesics in the osteoarthiritis management. • Meat tenderizing

  6. WHAT ELSE…?? Other effects include: • Hay fever • Treating a bowel condition that includes swelling and ulcer ulcerative colitis • Removing dead and damaged tissue after a burn debridement • Preventing the collection of water in the lung pulmonary edema • Relaxing muscles • Improving the absorption of antibiotics • Preventing cancer • Shortening labor • Help the body in reducing fats • Supplement may effect heart rate • systemic enzyme therapy

  7. DIASTASE • 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 AnselmePayen and Jean-François Persoz, chemists at a French sugar factory. • The name "diastase" comes from the Greek (diastasis) (a parting, a separation)

  8. ALPHA AMYLASE • EC NUMBER: is 1,4-a-D-Glucan glucanohydrolase • ALTERNATIVE NAME : glucogenase • Location: 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.

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


  11. INDUSTRIAL 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 grains • Checking out pancerititis the amylase levels are measured in the pancertic cells.


  13. HISTORY & SOURCE*** Pancreas TRYPSINOGEN TRYPSIN Bovine Pancreas expresses two forms of trypsin: dominant cationic form minor anionic form These protein sequences share 72% identity, while their coding regions share 78% identity. • 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

  14. CONVERSION*** TRYPSINOGENTRYPSIN {Ph 9.3} {ph 10.5} Trypsinogen is activated by removal of a terminal hexapeptide to yield single-chain β-trypsin. Limited autolysis produces other active forms having two or more peptide chains bound by disulfide bonds. Predominant forms are *α-trypsin, having two peptide chains and *β-, a single chain

  15. REACTION CATALYZED*** Cleavage occurs within the polypeptide chain rather than at the terminal amino acids located at the ends of polypeptides. Process catalyzed by trypsin *Trypsin Proteolysis* {Trypsinisation} Trypsin is considered as an endopeptidase* TRYPSIN SERINE PROTEASE HYDROLYZE PROTEINS

  16. USED FOR*** • Tissue dissociation • Mitochondria isolation • in vitro studies of proteins • Various hemagglutination procedures • DNA Fingerprinting • Environmental monitoring • Reduction of cell density in tissue culture • Cleavaging fusion proteins • Generating glycopeptides from purified glycoproteins

  17. What is Alkaline Phosphatase? • Alkaline phosphatase (EC 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 including • Intestinal (ALPI), Chromosome 2 • Placental (ALPP) • Liver/bone/kidney (ALPL) Chromosome 1 • It belongs to Alpha and Beta class of proteins

  18. STRUCTURE • Alkaline 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.

  19. I II General Mechanism III IV

  20. Properties AND FUNCTION • This enzyme was partially purified and studied by Kunitz (1960) • It is a hydrolase enzyme found in bacteria and mammals • Optimum pH: 8 – 9 • Activators: Mg2+ • Wide specificity • Inhibitors: 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 reaction • A phosphate monoester + H(2)O an alcohol + phosphate

  21. Alkaline Phosphatase Test One of the most important functions of alkaline phosphatase is as an indicator for disease.

  22. PEPSIN Classification EC number3.4.23.3 Member of the aspartate protease family First animal enzyme to be discovered Second to be crystallized Discovery – Theodor Schwann Northrop

  23. Structure: Two aspartate molecules at the active site Three sulphide bridges

  24. “A tricky business” Phenylalanine Tryptophan

  25. Activity and Stability: Temperature: 37°C-42°C pH: 1.5 – 2 Stable until pH 8- can be reactivated upon re- acidification Deficiency: Imbalance in pH Inability to digest protein

  26. Papaya Proteinase I Cysteine protease hydrolase PAPAIN

  27. Enzyme extraction

  28. Family & structure • Source: present in papaya (Carica papaya) and mountain papaya(Vasconcelleacundinamarcensis). • Cysteine protease (EC enzyme  • Family: members found in baculovirus, eubacteria, yeast, and practically all protozoa, plants and mammals, lysosomal or secreted • contains 345 amino acid residues, and consists of a signal sequence (1-18), a propeptide (19-133) and the mature peptide (134-345). The amino acid numbers are based on the mature peptide. The protein is stabilised by three disulfide bridges.

  29. Mechanism of action • mechanism by which it breaks peptide bonds involves deprotonation of Cys-25 by His-159 • 1. Deprotonation of thiol in cysteine by basic histidine • 2. Nucleophilic attack by deprotonatedcysteine on substrate carbonyl atom

  30. applications • The 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

  31. Introduction (Cellulase) Cellulase refers to an entourage of enzymes produced chiefly by fungi, bacteria and protozoans that catalyze cellulolysis (i.e. the hydrolysis of cellulose). However, there are also cellulases produced by a few other types of organisms, such as some termites and the microbial intestinal symbionts of other termites. Several different kinds of cellulases are known, which differ structurally and mechanistically.

  32. Complete vs. incomplete cellulases • 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.

  33. Types of reactions/ Classification General types of cellulases based on the type of reaction catalyzed: • Cleaves internal bonds at Endocellulase (EC randomly amorphous sites that create new chain ends. • Cellobiase (EC or beta-glucosidase hydrolyses the exocellulase product into individual monosaccharides. • Cellulose phosphorylasesdepolymerize cellulose using phosphates instead of water.

  34. Uses • Cellulase is used for commercial food processing in coffee. • It performs hydrolysis of cellulose during drying of beans. • 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 into bio fuels, although this process is relatively experimental at present. • Cellulase is used as a treatment for phytobezoars, a form of cellulose bezoars found in the human stomach.

  35. Succinyl coenzyme A synthetase Succinyl coenzyme A synthetase is an enzyme that catalyzes the reversible reaction of succinyl-CoA to succinate.

  36. Source • Bacteria e.g.E.coli • Mammals

  37. Chemical Reaction • SuccinylCoAsynthetase catalyzes the following reversible reaction: • SuccinylCoA + Pi + NDP ↔ Succinate + CoA + NTP Succinyl CoA succinate

  38. Mechanism The 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 of CoA from succinyl CoA by a nucleophilic inorganic phosphate molecule to form succinyl phosphate. The enzyme then utilizes a histidine residue 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.

  39. Mechanism

  40. Uses Succinyl-CoA synthetase plays a key role in • the citric acid cycle • ketone metabolism • heme synthesis

  41. Urokinasesources SOURCE ORGANISM • Human urine.  • much lower concentrations in human plasma. • Other organism may include rat, mouse, yeast etc. SOURCE TISSUE • Ovary • produced by kidney cells. • produced by a variety of tumor cells and involved in the formation of tumor metastasis. • Phagocytic cells

  42. MOLECULAR CHARACTERISTICS • 411-residue protein • three domains: 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 disulphide bond. • found in multiple molecular sizes. Low molecular weight (33-KDa) and high molecular weight (57-KDa). • Urinary Urokinase contained predominantly the LMW form

  43. REACTION CATALYSED PLASMINOGEN + H20  PLASMIN • Specific cleavage of Arg-Val bond in plasminogen to form plasmin. • Reaction type: Hydrolysis of peptide bond

  44. USES • used clinically for therapy of thrombotic disorders • used in medicine to dissolve blood clots. • employed in clinical medicine in the treatment of acute myocardial infarction and arterial blood clots in the legs and arms. • Used in peritoneal dialysis.

  45. LuciferaseSource • Luciferase is a generic term for the class of oxidative enzymes used in bioluminescence • 62 kDa molecular weight • pH optimum of 7.8 • A variety of organisms regulate their light production using different luciferases • bacteria • Fireflies • Jack-O-Lantern mushroom • Metridialonga (marine copepod) • Dinoflagellate, etc

  46. Chemical reaction The chemical reaction catalyzed by firefly luciferase takes place in two steps: luciferin + ATP → luciferyladenylate + PPi luciferyladenylate + O2 → oxyluciferin + AMP + light

  47. Mechanism

  48. Structure • The protein structure of firefly luciferase consists of two compact domain • The N-terminal domain • The C-terminal domain

  49. USES • gene report and detection • RNAi system research • interaction between proteins • cell analysis • detection of Microorganism

  50. Asparaginase