Study of Bromelain, Urokinase, Streptokinase

Study of Bromelain, Urokinase, Streptokinase

Study of Bromelain, Study of Urokinase, Study of 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

biological source of 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

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

                   Dietary Supplement and Nutraceuticals