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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 40^oC 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

Plasmin

Degrades various blood clotting factors including fibrin

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

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