FACULTY OF INDUSTRIAL SCIENCES AND TECHNOLOGY

BSB2452 ENZYME TECHNOLOGY LABORATORY

SEMESTER II 2021/2022

PRACTICAL 1

ISOLATION OF PROTEASE FROM PINEAPPLE

NAME MATRIC ID Part of contribution

NUR LIYANA BINTI ISMAIL SB20091 METHODOLOGY,
RESULT

NUR AISYAH BINTI ZULKIFLI SB20093 INTRODUCTION,

REFEERENCES

MUHAMMAD HAMZAH BIN MUSTAFFA KAMAL SB20056 OBJECTIVE,

CONCLUSION,
APPENDIX

AIDA SYAFINI BINTI MOHD AKHIR SB20012 DISCUSSION

SECTION 01

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
1.0 INTRODUCTION
A protease also known as a peptidase is an enzyme that catalyses, allowing proteins to be
broken down into smaller polypeptides or single amino acids and allowing new protein
products to be formed. Hydrolysis, a reaction in which water breaks bonds, is used to cleave
the peptide bonds within proteins. Proteases play a role in a variety of biological processes,
including protein digestion, protein catabolism which was the breakdown of aged proteins,
and cell signalling.

Figure 1 shows structure of protein

In the absence of functional accelerants, proteolysis would be very slow, taking hundreds of
years. Proteases can be found in all forms of life and viruses. They have independently
evolved multiple times, and different classes of protease can perform the same reaction by
completely different catalytic mechanisms

Enzyme assay can be divided by two which was stopped assay and continuous assay. To
determine an enzyme's activity, one must determine how much product is produced in a given
amount of time or, in some situations, how much substrate has been consumed, which should
be the same thing. As a result, a method for measuring one product or substrate while the
other is present is perfect. There are numerous methods; this section will focus on what are
called as ‘stopped assays.' Stopped assays entail stopping the reaction after a certain amount
of time has passed and then measuring the amount of product generated. Any method, from
chemical to enzymatic to bioassay, is possible, and the simplest is usually chosen if it is
dependable. In many circumstances, a selective approach can discriminate between the
substrate and the result, eliminating the need for a separating step. The usual
phosphomolybdate technique, for example, can be used to detect phosphate release from a
phosphate ester. Separation of unwanted substrate from product may be required otherwise.
This is particularly important in radiochemical assays because the measurement is of
radioactivity rather than a specific test for the product. Chromatographic, solubility, and
partition techniques are examples of separation methods. Denatured enzymes, such as strong
acid, alkali, or detergent; heat; or treatments with irreversible inhibitors, such as heavy metal
ions, are all methods for stopping the reaction. The enzyme can be halted in certain situations
by adding a complexing agent like ethylenediaminetetraacetic acid (EDTA), which eliminates
metal ions that are required for activity; in other cases, freezing on ice may be sufficient.
Stopped assays should be tested at least once with varied incubation times to ensure that the
rate is linear during the period chosen for the standard procedure.

A continuous assay, which tracks the course of the reaction as it occurs, is an alternative to a
halted test. Continuous assays are significantly more convenient because the result is visible
right away, and any variation from linearity in the beginning rate may be detected. On the
other hand, not all enzymes have a continuous-observation assay technique. The most basic
continuous test is one in which changes in absorbance example NAD(P)H at 340 nm with
dehydrogenases, fluorescence, viscosity, pH, or one of several other physical parameters can
be monitored while the enzyme is in activity. An artificial substrate that releases a coloured
or fluorescent product is employed in several hydrolase experiments. However, most
enzymes do not change a clearly visible physical property as a result of their activity. A
connected continuous technique can be used to solve this problem. In this procedure, the
product is further acted on until an end product is created that can be physically observed.
Coupled assays provide the advantage of removing the product, which helps to maintain the
observed rate consistent throughout time by eliminating product inhibition and reaction
reversal.
 Figure 2 shows Four different ways of assaying the enzyme invertase

2.0 OBJECTIVE
1. To investigate basic principle of protease
2. To prepare the standard curve of tyrosine with different desired concentration.
3. To calculate the concentration of tyrosine in sample using the standard curve
4. To determine colour development of reaction between Folin & Ciocaltue’s reagent.
5. To determine the amount of protease that has been isolated based on concentration of
tyrosine in sample
3.0 MATERIALS AND METHODOLOGY
3.1 REAGENTS:
A. 50mM Potassium Phosphate buffer, pH 7.5 at 37⁰C
Potassium Phosphate, Dibasic, Trihydrate was added to 200mL of deionized water
and was adjusted to pH 7.5 at 35⁰C using 1 M HCL
B. 0.65% (w/v) Casein Solution (Casein)
0.8125 g of Casein was added in 125 mL of reagent. The solution was heated gently
to 80-90⁰C for 10 minutes with stirring. The pH was adjusted to pH 7.5 at 37 ⁰C using
either 1 M NaOH or 1 M HCL.
C. 110 mM Trichloroacetic Acid Reagent (TCA)
9 mL of TCA (6.1 N, approximately 100%, w/v) was diluted to 500 ml of deionized
water.
D. Folin & Ciocalteu’s Phenol Reagent (F-C)
1 mL of Folin & Ciocalteu’s Phenol Reagent was diluted to 4 mL with deionized
water.
E. 500 mM Sodium Carbonate Solution (Na2CO3)
26.5 g anhydrous Na2CO3 was dissolved in 500 mL of deionized water.
F. 1.1 mM L-Tyrosine Standard (Standard Solution)
L-Tyrosine, free base was added in 100 mL of deionized water. The solution was
heated gently until Tyrosine was fully dissolved and cooled to room temperature.
G. Protease Enzyme Solution
Crude protease extracted from pineapple in Practical 1 used.
3.2 MATERIALS & APPARATUS:
Vials/ falcon tubes, test tubes, beaker, spatula, test tube rack, incubator, P100
micropipettes with tips and P1000 micropipettes with tips, cuvettes,
spectrophotometer, thermometer, timer and deionized water.
3.3 PROCEDURES:
3.3.1 Preparations of standard curve
1. 5 vials were prepared and labelled as blank, standard 1, standard 2, standard 3, and
standard 4.
2. 5 ml of reagent E and 1ml of reagent D were added into all the 5 vials.
3. 0.05ml of reagent F was added into the vial labelled as standard 1, then followed
by 1.95ml of deionized water.
4. 0.10ml of reagent F was added into the vial labelled as standard 2, then followed
by 1.90ml of deionized water.
5. 0.20ml of reagent F was added into the vail labelled as standard 3, then followed
by 1.80ml of deionized water.
6. 0.40ml of reagent F was added into the vial labelled as standard 4, then followed
by 1.60ml of deionized water.
7. 2ml of deionized water was added with the solution in the vial labelled as blank.
8. 2ml of mixture solution was pipetted into the cuvette from each vial.
9. The reading of absorbance at 660nm for each of the vials were recorded and
tabulated.
10. A graph of the standard curve was plotted.

Tube Std 1 Std 2 Std 3 Std 4 Blank

Volume of Reagent F (std soln)
0.05 0.10 0.20 0.40 0.00
(mL)
Volume of deionized water (mL) 1.95 1.90 1.80 1.60 2.00
Volume of Reagent E (Na2CO3) 5 5 5 5 5
Volume of Reagent D (F-C) (mL) 1 1 1 1 1
3.3.2 Colour Development for Samples
1. 3 suitable tubes were prepared and labelled as tube 1, tube 2 and blank.
2. 5ml of reagent B was added into the tubes which labelled tube 1, tube 2 and blank
respectively.
3. 1 ml of reagent G was then added into the vial labelled tube 1 and tube 2.
4. All tubes were mixed by swirling and then incubated at 37⁰C for exactly 10
minutes.
5. 5ml reagent C was then added into each of the tubes.
6. Then added 1 ml of reagent G which only was added into the tube labelled blank.
7. The mixture solution in the tubes were mixed by swirling and incubated at 37⁰C
for about 20 minutes.
8. The tubes were then centrifuged at 6000rpm and supernatant was collected to use
in colour development.

Test 1
Tube Blank 1
Tube 1 Tube 2

Volume of Reagent B (Casein) (ml) 5.00 5.00 5.00

Volume of Reagent G (Enzyme Solution) (ml) 1.00 1.00 0.00

Volume of Reagent C (TCA) (ml) 5.00 5.00 5.00

Volume of Reagent G (Enzyme Solution) (ml) 0.00 0.00 1.00

9. 2 ml of supernatant from tube 1, tube 2 and blank tube was transferred into
another new tube respectively for colour development.
10. 5ml of reagent E was added into all tubes, followed by 1ml of reagent D.
11. All tubes were mixed by swirling and incubated at 37⁰C for 30 minutes.
12. All the tubes were centrifuged at 6000 rpm, then the absorbance readings at 600
nm were recorded for each tube in suitable cuvettes
13. The colour development was determined.
14. Concentration of enzymes was determined from the standard curve.

Test 1
Tube Blank 2
Tube 1 Tube 2

Volume of test filtrate (ml) 2.00 2.00 0.00

Volume of blank filtrate (ml) 0.00 0.00 2.00

Volume of Reagent E (Na2CO3) (ml) 5.00 5.00 5.00

Volume of Reagent D (F-C) (ml) 1.00 1.00 1.00

4.0 RESULTS
Table 4.1 Volume of reagent B, C and G added to duplicate of Test 1 and Blank 1 to
determine the enzymatic activity of crude enzyme prepared from practical 1.
Test 1
Tube Blank 1
Tube 1
Volume of Reagent B (Casein) (ml) 5.00 5.00
All tubes were equilibrated to 37⁰C.
Volume of Reagent G (Enzyme Solution) (ml) 1.00 0.00
Solution in tubes were mixed by swirling and incubated at 37⁰C for exactly 10 minutes.
Volume of Reagent C (TCA) (ml) 5.00 5.00
Volume of Reagent G (Enzyme Solution) (ml) 0.00 1.00
Total volume (ml) 11.00 11.00

Figure 4.1 Supernatant and pellet formation of duplicate Test 1 (Tube 1-left) and Blank (left)
after centrifugation process at 6000rpm at
Preparation of standard curve
Table 4.2.1: Volume of Reagent D, E, F and deionized water required for standard curve
preparation with different concentration of Reagent F (L-Tyrosine standard solution).

Tube Std 1 Std 2 Std 3 Std 4 Blank

Volume of Reagent F (std soln) (ml) 0.05 0.10 0.20 0.40 0.00
Volume of deionized water (ml) 1.95 1.90 1.80 1.60 2.00
Volume of Reagent E (Na2CO3) (ml) 5 5 5 5 5
Volume of Reagent D (F-C) (ml) 1 1 1 1 1
Total Volume 8.00 8.00 8.00 8.00 8.00
 Figure 4.2.1: Colour development of 5 tubes (left to right) of tyrosine
from 0.000 to 0.440 μmoles.

Table 4.2.2: Absorbance reading at 660nm if different μmoles of tyrosine

Tube Std 1 Std 2 Std 3 Std 4 Blank
μmoles of Tyrosine 0.055 0.110 0.220 0.440 0.000
∆A660nm Standard (A) 0.213 0.361 0.415 0.789 0.000

Plot the graph for standard curve and calculate the R2 value.

Figure 4.2.2: Graph of ∆A660nm Standard (A) vs μmoles of Tyrosine

Sample Determination

Table 4.3.1: Absorbance reading at 660nm of test 2 and blank 2.

Tube Test 2
Blank 2
Tube 1
∆A660nm Standard (A) 0.158 0.157

Figure 4.3.1: colour development of Test 2 (T-left) and blank (B-right)

Calculation:
Preparation of standard curve
The absorbance value of standard tube is calculated using formula:
∆A660nm Standard = A660nm Standard - A660nm Standard Blank

Tube ∆A660nm Standard (A)

Std 1 0.213-0.000 = 0.213
Std 2 0.361-0.000 = 0.361
Std 3 0.415-0.000 = 0.415
Std 4 0.789-0.000 = 0.789

Measure the concentration of tyrosine that was used during the standard curve preparation.
When preparing for standard curve with different concentration of reagent F, L-tyrosine
standard solution, the formula n = MV was used to calculate the number of moles (μ) of
reagent F in sample solution.
n = number of moles of tyrosine
M = concentration of reagent F (1.1mM)
V = volume of reagent F
a. Volume of Reagent F = 0.05 mL b. Volume of Reagent F = 0.10 mL
n = 1.1 x 10-3(0.05x10-3) n = 1.1 x 10-3(0.10x10-3)
= 5.5 x 10-8 moles = 1.10 x 10-7 moles
= 0.055 μmoles = 0.110 μmoles

c. Volume of Reagent F = 0.20 mL d. Volume of Reagent F = 0.40 mL

n = 1.1 x 10-3(0.20x10-3) n = 1.1 x 10-3(0.40x10-3)
= 2.2 x 10-7 moles = 4.4 x 10-7 moles
= 0.220 μmoles = 0.440 μmoles

e. Volume of Reagent F = 0.00 mL

n = 1.1 x 10-3(0.00x10-3)
= 0.000 μmoles
The table below indicates the summary of concentration of tyrosine that were used during
standard curve preparation:

Concentration of tyrosine
Tube Volume of Reagent F (ml)
(μmoles)

Std 1 0.05 0.055

Std 2 0.1 0.110
Std 3 0.2 0.220
Std 4 0.4 0.440
Blank 0 0.000

Sample Determination
The absorbance value of sample tube is calculated using the formula:
∆A660nm Sample = A660nm Test - A660nm Standard Blank

Tubes ∆A660nm Standard

Tube 1 0.157 - 0.158 = 0.001

Average 0.001
According to the standard curve plotted by using Microsoft Excel, the equation of the
linear graph obtained is y = 1.9052x which fulfil the general equation of a straight-line
y = mx + c. However due to error that occur during the experiment, the value of the
absorbance that is used to calculate the unknown concentration of tyrosine equivalent
liberated is not accurate and it does not obey Beer-lamberts law.
0.001 = 1.9052x
x = 5.249 x 10-4 μmoles (moles of unknown concentration of tyrosine equivalent)
 Measure the proteolytic activity (U/ml). Explain your answers:
( μmoles of tyrosine equivalent released ) ×11
Unit/ml enzymes =
1 ×10 × 2
11 = Total volume of assay (ml)
10 = Time of assay as per the unit definition (minutes)
1 = Volume of enzyme used (ml)
2 = Volume used in Colorimetric Determination

( 5.249× 10−4 ) × 11
Unit/ml enzymes =
1 ×10 ×2
= 2.887 x 10-4 U/ml
Thus, enzyme unit calculated is 2.887 x 10-4 U/ml indicates that 2.887 x 10-4 μmoles of
enzyme protease catalyses the conversion of one μmoles of substrate (casein) into product
(tyrosine) and produces colour equivalent to one μmoles of tyrosine per minute at pH 7.5 at
37℃ .
5.0 DISCUSSION

Firstly, this practical was continued from the previous practical which isolation of
enzyme from pineapple to measure the reaction activities. In this case, it used UV- Vi’s
spectrophotometer. The spectrophotometer was measuring the quantity of the substance
which in liquid, gas and solid. It will absorb the light that passing through the medium, and
will resulted the production of the intensity from wavelength that create the spectrum. In this
experiment, the casein was act as substrate which very special known as dye- labelled protein
substance. Protease is an enzyme that responsible for the protein hydrolysis by breaking
down the peptide bonds. This is because it is high of specific and has a wide range of
application in medicine, detergent and others. There are many types of assay method to
measure protease activity. Such as method to identify the enzyme protease by the appearance
of coloured compound that known as colorimetric method. The protease will react with the
casein and the amino acid will hold together with the other fragments. A standard curve is a
calibration curve which used as a quantities of research technique. It will present the
relationship between two quantities.

Next the purpose of this experiment is to determine the concentration of tyrosine in

unknown sample by using the standard curve and also to development of the colour reaction
between F-C reagent and tyrosine. The experiment was first started by pipetted the reagent B
which is 0.65 % of Casein solution. It is already prepared by add 0.815g of casein in 125ml
of the reagent A (Potassium Phosphate buffer, pH 7.5). This solution was heated gently 80°C
to 90°C for 10 minutes with stirring and the pH was 7.5. The reagent B was equilibrated in
37°C. The test tube was filled 5ml and for the blank tube 5ml too. Then, the reagent B was
added into the reagent G which enzyme solution from the previous experiment. It was put
into the test tube for 1ml and the mixture was swirling and incubated at 37°C for 10 minutes.

Next, the reagent C which 110mM Trichloroacetic Acid Reagent (TCA) was added
into the enzymatic reaction in the tubes and was denature the casein substrate. On the other
hand, the solution in test tube was 5ml and blank tube 5ml. Moreover, reagent G (enzyme
solution) was added into test tube 1ml. The mixture was mix by swirling and incubated at
37°C for 20 minutes. However, there will be no tyrosine produced in the blank because no
enzymatic reaction and it will act as controlled reaction. Thus, it was centrifuge at 6000g and
the supernatant were used in colour development. It is centrifuge to remove the insoluble
casein and to get the clear supernatant.
 For the preparation of standard curve for colour development, it was divided into 5
different vials that had been prepared and labelled as standard 1, standard 2, standard 3,
standard 4 and blank. For each vial were added reagent F that will be active to produce
purple- blue coloured chromosphere with different intensity by used spectrophotometer.
Moreover, deionized water, reagent E and reagent D. For blank tubes, there was no reagent F
added to became as a control sample. For each standard and blank were put into the cuvette to
read the absorbance. The test for standard curve was calculated by used the formula:

∆A660nmStandard = A660nmStandard - A660nmStandard Blank

The result for standard curve of standard 1 was 0.213A. Next the result for standard 2
0.213A, standard 3 0.415A, and standard 4 was 0.789A.

Next, for the sample 2 different vials which test and blank. For the test it was added
test filtrate solution in 2ml and not for blank. Next for blank it was added blank filtrate in
2ml. Other than that, reagent B and reagent D were added into both tubes for 5ml and 1ml.
After that, both mixtures were mix by swirling and incubated at 37°C for 30 minutes. The
weight for test tube was 14.67 g and blank 14.54g. It was centrifuge at 6000g and the
absorbance of the tubes were measure at wavelength 660nm.

The result for standard curve of standard 1 was 0.213A. Next the result for standard 2
0.213A, standard 3 0.415A, and standard 4 was 0.789A. Next, the result for sample was
0.157A and 0.158A.

Finally, there were had error and precaution during this experiment that could affect
the result. The error that happens was the absorbance of blank for sample development. The
blank result was 0.158A. It should be 0 A because it acts as controlled sample. This happen
may be because of we do add the reagent E and reagent D while waiting for test filtrate and
blank filtrate done incubated. It causes the affect for measurement of the absorbance. Next
the precaution is the cuvette must wipe in and out by used the tissue paper and not scratch the
surface to avoid wrong result of absorbance value. Other than that, the high temperature of
pH for heating is lower than 7. It is because the protein and substrate would be denatured and
will resulted no reaction.
6.0 CONCLUSION

To conclude, we were able to investigate the basic principles of protease activity, which
is the breakdown of substrate under specific pH and temperature conditions to create the
amino acid tyrosine. We had able to calculate the concentration of unknown tyrosine in the
sample using a standard curve of A660mm standard against moles of tyrosine. The standard
curve of standard 1 to have of 0.213A. The results for standard 2 was 0.213A, standard 3 was
0.415A, and standard 4 was 0.789A. Following that, the sample results was 0.157A and
0.158A.

The purple-blue intensity of each soluzion could not be observed based on the colour
development of the reaction between Folin's reagent and tyrosine, but however the
absorbance value was able to be read using a spectrophotometer. The higher the absorbance
value of a sample, the higher the concentration of tyrosine present, and hence the higher the
catalytic capability of the enzyme. In this study, we used a spectrophotometer to measure the
absorbance readings at 660nm if different moles of tyrosine were present to assess the
amount of protease that had been separated based on the concentration of tyrosine in the
sample. The absorbance values of tubes 1 was 0.158A and have average of 0.001.

There might have been some error in this experiment where the reagents E and D were
exposed to air for a longer period of time while waiting for the filtrates to incubate, which
could have influenced the results.
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APPENDIX

Four standard solutions and one blank

solution were prepared.

5 mL of reagent E and 1 mL of reagent D

were added in each of the five vials.

The solution and blank are both transferred

to the cuvette.

Test 1 and a blank tube have been prepared.

 The tubes are weighed before being
centrifuged to ensure that the centrifuge is
balanced.

The tubes are centrifuged at a speed of 600

revolutions per minute.