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Even though Papain has wide application in many industries still there are very few industries, which are producing Papain so this paper helps to understand the complex procedure of refined Papain production in simplest possible way.

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iotechnology has s-pread its wings in the 21 st century and its allied products are playing a major role in chemical industry. The enzymes catalyze many reactions and make complex procedures simple. With the enzymes taking over harmful chemicals they also help to save environment. The enzyme 'Papain' has many applications. Isolation of Papain is done from ample amount of natural fruit papaya. Even though Papain has wide application in many industries still there are very few industries. which are producing Papain so this paper helps to understand the complex procedure of refined Papain production in simplest possible way. With the high quality refined Papain production right from initial plant setup.

orientation of the catalyzed substrates. Like any catalyst, enzymes work by lowering the activation energy of a reaction, thus allowing the reaction to proceed to its steady state or completion much faster than it otherwise would; the enzyme remains unaltered by the completed reaction and can therefore continue catalysis. Enzyme classification EC 1 Oxidoreductases: catalyze oxidation/reduction reactions EC 2 Transferases: transfer a functional group (e.g. a methyl or

phosphate group) EC 3 Hydrolases: cara lyz ej hydrolysis of various bonds EC 4 Lyases: cleave various bon by means other than hydrolys is oxidation EC 5 Isomerases: catalyz isomerization changes within single molecule EC 6 Ligases: join two molecul with covalent bonds. In EC 3 Hydrolases class fallin Enzyme occurs, Papain Pepsin Pancreatin Ox Bile Serratiopeptidase. Papain Papain is botanical name of th latex extracted from the papaya. It i a cysteine protease (EC 3.4.22.2 present in papaya (Carica papaya! which is useful in tenderizing meat an other proteins. It consists of 21 amino acids stabilised by rhre disulphide bridges.

Enzyme An enzyme (in Greek en = in and zyme = leaven) is a protein, or protein complex. that catalyzes a chemical reaction and also controls the 3D
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Isolation of paJlain: Papain is obtained by cutting the skin of the unripe but almost mature papaya and then collecting and drying the latex. which flows from the cuts. The isolation process is explained in detail in manufacturing methods .

Properties

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Alternate names = Papayapeptidase I. Specificity = Cleaves somewhat nonspecificallyat exposed residues . Source = Carica papaya latex. Molecular weight =23.000 Storage conditions = Store at 4e. Inhibitors = heavy metals. carbonyls. NEM. p-chloromercurobenzoate. Extinction coefficient = E196278 = 25.0 Isoelectric point: pH 9.6 Activators: Papain is activated by cysteine. sulphide. sulphite. etc. It is enhanced when heavy metal binding agents such as EDTAare also present. Kirschenbaum (1971) indicated that N-bromosuccinimide enhances the activity. Stability. Papain as a crystalline suspension is stable at 5C for 6-12 months. Stabilizing agents are EDTA. cysteine and dimercaptopropanol. Reaction conditions = pH 6-7. 20-

mm risk juices and starch from the fruit pulp mixing with the latex. which lowers the, quality. Fruits should be tapped at intervals of about 4-7 days and for the first tapping it is usually sufficient to make only one cut. On subsequent tapings the two or three cuts are spaced between earlier ones (as explained above). After about 4-6 minutes the flow oflatex ceases. A dish is used to collect the latex and the latex is then scraped into a polythene lined box with a close fitting lid; such a box should be stored in the shade. The use of a close fitting lid and keeping the box in the shade are both important because they reduce the reactions. which cause the loss of enzyme activity. Foreign matter such

will cause burning. Neither sh~ come into contact with heavy ''such as iron. copper or brass causes discoloration and los', activity. Pots. knives and sp" should not be used unless they are I" from plastic or stainless steel. latex does not keep well and s be dried to below 5 per cent mol (when it will have a dry and c texture) as soon as possible. After two or three months the are ripe and should be removed the tree. The ripe fruits are edible have very little sale value because their scarred appearance. However. skin of the green ripe papaya d contain about 10 per cent pectin ( weight) and the fruits could processed to extract this.

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Step 1 - Collection and extraction of papaya latex: Papain is obtained by cutting the skin of the unripe but almost mature papaya and then collecting and drying the latex. which flows from the cuts. Tapping of the fruit should start early in the morning and finish by mid-late morning (i.e. during periods of high humidity). At low humidity the flow of latex is low. Two or three vertical cuts (except the first cut) 1-2 mm deep are then made. meeting at the base 9fthe fruit. The incisio'ns are made using a stainless steel razor blade set into a piece of rubber attached to a long stick. The blade should not protrude more than about 2 mm as cuts deeper than 2
401 June 2007

Ribbon structure of papain

as dirt and insects in the latex should be avoided. Latex adhering to the fruit should be carefully scraped off and transferred to .the collecting box. However. dried latex should not be mixed with fresh latex as this lowers the quality. When handling fresh latex. care should be taken to ensure that it does not come into contact with skin as it

Manufacturing

process

Step 2 - Papain (IP) 1) Papaya latex is mixed wit potassium metabisulphate (K2S20S)' 2) Sieved to remove extranecv materials and then blended wit activated z e oca r b catia exchange resin. 3) The mixture is then centrifugt> to separate out the resin from tll
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In the second method to determine the activity of a papain sample. a known amount of papain sample is mixed with a fixed amount of casein (the protein found in milk). The reaction is allowed to proceed for 60 minutes at 40C. After this time. the reaction is stopped by-the addition of a strong acid. The product of the reaction is known as tyrosine. which is known to absorb ultra-violet light (invisible to the human eye). The solutions containing the tyrosine are prepared for analysis using the spectrophotometer. The amount of ultra-violet light absorbed by the solution can be related to the number of tyrosine units produced by the papain sample. Hence, the greater this number, the greater the activity of the papain sample. Applications Papain has a wide application in different industries, especially in food industry. 1) It is being used to tenderize meat and meat products, 2) Textile industry and tanning industry. 3) In the manufacture of protein hydrolysate, 4) In confectionery industry (to prepare chewing gum) 5) In brewing industry to remove cloudiness in beer, 6) In dairy industry (for cheese). Similarly, 7) It is used in pharmaceutical industry. 8) It is further used in aroma and perfume industries and also for effluent treatments. Case-l "Papain in tenderizing meat:" The wide application of papain is in tenderising meat. The relative effects of an aspartic proteinase (AP) and papain cn meat proteins and beef tenderness were evaluated by measuring release of hydroxyproline in collagen, and breakdown of myofibrillar proteins. Tenderness was objectively measured byWarner-Bratzlershear. AP <show ed self-limiting hydrolysis of myofibrillar proteins resulting in 25 to 30 per cent improvement in meat tenderness and was not adversely
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affected by pH, salt. phosphate, and ascorbate concentrations often encountered in meat processing. Like papain, its tenderizing effect was expressed primarily during cooking and caused no ignificant changes (p > 0.05) in tenderness during frozen or refrigerated storage. It was also inactivated at cooking temperatures in excess of 60C, therefore eliminating any undesirable side effects that may Manufacturing process be asso.ciated with residual protease activity. Case-2 "papain in' textile industry": In textile industry the washing of the fabric is done after giving it the desired size and shape, in the washing of the fabric papain is used as papain is hydrolase class enzyme and its main function is in breaking the molecules. While washing the fabric papain reacts with the stains on the fabric and breaks the molecules i.e., it breaks the bond between two molecules and due to which stains are washed out from the fabric and we get clean and glossary fabric for direct sell. World trade in papain The principal producers of crude papain are Zaire, Tanzania, Uganda and Sri Lanka.Most of the spray dried papain comes from Zaire. The principal importing countries are the United States, Japan, United Kingdom, Belgium and France. Almost aU the best quality papain goes to the United States.

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Crude papain is used, in Sri the brewing industry for chill p: beer and lager. However, the inci 'trend for additive free beers in by other European countries is effect in Britain and so this mal papain is declining. Another I papain is in the meat industry tenderization of meat an production of meat tende powders.

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Abhijit Rathi is student 01 ! Vidyapeeth University, Co! Enginl Pune - 43 and S V Godekor i Sharati Vidyapeeth Uni: College of Engineering. Pur
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liquefied latex. 4) The latex is again mixed with activated anion exchange resin. blended and centrifuged. 5) The liquefied latex is spread in trays and dried in vacuum shelf drier. and mixed with potassium metabisulphate. 6) Ground and diluted with lactose to get IP grade papain. Papain concentrate 1) Papaya latex is mixed with potassium metabisulphate and distilled! de-mineralized water. 2) Stirred thoroughly and stored in cold room for settling. 3) The supernatant liquid is siphoned off. filtered and the clear filtrate is mixed with denatured spirit Plant Parameters Capacity, TPA No. of Shifts / day Working days / Yr. Total land Area, m2 Covered Area, m2 9 1 300

and allowed to settle in cold room. 4) The supernatant liquid is decanted from the slurry. and the slurry is filtered. 5) The filtered cakes are washed with rectified spirit and dried first with compressed air and then in vacuum self dryer. 6) The dried material is mixed with potassium metabisulphite. 7) Ground and diluted with lactose to get concentrate grade papain. Enzyme activity If papain is to be exploited commercially for an export market or local food industry use. it is important to be able to determine the enzyme activity. The method is known as assaying. The assaying could be carried out by. for example. the National Standards office. Papain is used to hydrolyse (or breakdown) proteins. Therefore assays to measure papain activity are based on measuring a product of the hydrolysis. There are two main assay methods. The methods are: The first method relies on the ability of papain to clot milk. It is a low cost method but is time consuming. Also the lack of a standard method to find the clotting point and variations in the milk powder used can introduce errors. In this method a known amount of papain sample (made by dissolving a known weight of papain in a known volume of a solution of acetic acid) is added to a fixed amount of milk (made by dissolving a known weight of milk powder in a known volume of water) which has been warmed to 30 C in a water bath. The contents are thoroughly mixed and then observed until the first signs of clotting (formation of lumps) are detected. The time taken to reach this stage. from when the papain was added to the milk. is recorded. The experiment is then repeated using different known amounts of papain solutions. The
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Material Latex Pot. Metabisulphate Lactose powder Denatured spirit Zeocarb, Ltr Manpower ManaQerial Skilled Unskilled Utilities Power,KwH Water, GL

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Presumed plant parameters

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different amounts of papain used should give a range of ( times between 60 and 300 sece optimum results. The activity of the papain sa then calculated by plotting a finding the time taken to clot an infinite concentration of pap then using that value in a fon calculate the activity. To intrcduce a rnea s t standardization the amount of n be fixed at a certain concentration. This is done by r a known concentration of higl papain with the milk. The concentration of milk I solution can then be adjusted te the desired clotting time unde reaction conditions. The 'acti pure papain' at this known am milk can then be calculated. Test sample papain under the same r conditions and sam.e (known) c of milk will then give an a relative to the pure papain. The second method is based science of the absorption 0 known as absorptiometry. This analytical technique for measur amount of radiation (or 'colour'( absorbed by a chemical solutio known that. for example. a 'i coloured solution will absor light. (Blue is the 'complement to yellow). The greatel concentration of yellow colour solution the more is the absorp blue light. This is a useful dis because certain products of ch reactions are colored. The more i the colour is. greater the concen of product. Therefore. by shini relevant complement colour tl the sample liquid the amount ( absorbed can be related t concentratkm of product. Not all 'colours' (or radiati light) are visible to the human e1 technique used when the 'Colours' beyond the visible spectrum is as spectrophotometry anc instrument used is call spectrophotometer.