To continue, I’ll talk about the extraction process of papain from papaya.

First we have to
know the method of obtaining papain, then i’ll tell you how to refine these papain. I’ll
start with the materials and equipment to carry out this process. We need ... and in the
equipment... Now, to obtain the papain, firstly, we need to extract the latex papaya. It is
contained in latex pipeline across entire the trees, except the roots. Latex is the mixture
consisting of papain, chymopapain A (amino acids is glutamic acid end), chymopapain B
(original amino acid in the tyrosine), proteinase III, IV proteinase, thilprotesase, which
account for 95% papain. Latex have to be taken in these condition: Trees are growing
well, aren’t diseased and have stout trunk, we take the green fruit, whose weight from
0.3- 1 kg, and 10 weeks old is the best. The ideal time for taking is in early morning.
To obtain the latex, first we take the latex of the green fruit still on the tree, and use the
steel knife to split a few road at the highest diameter, to be separated from 3-5 cm (the
incision is not deeper than 2cm to keep the water and the starch in the fruit from being
mixed into the latex. It can reduce the latex’s quality
Then we catch the latex by the colored wide- mouth glass jar for 4- 6 minutes. After
catching the latex, we need to cover the lid, keep it in the dark, and cold preservation for
the next processing. The aim of this phase is to avoid the prolonged exposure of latex to
the atmostphere, make sure the active papain in latex. When we take the latex, we need to
avoid the dirt or insect in. Note that: Do not mix dry latex and fresh latex because it can
reduce the latex’s quality.
Fruit can be mix with latex during 4-7 days, the first time, it only needs 1slit, the next
time we split 2- 3 times in the midline previous slits
Avoid the latex contacting the skin because it will cause burn. We need to avoid the
instrument which made of heavy metals such as iron, copper because it would cause the
discolourment and reduction of the enzyme activity. Jars, knives, spoons, ... must be
made of plastic or stainless steels.
The fresh latex is unstable so it should be dried to 5
 Because latex papaya contain a number of enzymes, proteins, fats, ...so in order to have a
efficient extraction and purification process of papain from papaya, we have the
dissolving process which the contaminant is removed from papain. Here is the way: we
mix 180g latex papaya with 100g cetile and 150g clean sand, crushed with a solution of
200-300ml cysteine 0.04M (6.3g cysteine hydrochloride dissolved in 1000ml 0.054M
NaOH, alkali is used to extract pH to 5.7)
 When the latex form the suspension, we could remove the floating. The crushing and
extracted process are repeated with 300ml solution cysteine. Washing the mortar with
cysteine solution to adjust the volume to 11.
 Finally, the suspension is filtered on Whatmal filter paper 1. It can take a long time,
filtered water has a milky white or yellow green pH close to 5.7
 The remaining step of the enzyme purification process be conducted in cold conditions
 *) The next step is Removing the insoluble substances pH 9:
 Add slowly and stir approximately 110ml NaOH 1M to adjust the pH of the obtained
extraction above to pH 9.
 Filter or centrifuge at 2600 rev/ min for 1 hour to remove the grey precipitate
 *) Then is the segmentation process by (NH4)2SO4:
 Put (NH4)2SO4 into enzyme saturation 40%, after 1-2 hours, centrifuged at 2500 RPM.
 Collect precipitate, remove fluid or retain chymopapain. Precipitate is washed again with
400-500 ml solution of (NH4)2SO4 saturation 40%, remove the fluid.
 After that we move on to The segmentation process by NaCl:
 Dissolved 600ml cysteine 0.02M, the obtained solution has pH 7-7.5, then add slowly
60g solid NaCl.
 After 1 hour, centrifuge at 2500 RMP for 1 hour to collect the precipitate, remove the
liquid.
 Next is the Crystallization:
 Dissolved the precipitate with 400ml cysteine 0.002M obtained a suspension has pH
=6.5 at the room temperature.
 Keep it for 30 minutes to form the crystals, maintain at 40C overnight, then cold
centrifuged at 2500 RMP for 4-5 hour to collect the crystals
 then, we ‘ll look at the Recrystallization:
 Dissolved the crystals in distilled water at room temperature to create a protein
concentration of about 1%.
 Then put it slowly (stir at the same time) NaCl saturation (10ml/300ml solution of the
protein). When 75% NaCl solution is given in, papain started crystallized at room
temperature.
 The suspension is kept in 40C overnight, then cold centrifuged at 2500 RMP for 4-5
hours of receipt. The private activities of enzymes might increase slightly if we
crystallized it once again as above. In order to increase the enzyme activity, before
recystallization, we can dissolve the papain crystals in phosphate buffer solution contain
cysteine, then we conduct the ultrafiltration membranes 4000A0 and crystallize it. Finally
we dried at 400C to obtain the refined papain. To obtain the clean papain, we turn to the
The purification method of papain.There are two steps in this part, First of all is the The
electrophoresis:
 Use 12% polyacrylamide gel, buffer solution is beta-alanine 34mm, pH = 4.3 and the
constant current density is 4mM, the samples will be prepared in Tris-glycerol-beta-
mercatoethanol in boiling water for 40 seconds.
 The gel will be colored by Coomassie reagent-Blue R-250 and Brilliant Blue G-
colloidal. And the last one is the The filtration through Sephadex G-75:
 To determine the molecular weight of papain, we use the Sephadex G-75 having the size
column is 1.1x100 cm. The column is balanced before the buffer solution consisting of
sodium phosphate 0.1 M and the solution of EDTA (ethylenediamine-tetraacetic aicd) pH
=7

To finish up, let look at the The diagram shows the process of papain production.