BROMELAIN 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 3.4.22.32 Fruit bromelain - EC 3.4.22.33 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 temperature Optimal temperature Deactivation temperature 40-60 °C 50-60 °C above65 °C approx. α HELIXES AND HELICAL TURNS 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. 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. 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 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 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 DIASTASE ENZYME. DIASTASE Alpha amylase Diastase are any one of a group of enzymes which catalyses the breakdown of starch into maltose. first enzyme discovered. diastase It was extracted from malt solution in 1833 by Anselme Payen and Jean-François Persoz, chemists at a French sugar factory. Gamma Beta The name "diastase" Greek (diastasis) amylase comes from theamylase (a parting, a separation) ALPHA AMYLASE EC NUMBER: 3.2.1.1 is 1,4-a-D-Glucan glucanohydrolase • Ph-5.9-6.6 Conditions ALTERNATIVE NAME : glucogenase • Temperature opt for =37’C Location: it is secreted in saliva and pancreas, found in amylase 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. Chloride ion and Metal ions and activators Causes hydrolyses alpha-bonds of largeion alpha-linked bromide polysaccharides, such as starch and glycogen, yielding • Most effective. glucose and maltose. 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)8barrel. 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 βstrands. 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 AMYLOSE IN STARCH GLUCOSE RESIDUE CLEAVED BY 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. Trypsinogen is activated by removal of a terminal hexapeptide to yield singlechain 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 ALKALINE PHOSPHATASE 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 including Intestinal (ALPI), Chromosome 2 Placental (ALPP) Liver/bone/kidney (ALPL) Chromosome 1 It belongs to Alpha and Beta class of proteins 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. General Mechanism I Alkaline phosphatase binds substrate. Only the phosphate moiety of the substrate binds specifically to the enzyme. II A nucleophile attacks the phosphorous atom of the phosphate and induces cleavage of the oxygen-phosphorous bond. At alkaline pH, a nucleophilic hydroxide (from an active site water molecule) attacks the covalently bound phosphate, The noncovalently bound phosphate IV molecule is slowly released from the enzyme. III 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 Alkaline Phosphatase Test One of the most important functions of alkaline phosphatase is as an indicator for disease. High levels of alkaline phosphatase • bones, liver, bile system or malignancies,blood, reproductive tissues and GI tract Low levels of alkaline phosphatase • hypophosphatasia,malnutrit ion, celiac disease, magnesium and zinc deficiency, anemia PEPSIN Classification EC number 3.4.23.3 Member of the aspartate protease family First animal enzyme to be discovered Second to be crystallized Discovery – Theodor Schwann Northrop Structure: Two aspartate molecules at the active site Three sulphide bridges “A tricky business” PEPSINOGEN - primary structure has an additional 44 amino acids Released by chief cells in the stomach HCL causes activation Pepsinogen → pepsin TARGETS: (Amide autocatalysis in aromatic acidic env) bonds of amino acids like tryptophan, phenylalanine and tyrosine Phenylalanine Tryptophan 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 PAPAIN Papaya Proteinase I Cysteine protease hydrolase Enzyme extraction papaya fruit is first slit at the neck. latex that drips is either collected in a container or left to dry on the fruit latex that is collected is further allowed to dry into a crude format. material needs to undergo a purification process to get rid of all the contaminating products present in it. Family & structure Source: present in papaya (Carica papaya) and mountain papaya(Vasconcellea cundinamarcensis). Cysteine protease (EC 3.4.22.2) 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. 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 deprotonated cysteine on substrate carbonyl atom 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 Cellulase Introduction (source) 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. 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. Other Names Other names for 'endoglucanases' are: endo1,4-beta-glucanase, carboxymethyl cellulase (CMCase), endo-1,4-beta-D-glucanase, beta1,4-glucanase, beta-1,4-endoglucan hydrolase, and celludextrinase. The other types of cellulases are called exocellulases. Types of reactions/ Classification General types of cellulases based on the type of reaction catalyzed: 1. Cleaves internal bonds at Endocellulase (EC 3.2.1.4) randomly amorphous sites that create new chain ends. 2. Cellobiase (EC 3.2.1.21) or betaglucosidase hydrolyses the exocellulase product into individual monosaccharides. 3. Cellulose phosphorylases depolymerize cellulose using phosphates instead of water. Uses 1. Cellulase is used for commercial food processing in coffee. 2. It performs hydrolysis of cellulose during drying of beans. 3. Furthermore, cellulases are widely used in textile industry and in laundry detergents. 4. They have also been used in the pulp and paper industry for various purposes, and they are even used for pharmaceutical applications. 5. Cellulase is used in the fermentation of biomass into bio fuels, although this process is relatively experimental at present. 6. Cellulase is used as a treatment for phytobezoars, a form of cellulose bezoars found in the human stomach.