experiment 2 exploring the enzymatic activity of bromelain

Specifically, the experiment aims to determine the ability of thepineapple extract to hydrolyze casein and produce tyrosine as a product.Additionally, the experiment aims to quantify the

SEMESTER II, 2022-2023 INTERNATIONAL UNIVERSITY

VIETNAM NATIONAL UNIVERSITY HCMC

BIOCHEMISTRY LABORATORY REPORT

Instructor: Ms Nguyen Hong Lan EXPERIMENT 2:

EXPLORING THE ENZYMATIC ACTIVITY OF

BROMELAIN

Team members:

Grade: _

Comment: _

Table of Contents

4.2.3 Survey the enzymatic activity 5

I INTRODUCTION

1 Objectives:

The objective of this lab is to investigate the enzymatic activity of pineapple extracts Specifically, the experiment aims to determine the ability of the pineapple extract to hydrolyze casein and produce tyrosine as a product Additionally, the experiment aims to quantify the enzymatic activity using the Folin-Ciocalteu method

2 Abstract:

In this lab, students investigated the enzymatic activity of pineapple extracts by measuring the ability of the extract to hydrolyze casein and produce tyrosine Enzyme activity is the measure of the rate at which enzymes catalyze chemical reactions in biological systems During the experiment procedure, the ability of pineapple extract to hydrolyze casein and produce tyrosine is investigated to determine its enzymatic activity Hydrolysis is a chemical reaction in which bonds in a molecule are broken by adding water, usually with the help of an enzyme as a catalyst Casein is a protein found in milk that can be hydrolyzed

by enzymes such as those found in pineapple extract This hydrolysis results in the production of tyrosine, which can be measured to determine the extent of enzymatic activity The Folin-Ciocalteu method is used to quantify the amount

of tyrosine produced, which gives an indication of the enzymatic activity in the pineapple extract Therefore, by measuring the amount of tyrosine produced, students can understand the ability and extent of enzymatic activity present in pineapple extracts This information can be useful in exploring the potential applications of pineapple extracts as a natural alternative to enzymatic

hydrolysis

3 Experiment:

The experiment used test tubes containing water (as a blank), tyrosine standard solutions, and samples of pineapple extract These test tubes were used to perform a series of steps Overall, the experiment aimed to determine the ability and amount of enzymatic activity in pineapple extracts, which can provide

insights into its potential use as a natural alternative to enzymatic hydrolysis.

4 Material and Procedures

4.1 Material

4.1.1 Equipment:

- Blender or stone mortar

- Filter papers (11mm)

- Centrifuges

- Test tubes

- Pipettes (1 mL, 2 mL, and 5mL) and pump

- Beakers (500mL, 250mL and 100mL)

- Falcons (50mL)

- Spectrophotometer

4.1.2 Chemicals:

- Fresh, moderately ripe pineapple

- 2% Casein solution

- 5% Trichloroacetic acid solution

- 0.2M HCl solution

- Tyrosine solution

- Folin-Ciocalteu reagent

- 0.5M NaOH solution

4.2 Procedures

4.2.1 Preparation of sample:

Step 1: Select and cut fresh, ripe pineapple into pieces and then pulverize

it by blender or stone mortar

Step 2: Remove the remaining grounds out by filter papers or centrifuge

to get pineapple juice

4.2.2 Extraction:

Step 1: Prepare the experiment following the table below.

( mL )

Tubes of Tyrosine ( mL )

Tubes of sample ( mL )

Keep the tubes at room temperature

Pineapple solution containing

bromelain

Shake the tubes well and keep in 5 minutes

Pineapple solution containing

bromelain

Step 2: Shake each tube well and keep them still for 10 minutes at room temperature Step 3: Transfer 2mL of filtered solution of each tube into new tubes.

Step 4: Add 5mL of 0.5M NaOH solution and 1mL of Folin-Ciocalteu reagent.

Step 5: Shake the tubes well, then put the tubes at the stand for 15 minutes and

measure at the A620nm

4.2.3 Survey the enzymatic activity:

II RESULTS AND DATA ANALYSIS

1 Results:

Test tubes Tubes of water

( blank )

Tubes of Tyrosine ( standard solution)

Tubes of sample

Tube number 1

(Ctr1)

2 (Ctr2)

3 (Ctr3)

4 (St1)

5 (St2)

6 (St3)

7 (Un1)

8 (Un2)

9 (Un3)

OD 0.3533 0.3824 0.3377 0.6140 0.5641 0.5163 0.6750 0.5040 0.7457

Figure : The tubes are in order Tube of water, Tube of Tyrosine and Tube

of sample after completing step of adding 5mL of 0.5M Sodium Hydroxide (NaOH) solution and 1mL of Folin- Ciocalteu reagent (Step 12

of procedures).

2 Calculation:

The Average OD value of Tube of water (Tube 1,2,3):

𝑋 𝑖=1

𝑁

∑ 𝑋𝑖

𝑁 0.3533 + 0.3824 + 0.33773

The Average OD value of Tube of Tyrosine (Tube 4,5,6):

𝑋 𝑖=1

𝑁

∑ 𝑋𝑖

𝑁 0.6140 + 0.5641 + 0.51633

The Average OD value of Tube of sample (Tube 7,8,9):

𝑋 𝑖=1

𝑁

∑ 𝑋𝑖

𝑁 0.6750 + 0.5040 + 0.74573

→ The ∆OD is calculated by subtracting the OD value of water from the sample tube and the Tyrosine tube

The ∆OD value of Tube of water (Tube 1,2,3):

△𝑂𝐷 = 0 3578 − 0 3578 = 0

The ∆OD value of Tube of Tyrosine (Tube 4,5,6):

△𝑂𝐷 = 0 5648 − 0 3578 = 0 207

The ∆OD value of Tube of sample (Tube 7,8,9):

△0𝐷 = 0 6416 − 0 3578 = 0 2838

The concentration value of Tube of sample:

Csample= ∆𝑂𝐷 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑∆𝑂𝐷 𝑠𝑎𝑚𝑝𝑙𝑒 ×Cstandard(mg/mL) = 0.28380.207 x 0.45 = 0.617

The amount of tyrosine liberated is calculated by the formula below:

msample= ∆𝑂𝐷 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑∆𝑂𝐷 𝑠𝑎𝑚𝑝𝑙𝑒 ×Cstandard(mg/mL) ×Vstandard(mg/mL)

→ msample= 0.28380.207 x 0.45 x 0.5 = 0.308 (mg)

The number of moles of the sample tyrosine is:

→ n = 181 (𝑚𝑔/𝑚𝑚𝑜𝑙𝑒)𝑚 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑚𝑔) x 103= 0.308181 x 103= 1.702 (µ𝑚𝑜𝑙𝑒)

The enzyme activity of bromelain is then determined as the number of µmole

of tyrosine liberated from casein per minute:

Enzyme activity = 𝑝𝑟𝑜𝑡𝑒𝑜𝑙𝑦𝑡𝑖𝑐 𝑡𝑖𝑚𝑒𝑛 𝑠𝑎𝑚𝑝𝑙𝑒 = 10𝑚𝑖𝑛𝑠1.702 = 0.1702 ( mole/min) or (U)∞

As the volume of bromelain solution used in this experiment is V sample (0.2 mL), the enzyme activity of 1 mL bromelain solution is:

Enzyme activity = 𝑝𝑟𝑜𝑡𝑒𝑜𝑙𝑦𝑡𝑖𝑐 𝑡𝑖𝑚𝑒𝑛 𝑠𝑎𝑚𝑝𝑙𝑒 x 𝑉𝑠𝑎𝑚𝑝𝑙𝑒 (𝑚𝐿)1 = 10𝑚𝑖𝑛𝑠1.702 x 0.21 = 0.851 (U/mL)

catalysis in 1mL of solution or 1mg of bromelain mixture for 1 minute is used to measure the activity of bromelain enzyme

III DISCUSSION

Ripe pineapple was blended for juice instead of using stone mortar This will save a lot of time for students At the extraction step, bromelain from pineapple juice was extracted using centrifugation TA’s class used a big falcon for the whole class So, our team did not take photos for it

This method is based on the hydrolysis of enzyme Bromelain to peptide bond which is casein protein present in the all tubes Through the experiment, TCA ( trichloroacetic acid solution) is used for inactivation of the enzyme or

precipitate of the unhydrolyzed protein That is the reason why we can observe precipitation after step 3 in tubes of tyrosine and blank tubes (water)

For standard tubes, we did add 0.5ml tyrosine So, the steps of adding distilled water was to balance the Volume of the whole 9 tubes

For the sample tubes 7,8,9 , TCA was used at step 8 to make the enzyme Bromelain stop reacting to casein

Besides that, we continued to add Bromelain for tubes of tyrosine and blank (step 9) The results showed that pineapple juice extract seemed to be above So,

we think that it would not react a lot since TCA already reacts to unhydrolyzed Casein, then Bromelain added at the 9thstep cannot break down the peptide bond of Casein anymore

After transfer 2mL of filtered solution of each tube into new tubes and add 5mL

of 0.5M NaOHSodium Hydroxide solution and 1 mL of Folin-Ciocalteu reagent

The result after measuring the OPTICAL DENSITY (OD) is part II of this report ( above) - Bromelain has an enzymatic activity of 0.851 (U/mL) That value indicates that under 1 mg of bromelain combination for 1 minute, 0.851g

of Tyrosine will be liberated from Casein breakdown

IV CONCLUSION:

The experiment allowed us to analyze the enzymatic function of bromelain, and we

were able to calculate a value of 0.851 (U/mL), as the substance's ultimate enzymatic activity, by using the aforementioned equation Despite various ambiguities and missteps along the way, we were able to compute the final result and gain insights

about bromelain's activity One of the key findings we made was the importance of timing in the process and how important it is to administer the drugs at the right time Furthermore, we performed replications throughout the experiment to guarantee that the final results were more reliable