ENZYMES AND PROTEINS.pptx

Detailed study of Enzymes and proteins

1. ENZYMES AND PROTEINS

2. ENZYMES Enzymes are organic catalysts produced in the body by living organisms. They perform many complex chemical reactions that make up life processes. Enzymes are lifeless and when isolated, they still exert their characteristic catalytic effect Enzymes are found in combination with inorganic or organic substances that have an important part in the catalytic action.

3. Enzymes are obtained from plant and animal cells and many have been purified. They are used as therapeutic agents and as controlling factors in certain chemical reactions in industry. Pepsin , pancreatin , and papain are used therapeutically as digestants . Hyaluronidase facilitates the diffusion of injected fluids. Streptokinase and streptodornase dissolve clotted blood.

4. Properties of Enzymes 1. Enzymes are sensitive to heat and are denatured by excess heat or cold. 2. Enzymes are sensitive to pH, the rate at which they can conduct reaction is dependent upon the pH of where the reaction is taking place 3. Enzymes are reusable and some enzymes are capable of catalyzing many hundreds or thousands of reactions

5. 1 . PEPSIN: Biological Source : It is the enzyme prepared from the mucous membrane of fresh stomach pig Sus scrofa Linn, belonging to family Suidae .

6. METHOD OF PREPARATION Mucous lining of stomach scrapped off Cut into small pieces Pulp placed in water Add HCL for digestion Kept at 37°C

7. Autolysis takes place Clear liquid is obtained Contain peptones and pepsin Add NaCl NaCl precipitaes pepsin and peptone remain as such

8. Collection of ppts . Suspended in the water in dialyser Removel of salt by dialysis To the aqueous solution, add alcohol to bring ppt of pepsin Collection of ppt Drying and packing

9. Description: Colour : pale yellow Odor: odourless or with very faint odour , translucent grains Taste: slightly bitter It is best active at a temperature of 40°C with pH 2–4. Pepsin is unstable above pH 6. The enzyme gets denatured at a temperature of 70°C and in the presence of alcohol and sodium chloride. Pepsin can be stored for 1–2 years at 2–8°C.

10. Uses : 1. It is used in the deficiency of gastric secretion. 2. Pepsin is also used in the laboratory analysis of various proteins; in the preparation of cheese, and other protein-containing foods.

11. 2. UROKINASE Synonym : Uroquinase . Biological Source: Urokinase is serine protease enzyme isolated from human urine and from human kidney cells by tissue culture or by recombinant DNA technology.

12. Preparation: 1. Urokinase is a fibrinolytic enzyme produced by recombinant DNA using genetically manipulated E. coli cells. 2. It is produced firstly as prourokinase and then converted to active form by plasmin or kallikrein . 3. Urokinase used medicinally is also purified directly from human urine.

13. 4 . It binds to a range of adsorbents such as silica gel or kaolin which can be use to initially concentrate and purify the product. 5. It can be further purified by precipitation with sodium chloride or ethanol or by chromatography. 6. Human urokinase needs sterile filtration, aseptic filling and freeze drying.

14. Description : 1. Urokinase enzyme occurs in two different forms as single and double polypeptide chain forms. 2. It has a half-life of 10–16 minutes after intravenous administration. 3. It converts plasminogen to plasmin

15. Chemical Constituents: 1. Urokinase enzymes are serine proteases that occur as a single low molecular weight (33 kDa ) and double, high molecular weight (54 kDa ) polypeptide chain forms. They differ in molecular weight considerably. Uses : Urokinase is used in the treatment of pulmonary embolism, coronary artery thrombosis and for restoring the potency of intravenous catheters

16. 3.STREPTOKINASE Synonym : Estreptokinase , plasminokinase . Biological Source: It is a purified bacterial protein produced from the strains of β- haemolytic Streptococci.

17. Method of Preparation: 1. Streptokinase is a bacterial derived enzyme of serine protease group. 2. Streptokinase is produced by fermentation using streptococcal culture and is isolated from the culture filtrate. It is produced in the form of a lyophilized powder in sterile vials.

18. Description: Streptokinase is a bacterial protein with half-life of 23 minutes. Chemical Constituents: Streptokinase is the purified bacterial protein with about 484 amino-acid residues.

19. Uses: Streptokinase is the first available agent for dissolving blood clots. Streptokinase and anistreptase are both used in the treatment of pulmonary embolism, venous, and arterial thrombosis and coronary artery thrombosis.

20. 4.BROMELIN Synonyms : Bromelin , bromelain . Biological Source : Bromelin is a mixture of proteolytic enzymes isolated from the juice of Ananas comosus (pineapple), belonging to family Bromeliaceae . Geographical Source: It is grown in almost all parts of the world including India, China, Thailand, United States, Brazil, Philippines, Mexico, Hawaii, and Taiwan.

21. Cultivation, Collection, and Preparation 1. Bromelin is found in pineapple fruit juice and stem. 2. It is propagated through suckers, slips, and crowns. 3. Plantation month is august. 4. Flowering month – February-march 5. fruit ripens during July–October 6. When fruits turns orange then they are cut off. 7. The enzyme bromelin does not disappear as the fruit ripens. The enzyme from fruit and stem are known as fruit bromelin and stem bromelin , respectively. 8. It is isolated from pineapple juice by precipitation with acetone and also with ammonium sulphide .

22. Description: 1. The optimum pH of bromelain is 5.0–8.0. 2. In solution pH below 3.0 and above 9.5 inactivates the enzyme. 3. The optimum temperature is between 50 and 60°C. 4 . The moisture content should not exceed 6%. It is obtained in light brown coloured powder.

23. Chemical Constituents: 1. It is a mixture of sulphur -containing protein-digesting enzymes, called proteolytic enzymes or proteases. 2. It also contains several other substances in smaller quantities, including peroxidase, acid phosphatase, protease inhibitors, and calcium. Uses : 1. Bromelain is an effective fibrinolytic agent; bromelain inhibits platelet aggregation and have anti-inflammatory effect. 2. Enhance healing process of body 3. as a digestive enzyme 4. as an antihypertensive 5. It may even be useful in the treatment of AIDS to stop the spread of HIV.

24. 5.SERRATIOPEPTIDASE Synonym : Serrapeptase , serratiopeptidase . Biological Source: Serratiopeptidase is a proteolytic enzyme isolated from nonpathogenic Enterobacteria Serratia E 15. It is also produced by the larval form of the silk moth.

25. Preparation 1. Serratiopeptidase is produced by fermentation technology by using nonpathogenic enterobacteria species such as Serratia E 15. 2. The larvae of silk moth produce this enzyme in their intestine to break down cocoon walls. It can thus be obtained from the silk moth larvae. Description : 1. When consumed in unprotected tablet or capsule, it is destroyed by acid in stomach. However enteric coated tablets facilitate its absorption through intestine. Chemical Constituents: Serratiopeptidase is a proteolytic enzyme of protease type XXVI .

26. Uses: 1. As an anti-inflammatory enzyme. 2. It is also used as a fast wound healing agent. 3. It is traditionally used to treat rheumatoid arthritis and osteoarthritis. 4. It has wide ranging applications in trauma surgery, plastic surgery, respiratory medicine, obstetric and gynaecology .

27. 6.PAPAIN Synonyms : Papayotin , vegetable pepsin Biological Source Papain is the dried and purified latex of the green fruits and leaves of Carica papaya belonging to family Caricaceae .

28. Description: 1. Colour - white/brown colored amorphous powder 2. Typical odour and taste 3. Soluble in water and insoluble in alcohol 4. Maximum proteolytic activity at pH between 5 to 6.

29. Preparation 1. The latex is obtained by making two to four longitudinal incisions, by stainless steel or wooden sharp knives. 2. The exudate is collected in nonmetallic containers. 3. The latex is pass through sieves to remove dirt and unwanted material. 4. Then to the latex Potassium metabisulphite is added. 5. Mixture is spread on the trays and dried in the vaccum shelf dryer at 55 to 60°C for 4 to 5 hours. 6. The dried latex is called papain.

30. Chemical Constituents : Papain Proteolytic enzyme Peptase (clotting enzyme) Mixture of papain and chymopapain Renin ( used in preparation of cheese) Peptidase-I ( covert protein into dipeptide and polypeptide )

31. Uses: 1. In clarification of beverages 2. It is meat tenderizer. It breakdown protein and making the meat easier to chew. 3. In manufacturing of cheese as a substitute of renin. 4. Deconing of silk febric in textile industry. 5. As dehairing agent in leather industry. 6. Medicinally, anti-inflammatory agent 7. Used as an ingredient in cleansing solution for soft contact lenses

32. Marketed products: 1. Panafil 2. Papase 3. Soft lens enzymatic contact lens cleanser.

33. PROTEINS

34. GELATIN Synonyms : Gelfoam ; puragel ; gelatinum . Biological Source : Gelatin is a protein derivative obtained by evaporating an aqueous extract made from bones, skins, and tendons of various domestic animals. Some important sources are: Ox,  Bos taurus , and Sheep,  Ovis aries  belonging to family Bovidae .

35. Preparation The process of manufacture of gelatin vary from factory to factory. However, the general outline of the process is given below. Raw material Bones, skins, and tendons of Bovideans is collected and subjected to liming operation Liming Process The raw material is first subjected to the treatment known as ‘liming’. In this process, the skins and tendons are steeped for fifteen to twenty and sometimes for 40 days in a dilute milk of lime. During this, fleshy matter gets dis-solved, chondroproteins of connective tissues gets removed and fatty matter is saponified . The animal skin is further thoroughly washed in running water.

36. Defattying : In case of bones, the material is properly ground and defatted in close iron cylinders by treatment with organic solvents such as benzene. The mineral and inorganic part of the bone is removed by treatment with hydrochloric acid.

37. Extraction The treated material from bones, skins and tendons is boiled with water in open pans. The clear liquid runs of again and again and is evaporated until it reaches to above 45 per cent gelatin content.

38. Setting : The concentrated gelatin extract is transferred to shallow metal trays or trays with glass bottom. It is allowed to set as a semisolid jelly. Drying : The jelly is transferred to trays with a perforated wire netting bottom and passed through series of drying compartments of 30–60°C increasing each time with 10°C. About a month is taken for complete drying. Bleaching : In case of darker colour , finished product is subjected to bleaching by sulphur dioxide. Bleaching affords a light coloured gelatin.

39. Description: Color- colorless or pale yellow Odor: Characteristic Taste: Slight and broth like Solubility: insoluble in cold water, but swells and softens when immersed. Soluble in hot water. Insoluble in alcohol, chloroform and ether.

40. Chemical Constituents: Gelatin consists of the protein glutin which on hydrolysis gives a mixture of amino acids. The approximate amino-acid contents are: glycine, alanine, valine , cystine and cysteine the adhesive nature of gelatin is due to the presence of glutin .

41. Uses Gelatin is used to prepare pastilles, pastes, suppositories, capsules, pill-coatings, gelatin sponge; as suspending agent, tablet binder, coating agent, as stabilizer, thickener and texturizer in food; for manufacturing rubber substitutes, adhesives, cements, and printing inks, plastic compounds, artificial silk, photographic plates and films, for inhibiting crystallization in bacteriology, for preparing cultures and as a nutrient.

42. 2.CASEIN Biological Source: Casein is a proteolytic enzyme obtained from the stomachs of calves. It is extracted from the proteins of the milk; in the milk, casein is structured in voluminous globules. These globules are mainly responsible for the white colour of the milk.

43. Characteristics 1. The isoelectric point of casein is 4.6. 2. The purified protein is water insoluble. While it is also insoluble in neutral salt solutions, it is readily dispersible in dilute alkalis and in salt solutions such as sodium oxalate and sodium acetate. 3. Casein does not coagulate on heating. It is precipitated by acids and by a proteolytic enzyme (rennet).

44. Chemical Constituents 1. Milk consists of 80% of milk proteins (casein). 2. The major constituents of casein are alpha (s1) and alpha (s2)-caseins, β-casein and kappa-casein. These caseins are conjugated proteins with phosphate group(s) which are esterified into serine residues they have a low solubility at pH 4.6.

45. Uses 1. It is used in the manufacture of binders, adhesives, protective coatings, plastics (such as for knife handles and knitting needles), fabrics, food additives, and many other products. 2. It is commonly used by bodybuilders as a slow-digesting source of amino acids.