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Manufacturing process o f 'Papain: Even though Papain has wide application in many industriesstill there are very few industries, which are producing Papain

so this paper helps to understand the complex procedure ofrefined Papain production in simplest possible way.

Biotechnology has s-pread itswings in the 21st century andits allied products are playing amajor role in chemical industry. Theenzymes catalyze many reactions andmake complex procedures simple. Withthe enzymes taking over harmfulchemicals they also help to saveenvironment.

The enzyme 'Papain' has manyapplications. Isolation of Papain isdone from ample amount of naturalfruit papaya. Even though Papain haswide application in many industriesstill there are very few industries.which are producing Papain so thispaper helps to understand the complexprocedure of refined Papainproduction in simplest possible way.With the high quality refined Papainproduction right from initial plantsetup.

orientation of the catalyzed substrates.Likeany catalyst, enzymes work by

lowering the activation energy of areaction, thus allowing the reaction toproceed to its steady state or completionmuch faster than it otherwise would; theenzyme remains unaltered by thecompleted reaction and can thereforecontinue catalysis.Enzyme classification EC 1 Oxidoreductases: catalyze

oxidation/reduction reactions EC 2 Transferases: transfer a

functional group (e.g. a methyl or

EnzymeAn enzyme (in Greek en = in and

zyme =leaven) is a protein, or proteincomplex. that catalyzes a chemicalreaction and also controls the 3D

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phosphate group) EC 3 Hydrolases: cara lyzej

hydrolysis of various bonds EC 4 Lyases: cleave various bon

by means other than hydrolys isoxidation

EC 5 Isomerases: catalyzisomerization changes withinsingle molecule

EC 6 Ligases: join two moleculwith covalent bonds.In EC 3 Hydrolases class fallin

Enzyme occurs, Papain Pepsin Pancreatin Ox Bile Serratiopeptidase.Papain

Papain is botanical name of thlatex extracted from the papaya. It ia cysteine protease (EC 3.4.22.2present in papaya (Carica papaya!whichis useful in tenderizing meat another proteins. It consists of 21amino acids stabilised by rhredisulphide bridges.

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Isolation of paJlain: Papain isobtained by cutting the skin of theunripe but almost mature papaya andthen collecting and drying the latex.which flows from the cuts. Theisolation process is explained in detailin manufacturing methods .

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Properties of PapainAlternate names = Papayapeptidase I.Specificity = Cleaves somewhat

nonspecificallyat exposed residues .Source =Carica papaya latex.Molecular weight =23.000Storage conditions =Store at 4e.Inhibitors = heavy metals.

carbonyls. NEM. p-chloromercuro-benzoate.Extinction coefficient = E196278= 25.0Isoelectric point: pH 9.6Activators: Papain is activated by

cysteine. sulphide. sulphite. etc.It is enhanced when heavy metalbinding agents such as EDTAare also

present. Kirschenbaum(1971)indicatedthat N-bromosuccinimide enhances theactivity.

Stability. Papain as a crystallinesuspension is stable at 5C for 6-12months. Stabilizing agents are EDTA.cysteine and dimercaptopropanol.Reaction conditions =pH 6-7. 20-

3rC

Manufacturing methodsStep 1 - Collection and extractionof papaya latex: Papain is obtained by

cutting the skin of the unripe butalmost mature papaya and thencollecting and drying the latex. whichflows from the cuts. Tapping of thefruit should start early in the morningand finish by mid-late morning (i.e.during periods of high humidity). Atlow humidity the flow of latex is low.Twoor three vertical cuts (except

the first cut) 1-2 mm deep are thenmade. meeting at the base 9fthe fruit.The incisio'ns are made using astainless steel razor blade set into apiece ofrubber attached to a long stick.The blade should not protrude morethan about 2 mm as cuts deeper than 2401 June 2007 IChemical Products Finder

mm risk juices and starch from thefruit pulp mixingwith the latex. whichlowers the, quality.Fruits should be tapped at intervals

of about 4-7 days and for the firsttapping it isusually sufficient to makeonly one cut. On subsequent tapingsthe two or three cuts are spacedbetween earlier ones (as explainedabove).After about 4-6 minutes the flow

oflatex ceases. Adish isused to collectthe latex and the latex is then scrapedinto a polythene lined boxwith a closefitting lid; such a box should be storedin the shade. The use of a close fittinglid and keeping the box in the shadeare both important because they reducethe reactions. which cause the loss ofenzyme activity. Foreign matter such

Ribbon structure of papainas dirt and insects in the latex shouldbe avoided. Latexadhering to the fruitshould be carefully scraped off andtransferred to .the collecting box.However. dried latex should not bemixed with fresh latex as this lowersthe quality.When handling fresh latex. care

should be taken to ensure that it doesnot come into contact with skin as it

will cause burning. Neither sh~come into contact with heavy '-'such as iron. copper or brasscauses 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 sbe dried to below 5 per cent mol(when it will have a dry and ctexture) as soon as possible.After two or three months the

are ripe and should be removedthe tree. The ripe fruits are ediblehave very little sale value becausetheir scarred appearance. However.skin of the green ripe papaya dcontain about 10 per cent pectin (weight) and the fruits couldprocessed to extract this.

Manufacturing processStep 2 - Papain (IP)-1) Papaya latex is mixed wit

potassium metabisulphate (K2S20S)'2)Sieved to remove extranecvmaterials and then blended witactivated zeoca r b catiaexchange resin.3) The mixture is then centrifugt>

to separate out the resin from tllI I

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In the second method to determinethe activity of a papain sample. a knownamount ofpapain sample is mixed witha fixed amount of casein (the proteinfound in milk).The reaction is allowedto proceed for 60 minutes at 40C.Afterthis time. the reaction is stopped by-theaddition of a strong acid.The product of the reaction isknown

as tyrosine. which is known to absorbultra-violet light (invisible to the humaneye). The solutions containing thetyrosine are prepared for analysis usingthe spectrophotometer. The amount ofultra-violet light absorbed by thesolution can be related to the number oftyrosine units produced by the papainsample. Hence, the greater this number,the greater the activity of the papainsample.

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ApplicationsPapain has a wide application in

different industries, especially in foodindustry.1) It is being used to tenderize meat andmeat products,

2) Textile industry and tanning industry.3) In the manufacture of proteinhydrolysate,

4) In confectionery industry (to preparechewing gum)

5) In brewing industry to removecloudiness in beer,

6) In dairy industry (for cheese).Similarly,

7) It is used inpharmaceutical industry.8) It is further used in aroma andperfume industries and also foreffluent treatments.Case-l "Papainin tenderizing meat:"

The wide application of papain is intenderising meat. The relative effectsof an aspartic proteinase (AP) andpapain cn meat proteins and beeftenderness were evaluated bymeasuring release of hydroxyproline incollagen, and breakdown ofmyofibrillarproteins. Tenderness was objectivelymeasured byWarner-Bratzlershear. AP 0.05) intenderness duringfrozen orrefrigeratedstorage. It wasalso inactivatedat cookingtemperatures inexcess of 60C,thereforeeliminating anyundesirable sideeffects that may Manufacturing processbe asso.ciatedwith residual protease activity.Case-2 "papain in' textile

industry": In textile industry thewashing of the fabric is done aftergiving it the desired size and shape,in the washing of the fabric papainis used as papain is hydrolase classenzyme and its main function is inbreaking the molecules.While washing the fabric papain

reacts with the stains on the fabric andbreaks the molecules i.e., it breaks thebond between two molecules and dueto which stains are washed out fromthe fabricand we get clean and glossaryfabric for direct sell.World trade in papainThe principal producers of crude

papain are Zaire, Tanzania, Uganda andSri Lanka.Most ofthe spray dried papaincomes from Zaire. The principalimporting countries are the UnitedStates, Japan, UnitedKingdom, Belgiumand France. Almost aU the best qualitypapain goes to the United States.

Crude papain is used, in Srithe brewing industry for chill p:beer and lager. However, the inci'trend for additive free beers iby other European countries iseffect in Britain and so this malpapain is declining. Anotherpapain is in the meat industrytenderization of meat anproduction of meat tendepowders.

Abhijit Rathi is student 01Vidyapeeth University, Co!

EnginlPune - 43 and S V Godekor

Sharati Vidyapeeth Uni:College of Engineering. Pur

CorrigendumThe title of the article by Sheetal Vatturkar apjthe April issue of Chemical Products Finderwrongly printed as "Phase Equilibria StUMulticomponent Systems". The tit le should actuas "Remove the Bottleneck of the RO PlantsTechno-Economical Performance",The error is regretted.

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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 intrays and dried in vacuum shelf drier.and mixed with potassiummetabisulphate.

6) Ground and diluted with lactoseto get IP grade papain.Papain concentrate

1) Papaya latex is mixed withpotassium metabisulphate anddistilled! de-mineralized water.

2)Stirred thoroughly and stored incold room for settling.

3) The supernatant liquid issiphoned off. filtered and the clearfiltrate is mixed with denatured spiritPlant ParametersCapacity, TPA 9No. of Shifts / day 1Working days / Yr. 300Total land Area, m 2 600Covered Area, m 2 200Raw (Tonne per TonneMaterial of Product)Latex 5.0Pot. Meta-bisulphate 0.015Lactose powder 0.56Denatured spirit 0.025Zeocarb, Ltr 0.47

ManpowerManaQerial 1Skilled 3Unskilled 6

Utilities (Per Tonneof Product)

Power,KwH 2.3Water, GL 6.6Cooling water, GL 26.6Rawwater, GL 13.2Steam, Kg 6.6

Pr esu m ed plan t parame ters of papain plant

and allowed to settle in cold room.4) The supernatant liquid is

decanted fromthe slurry.and the slurryis filtered.

5) The filtered cakes are washedwith rectified spirit and dried firstwith compressed air and then invacuum self dryer.

6) The dried material is mixed withpotassium metabisulphite.7) Ground and diluted with lactoseto get concentrate grade papain.Enzyme activity

If papain is to be exploitedcommercially for an export market orlocal food industry use. it isimportant to be able to determine theenzyme activity. The method is knownas assaying. The assaying could becarried out by. for example. theNational Standards office.

Papain is used to hydrolyse (orbreakdown) proteins. Therefore assaysto measure papain activity are basedon measuring a product of thehydrolysis. There are two main assaymethods.

The methods are: The first method relies on the

abilityof papain to clot milk. It is a lowcostmethod but is time consuming.Also the lack of a standard method tofind the clotting point and variationsin the milkpowder used can introduceerrors.

In this method a known amount ofpapain sample (made by dissolving aknown weight of papain in a knownvolume of a solution of acetic acid) isadded to a fixed amount of milk (madeby dissolving a known weight of milkpowder in a known volume of water)which has been warmed to 30DC in awater bath. The contents arethoroughly mixed and then observeduntil the first signs of clotting(formation of lumps) aredetected. Thetime taken to reach this stage. fromwhen the papain was added to themilk. is recorded. The experiment isthen repeated using different knownamounts of papain solutions. The

different amounts of papainused should give a range of (times between 60 and 300seceoptimum results.

The activity of the papain sathen calculated by plotting afinding the time taken to clotan infinite concentration ofpapthen using that value in a foncalculate the activity.To intrcduce a rnea s tstandardization the amount of nbe fixed at a certainconcentration. This is done by ra known concentration of higlpapain with the milk.

The concentration of milk Isolution can then be adjusted tethe desired clotting time undereaction conditions. The 'actipure papain' at this known ammilkcan then be calculated. Testsample papain under the same rconditions and sam.e (known) cof milk will then give an arelative to the pure papain. The second method isbasedscience of the absorption 0known as absorptiometry. Thisanalytical technique for measuramount of radiation (or 'colour'(absorbed by a chemical solutioknown that. for example. a 'icoloured solution will absorlight. (Blue is the 'complementto yellow). The greatelconcentration of yellow coloursolution the more is the absorpblue light. This is a useful disbecause certain products of chreactions are colored. Themore ithe colour is. greater the concenof product. Therefore. by shinirelevant complement colour tlthe sample liquid the amount (absorbed can be related tconcentratkm of product.Not all 'colours' (or radiatilight)are visible to the humane1technique usedwhenthe 'Colours'beyond the visible spectrum isas spectrophotometry ancinstrument used is callspectrophotometer.