Study of Bromelain, Urokinase, Streptokinase
Study of Bromelain, Urokinase, Streptokinase
Objectives
At the end of the lecture, the students will be able to
• Explain the method of production of Bromelain, urokinase and Streptokinase
• Describe the applications of Bromelain, Urokinase and Streptokinase
Bromelain
Synonym: Ananase, Inflamen
Biological Source: Bromelain is a mixture of proteolytic enzymes obtained from the fruits and stems of Ananas comosus belonging to the family Bromeliaceae
Fruits – 0.08 to 0.125%
Stem – 0.1 to 0.6%
Stalk – 0.04 to 0.06%
Skin – 0.05 to 0.07%
Methods of production:
i. From fruits (Greenderg method):
• Collect the fruits & cut it into small pieces
• Press or grind the pieces to obtain the juice and filter through muslin cloth
• Adjust the pH of the filtrate to 6
• Add ammonium sulphate [(NH4)2SO4] until saturation to precipitate bromelain
• Precipitate of bromelain is obtained
• Partially purified by dissolving in sodium cyanide and repeatedly precipitating with 0.6% of ammonium sulphate & then with acetone
• Precipitate – washed with acetone and ether and dried by using vacuum or spray drying
ii. From stems:
• Collect the stems and make free of leaves, then cut it into small pieces
• Stems were collected after final fruit harvest and freed sucker stems from leaves
• Press or grind the pieces to obtain the juice and filter
• Adjust the pH of the filtrate to 6
• Acetone is used to precipitate the enzyme
• Add two volume of acetone to two volume of filtrate
• Precipitate obtained is discarded because of low enzymatic activity, poor colour and poor stability
• Add one volume of acetone to one volume of filtrate
• Enzyme fraction was centrifuged – 20,000 g for 25 minutes
• Collect the precipitate and wash it with acetone and ether & dried in vacuum oven
Properties:
• White / slightly yellowish white / buff colored powder
• Odourless with irritating taste
• Slightly soluble in water, insoluble in organic solvents like ether, chloroform etc.,
• Exhibits its maximum activity at pH 5.5-6.5
• Fruit Bromelain is acidic & stem is basic in nature
Uses:
• Anti-inflammatory and in the treatment of oedem
• To tenderize meat
• Used for dehairing of skin in leather industry
• Anti-arthritis
• Anti-gout
• In the treatment of Upper Respiratory Tract Infections
Activity of Bromelain:
• Determined by Milk Clotting Unit (MCU)
• One MCU is the amount of enzyme required to clot the milk in one test tube in 1 min.
Method:
• 10 g of dried milk powder – diluted to 100 ml with acetate buffer at pH 4.6
• Solution of Bromelain is prepared by triturating the crude enzyme in to a smooth paste with distilled water,
• Prepare different dilutions of bromelain solution
• To 1 ml of each dilutions, add 10 ml of milk preparation and incubate at 40oC and note down the time required to clot the milk
• Determine the concentration for the activity by using the formula
E=K/t
Where E = Enzyme concentration; K = Constant; t = time (min)
Streptokinase
• Fibrinolytic enzyme secreted in several species of Streptococci
• Used as thrombolysis medication – myocardial infarction and pulmonary embolism
Mechanism of action
Streptokinase binds – plasminogen – activate
â
Plasmin
â
Degrades various blood clotting factors including fibrin
â
Lysis of fibrin
Method of Production
• Preparation and sterilization of media
• Inoculation of medium
• Purification of streptokinase
Preparation and sterilization of media
• Casein digest media – ß haemolytic Streptococci
• Casein digest solution – dissolving 10.17 kg of caesin in 50 lts of distilled water – heat to 100˚C, continue heating till clear solution and Cool to 15˚
• Filter, add small quantity of toluene as preservative and store for 4 days at 2˚C
• Again filter to remove any insoluble impurities and add the following nutrients
– Macronutrients: Pottassium dihydrogen phosphate, KHCO3 in distilled Water
– Micronutrients: Manganese chloride, Zinc sulphate, Copper sulphate, Ferrous sulphate
– Amino acids: Glycine, Cysteine, Tryptophan
– Vitamins: Riboflavin, Nicotinic acid, Calcium pentothionate
– Miscellaneous: Uracil, Adenine sulphate, Thioglycollic acid, 50 % Dextrose
• Adjust pH to 7.8 and sterilise by membrane filtration method
Inoculation of Media
• Sterlized media is inoculated with bacterial suspension – 20 billion/ml bacterial count
• Fermentation at 37 ˚C – no pH adjustment – 14 hrs
• Then adjust pH with 5N NaOH and add 50 % dextrose at 15 min interval
• Repeated after 8 hrs
• When the growth of bacteria ceases, stop the fermentation
• Fermented medium – 1000/ units/ml
Purification of Streptokinase
Dialysis
• Phosphate buffer – outer solution
• Contents of cellulose bag – column chromatography – cellulose as adsorbent
• Eluted with phosphate buffer in the order of molarity of 0.005M to 1M) and pH (5.5-8.5)
• pH 8 and 0.75 M – impurities elute out from column
• pH 8 and 1 M – streptokinase elute out, treated with phosphate buffer and cellulose – stir for 1 hr and filter
• Filter cake is suspended in 0.1 M phosphate buffer for 1hr and filter
• Concentrated to obtain pure streptokinase
Streptokinase Applications
• Act as fibrinolytic agent – acute myocardial infarction
• In vein thrombosis
• Treatment of bone abscession
• Treatment of burns
Urokinase
• Urinary plasminogen activator (uPA or u-PA)
• Originally isolated from human urine
• Cultured human kidney cells
• Deep venous thrombosis, pulmonary embolism, myocardial infarction
Method of production
• Precipitation of urokinase
• Adsorption on benzoic acid crystals
• Treatment with acetone
• Adsorption on celite 505
• Treatment with phosphate buffer
• Ion exchange chromatography and dialysis
Urokinase – Production
1. Precipitation
Collect 20lts of human urine
â add 1.2 kg sodium benzoate + 2 lts dil. HCl – pH 4.5
Precipitation occurs
â Stir – 20 min
Allow to stand for 30 min
2. Adsorption on benzoic acid crystals
• Filter the suspension through Buchner’s funnel precoated with benzoic acid crystals
• Filter cake – weigh 2 kg – benzoic acid + urokinase
3. Treatment with acetone
• Acetone – 1.5 times weight of filter cake
• Acetone dissolves benzoic acid crystals and urokinase
flocculates out
• Add 1.5 times weight of sodium benzoate to enhance the rate of precipitation
4. Adsorption on Celite 505
• Pass the suspension through celite 505 precoated on buchner funnel
• Celite adsorbs urokinase
• Celite adsorbate washed with acetone and ether
• Adsorbate weighs around 2.2 g
5. Treatment with phosphate buffer
• Many batches of urokinase are prepared and pooled to get 25 gms
– filter cake
• Dissolve in 750 ml 0.1M phosphate saline buffer
• Celite 505 is removed by centrifugation
• Residue is again treated with phosphate buffer and centrifuged
• Combine both supernatent liquids
6. Ion exchange chromatography and dialysis
• Amberlite XE-64 ion exchange resin
• Column – eluted – 5N sodium chloride solution
• Eluent obtained from column is subjected to dialysis through cellulose membrane, distilled water as outer solution
• Contents of dialysis bag – Lyophilization
• Yield – 166 units/mg
1 unit Urokinase: Amount of urokinase required to induce sufficient fibrinolysis to dissolve 1 ml of 0.1% bovine clot in 10 min at 37˚C in physiological solution buffered with phosphate at pH of 2
• Used to dissolve blood clot in acute myocardial infarction
• Deep vein thrombosis
• In the treatment of Angina pectoris
Summary
• Bromelain – proteolytic enzymes, Ananas comosus, Bromeliaceae
• Activity of bromelain – MCU
• Streptokinase – Fibrinolytic enzyme – secreted – several species of streptococci –
• Used – thrombolysis medication – myocardial infarction and pulmonary embolism
• Urokinase – Urinary plasminogen activator (uPA or u-PA)
• Originally isolated from human urine
• Deep venous thrombosis, pulmonary embolism, myocardial infarction