FACULTY OF INDUSTRIAL SCIENCES AND TECHNOLOGY

BSB2452 ENZYME TECHNOLOGY LABORATORY

SEMESTER II 2021/2022

PRACTICAL 1

ISOLATION OF PROTEASE FROM PINEAPPLE

SECTION 01
PERSON IN CHARGE:

NAME MATRIC ID Part of contribution

MUHAMMAD HAMZAH BIN MUSTAFFA SB20056 INTRODUCTION,

KAMAL REFEERENCES

NUR AISYAH BINTI ZULKIFLI SB20093 METHODOLOGY,

RESULT

NUR LIYANA BINTI ISMAIL SB20091 DISCUSSION

AIDA SYAFINI BINTI MOHD AKHIR SB20012 OBJECTIVE,

CONCLUSION,
APPENDIX

Marks Distribution:

Bil. Lab report section Mark Marks obtained

distribution
1 Introduction 10
2 Objective(s) 5
3 Materials and Methods 10
4 Results and Answers to questions 20
5 Discussion 40
6 Conclusion 5
7 References 5
8 Appendix 5
TOTAL
Introduction :
Bromelain is a generic term used to proteolytic\senzymes found in plant tissues.
Phosphatases, glucosidases, peroxidases, cellulases, glycoproteins, and sugars are all present
in this crude aqueous extract from pineapple stems and immature fruits. Bromelain is one of
two protease enzymes derived from the Bromeliaceae plant family. Bromelain extract is a
combination of Proteases, protein-digesting enzymes, and a few additional compounds in lower
amounts. Because a free sulfhydryl group on a cysteine side chain is required for the function,
the proteolytic enzymes are known as Sulfhydryl proteases. We used pineapple proteases
because the enzyme isn't present in the early phases of fruit growth, but it quickly grows and
stays high until maturity, when it drops somewhat. In its full state, the pineapple is the only
fruit with a moderately high protease content.

Diagram 1 : Isolation of Protease from Pineapple

Precipitation is a chemical process in which two ions join together to produce an
insoluble salt called the precipitate in an aqueous solution. When two solutions containing
different salts are combined, a cation/anion pair produces an insoluble salt in the resultant
combined solution, which then precipitates out of solution. To purify proteins by precipitation,
salt precipitation can be a very strong method. Ammonium sulphate is commonly used as a salt
because it is inexpensive, extremely soluble in water, and can become considerably more
hydrated than virtually any other ionic solvent.

Diagram 1 : Salt Precipitation Process

 Proteins can be reduced in solubility but not denatured by ammonium sulphate
precipitation because ammonium sulphate does not denature them. Proteins can be
concentrated by extracting the leftover ammonium sulphate solution and then resolubilized in
normal buffers or at a lower concentration of ammonium sulphate since ammonium sulphate
precipitation simply affects the solubility of proteins and does not denature them. The protein
solution can subsequently be purified even further using hydrophobic interaction
chromatography or gel filtration chromatography. In practice, to precipitate required proteins,
ammonium sulphate is either supplied as a solid or as a typically saturated solution.
When adding more solute to a solution does not raise the concentration of the solution,
it is said to be saturated. The degree of solubility varies greatly across substances, ranging from
infinitely soluble (totally miscible) ethanol in water to slightly soluble silver chloride in water.
Poorly soluble chemicals are sometimes referred to as "insoluble." The equilibrium solubility
can be surpassed under certain conditions, resulting in a supersaturated solution. Particle size
has little effect on solubility; given enough time, even massive particles will dissolve.

Objective:

1. To determine the total of protein from pineapple by precipitation of protein as part of

purification.
2. To understand the salt precipitation technique of protein.

Material and Apparatus:

I. Reagents

1. Ammonium sulphate crystals

2. 0.01M Sodium phosphate buffer (pH 6.5)

II. Apparatus
1. Muslin cloth

2. Food homogenizer
3. Dialysis bag

4. Falcon tube
Methodology:
Freshly pineapple was peeled and cut into small pieces then the flesh was put into a
clean beaker. After that, the pineapple flesh was homogenized by using food homogenizer and
the mixture was filtered using muslin cloth. The pomace and insoluble material were discarded.
The protein content in the supernatant is determined by taking OD at 280 nm and then brought
to 100% saturation with solid (NH4)2SO4 means add salt until fully dissolved. Next, the salt-
enriched solution was slowly stirred. The precipitate was collected by centrifugation then the
precipitate was dissolved in small volume of 0.1M Phosphate buffer pH 6.5. The enzyme was
dissolved in water and dialyzed 5h or overnight at 4 °C against three changes of buffer. Lastly,
the dialysate was collected.

Result:

Mass of empty centrifuge tube = 12.3600g

The final mass of centrifuge tube with crude enzyme and supernatant = 22.4400 g
The final mass of crude enzyme and supernatant = 10.0800 g

Result Observation

Yellow precipitants were obtained after first

centrifuge at 6000 rpm at 4⁰C for 10
minutes. The supernatant was removed.
The dialysis tube which was taken out after
immersed in a beaker of sodium phosphate
buffer overnight.
The solution in dialysis tube remains
yellowish after immersed in the sodium
phosphate buffer overnight.

The crude enzyme was then weighed and

recorded 22.44 g.

The crude enzyme was centrifuge at 6000

rpm and 4⁰C for 10 minutes.
The supernatant and platelet were clearly to
be seen.

The supernatant shows slightly cloudy, and

the yellow platelet was at bottom of tube.

The slightly cloudy supernatant transferred

to a clean falcon tube. Then, supernatant was
stored in – 20 for lab 2.
Discussion:
A. Bromelain.

Bromelain is a type of enzyme that can be easily found in pineapple juice and
pineapple stem. This enzyme is a proteolytic enzyme or also known as proteases is an
enzyme that breaks down protein into smaller polypeptides or single amino acid. Due
to their ability to break down protein, this enzyme is one of the most important enzymes
needed in our body as it helps aid digestion more efficiently. In this experiment, we
want to extract bromelain from pineapple. However, due to the presence of other
substances such as cellulases, glycoproteins, carbohydrates and many more in the
pineapple, we need to undergo methods in order to achieved only bromelain mixture.
For the first part of procedures for this practical, the pineapple was peeled and
cut into cubes before it is being homogenized by homogenizer which in this case, we
use food blender as the homogenizer. The juice that has been blend contains enzyme
bromelain which the enzyme that we want to extract from the pineapple. By using
blender as homogenizer, we can break down and disrupt the cells and fibers of the fruit
until it is soft enough to be filtered for its pure juice and separate the debris by using
muslin cloth. Thus, the extraction of bromelain will result in increases of breakdown of
cystine and other protein substances through hydrolysis. This mechanism will be put to
rest and use later for practical 2.

B. Salting Out with Ammonium Sulphate.

Proteins and enzyme usually have both positively and negatively charged
regions in which when they are exposed at extremely low salt concentration these
molecules will self-aggregate and move closer to each other making it less soluble.
However, with the addition of small amount of salt will increase the its solubility and
allow the ions that enters the solution to neutralize the charged regions of the enzyme.
In this case, when bromelain is neutralized, it will not self-aggregate and become
soluble. This is what we called process of salting in. By adding salt into the solution
will make the charge of the proteins increases. Thus, instead of neutralize it, this process
will make it self-aggregate again and becomes less soluble which is called salting out
process. In this experiment, bromelain that contain in pineapple juice contains 1mg of
sodium per 100g of pineapple fruit which means there is presence of charged ions in
the juice that makes bromelain soluble but, in this experiment, we need it to be insoluble
in order for it to be able for extraction, hence why ammonium sulphate is important in
this practical as it is the most efficient salt that can be used for salting out process.
Furthermore, by adding ammonium sulphate to bromelain precipitate is an
important step to extract bromelain effectively. This is because during a process called
centrifugation, all other small molecules such as enzymes might not be separated
efficiently it were to be soluble. Thankfully, with the help of ammonium sulphate in
salting out process, it helps by lowering the enzyme solubility thus, making is easier to
separate during centrifugation process. Protein is able to aggregate because of hydration
 barrier that breaks between protein molecules. Therefore, salting out is a part of most
crucial step when we want to do extraction or separation.

C. Centrifugation.
Centrifugation is a mechanical process which involves the use of the
centrifugal force to separate particles from solution according to their size, shape,
density and medium viscosity. This technique is very helpful to separate mixture by
applying centrifugal force. In This practical, the centrifugation process was carried in
at 4⁰C for 10 minutes for 2 times. It is done in 4⁰C to prevent enzyme from denature
and it is important for enzymes as it can easily denature when exposed to high
temperature. In this practical, first centrifugation was done for 10 minutes and the
supernatant was removed in which left the platelet that contains bromelain. This platelet
is added with sodium phosphate buffer. The centrifugation was done perfectly as we
can see the separation of supernatant and enzyme precipitate clearly. After the addition
of buffer, the sample was then let to rest for 24 hours in water bath which is known as
dialysis process. It is further being centrifuged for the second time to get the crude
enzyme.

D. Addition of Phosphate buffer pH 6.5.

After the first centrifugation process is done, the supernatant is discarded

immediately to avoid the target enzymes which contains in the pellet mix together again
with the supernatant if it is left for a while. A buffer known as sodium phosphate buffer
with pH of 6.5 were added into the falcon tube together with pallet. This purpose is to
let the pellet dissolve with the buffer before it is transferred into dialysis bag. After it is
transferred, the dialysis bag was then put into 500ml of water bath and let to rest for 24
hours before it undergoes for the second centrifugation later.
Sodium Phosphate buffer with pH of 6.5 is used in this practical due to
bromelain has an optimum pH of 6.5 to 7. If we were to use other buffer with higher or
lower pH, it might result in denature of enzyme as the shape and configuration of
bromelain is now changed and loss its function. Other than that, if the pH is extreme
and far from optimum it will interfere with the hydrogen bonds as it plays important
role in maintaining the shape and function of the enzyme. Furthermore, buffer has the
ability to enter semi permeable membrane of dialysis tube due to process called osmosis
in which water molecules moving in and out of the membrane cell that makes the
ammonium sulphate or other molecules increase or decrease the pH will neutralize the
buffer to balance the pH and makes it at optimum pH for bromelain.
After the dialysis process, the second centrifugation is done to get the crude
enzyme. After the centrifugation is we can see the supernatant clearly separate from the
crude enzyme. Both supernatant and crude enzyme is stored in -20⁰C to let bromelain
to be stable.
Conclusion:
In conclusion, the purpose of this experiment was to determine the total of protein by
precipitation of protein as part of purification and understand salt precipitation technique of
protein. Salt precipitation is used for protein purification to separate the proteins by altering
the solubility in the presence of high salt concentration. The hypothesis that the separation of
proteins by altering their solubility in the presence of a high salt concentration. This is
because the protein is less soluble in solution that high salt concentration because of the salt
ions that shield the protein with the multi ion charges. Bromelain is proteolysis protease that
catalyse the breakdown of protein into the amino acid building blocks through a hydrolysis
reaction. For the future, the dialysate will be used in practical two. Based on the result, the
yellow precipitate are obtained after centrifuge. Lastly, the supernatant slightly cloudy and
the yellow platelet was at the bottom tube.

References:

Structural biochemistry/proteins/purification/salting out. Wikibooks, open books for an open

world. (n.d.). Retrieved March 19, 2022, from
https://en.wikibooks.org/wiki/Structural_Biochemistry/Proteins/Purification/Salting_O
ut

Wingfield, P. (2001, May). Protein precipitation using ammonium sulfate. Current protocols
in protein science. Retrieved March 19, 2022, from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4817497/

Bresolin, I. R. A. P., Bresolin, I. T. L., Silveira, E., Tambourgi, E. B., & Mazzola, P. G.
(2013, December 1). Isolation and purification of bromelain from waste peel of
pineapple for therapeutic application. Brazilian Archives of Biology and Technology.
Retrieved March 19, 2022, from
https://www.scielo.br/j/babt/a/9SD7YsdQLxvhz8KpJs3LSqv/?lang=en

Salting Out - an overview | ScienceDirect Topics. (2018). Dezhi Qi.

https://www.sciencedirect.com/topics/chemistry/salting-out

Man, T. P. (2017, July 1). Ammonium Sulfate Protein Precipitation: The key to Salting-Out.
G-BIOSCIENCES. https://info.gbiosciences.com/blog/ammonium-sulfate-protein-
precipitation-the-key-to-salting-
out#:%7E:text=Salt%20precipitation%20can%20be%20a,almost%20any%20other%2
0ionic%20solvent.

Bresolin, I. R. A. P. (2013, December 1). Isolation and purification of bromelain from waste
peel of pineapple for therapeutic application. scielo.
https://www.scielo.br/j/babt/a/9SD7YsdQLxvhz8KpJs3LSqv/?lang=en
Appendix A: The step of preparation

The pineapple was blended to get the juice.

The mixture was filtered to separate the

extract and the juices by use muslin cloth.

The ammonia sulphate was added bit by bit

until it fully dissolved and slowly stir.
Next, put the solution in the tube to
centrifuge in 10 minutes.

The mass of solution before centrifuge.

The tube was centrifuge in 10 minutes.

 After centrifuge, the precipitate was
occurred. The precipitate is collected and
dissolve it in volume of 0.1 M phosphate
buffer pH 6.5.

The enzyme is dissolved in water and

dialyze.

Appendix B: The sources of purified of bromelain

Bromelain can be extract by:

- Add ammonium sulphate into the pineapple juice to obtain 100% saturation. The
ammonium sulphate stirs slowly.
- The precipitate form after centrifuge.