Experiment To Investigate Rate

Published Date: 02 Nov 2017

Enzyme Activity

Aim: To investigate how varying concentration of lipase affects the rate at which lipase catalyses the hydrolysis of the lipids found in full fat milk.

Introduction

Enzymes are bio-catalyst which speed up the rate of biochemical reactions. Enzymes are globular protein. They possess an active site which binds specifically to a substrate. They are required in small amount as they have rapid turnovers. The molecules have a small functional group known as the ‘active site’. This ‘active site’ is made up of the following:

Contact amino acid – to provide specificity to enzyme.

Catalytic amino acid- to catalyze reactions and lower down the activation energy (Ea).

The 3-D structure of the enzyme is made of amino acid which carries out the following roles :

Maintain the general globular shape of the enzyme.

Maintain the shape of the active site.

Contribute to solubility of enzyme.

Enzyme catalyzed reactions are affected by many factors. These are :

Temperature

pH

Presence of inhibitors

Substrate concentration

Enzyme concentration

The way in which enzymes operate is the ‘Lock and Key’ hypothesis postulated by Emil Fisher in 1984. This hypothesis describes how the substrate binds with the active site of the enzyme. The enzymes being the larger molecule represent the ‘lock’ and the substrate the ‘key’. The hypothesis states that when the active site is complementary to the substrate. It will fit exactly into the active site of the enzyme and form the ‘Enzyme-Substrate Complex’ (also denoted by ESC). Reaction occurs and product will be formed. The product is formed is not complementary to active site of the enzyme.

Lipase is a type of enzyme which hydrolyzes only triglycerides (substrate) into fatty acids and glycerol. Fatty acids are organic acids consisting of 14-24 carbon atoms with a carboxyl group (-COOH) joined at one end. The acid’s tail is hydrophobic i.e. why lipids are insoluble in water and are transported by lipoproteins by blood.

Lipase oxidizes triglycerides using 3 molecules of water to break 3 ester bonds and form a glycerol molecule and 3 fatty acids.

Digestion of fats in humans takes place in the stomach and duodenum of the small intestine. Lipase enzyme is sensitive to pH alteration. Every enzyme works most efficiently at an optimum pH as the structure of the active site of the enzyme is arranged by ionic and hydrogen bonds between amino and carboxyl groups of the polypeptides.

Variables

Independent: Concentration of the substrate

Dependent:: Enzyme activity measured by decrease in pH over time.

Controlled:

Variable

How was it controlled

Explanation

Concentration of Enzyme

1%

To have same amount of active site.

Time for enzyme to act

10 minutes

To have same reaction period.

Brand of full cream milk

Red Cow

To have same amount of lipids

Volume of milk used

3ml

To have same substrate concentration.

Experiment was set at different temperature

10oC,28oC,40oC,60oC

-The 10oC temperature was done in the refrigerator

-The 28oC was at room temperature.

-The 40oC and 60oC was set using a waterbath.

Apparatus needed:

Beaker

pH probe

test tubes

pipette

stop watch

Lipase (1 %)

Milk (full cream)

Data Logger

Procedure:

Using a pipette , pour 3 ml of full cream milk in a test-tube.

Place test-tube rack in the water bath.

Set temperature of water bath at 40oC.

Place the test-tube containing the content in a test-tube rack using a test-tube holder

Add enzyme lipase after 2 minutes with an interval of 20 seconds between each repeats done.

Check the initial pH of each test-tube using a data logger and a pH probe and record in a table.

Place the test-tube in the water bath for 10 minutes still having an interval of 20 seconds between each test-tube.

Then measure the final pH of each test-tube and record them in the table.

Repeat the experiment for the following temperatures: 10oC, 28oC,40oC, 60oC

Note:

Taking into consideration that each temperatures have 5 repeats done.

The first two temperatures were not done using a water bath as:

For 10oC, the experiment was carried out in the fridge.

For 28oC, the experiment was carried out at room temperature.

Data Collection and Processing

Results:

At 10oC, pH

Trials

Initial

Final

Percentage change in pH

1

6.60

6.34

-3.939393939

2

6.79

6.35

-6.48011782

3

6.85

6.36

-7.153284672

4

6.81

6.33

-7.04845815

5

6.83

6.43

-5.856515373

Mean Percentage change

-6.095553991

Standard Deviation

1.311571147

Table 1: Showing results of mean percentage change in pH at 10oC.

At 28oC, pH

Trials

Initial

Final

Percentage change in pH

1

6.65

6.12

-7.969924812

2

6.81

6

-11.89427313

3

6.82

5.98

-12.31671554

4

6.83

6.03

-11.71303075

5

6.82

6.05

-11.29032258

Mean Percentage change

-11.03685336

Standard Deviation

1.753619763

Table 2: Showing results of mean percentage change in pH at 28oC.

At 40oC, pH

Trials

Initial

Final

Percentage change in pH

1

6.57

5.81

-11.56773212

2

6.72

5.84

-13.0952381

3

6.76

5.85

-13.46153846

4

6.73

5.85

-13.07578009

5

6.74

5.86

-13.05637982

Mean Percentage change

-12.85133372

Standard Deviation

0.736867072

Table 3: Showing results of mean percentage change in pH at 40oC.

At 60oC, pH

Trials

Initial

Final

Percentage change in pH

1

6.60

6.00

-9.090909091

2

6.66

5.95

-10.66066066

3

6.62

5.92

-10.57401813

4

6.64

5.89

-11.29518072

5

6.62

5.90

-10.87613293

Mean Percentage Change

-10.49938031

Standard Deviation

0.835256572

Table 4: Showing results of mean percentage change in pH at 60oC.

Temperature/oC

Mean Percentage change in pH

Standard Deviation

10

-6.095553991

1.31157115

28

-11.03685336

1.75361976

40

-12.85133372

0.73686707

60

-10.49938031

0.83525657

Table 5: Showing results of mean percentage change of pH at different temperatures.

Graph 1: Showing the Effect on Temperature on pH

When there is an increase in temperature from 10-40oC, there is a decrease in pH of the reaction mixture. For example at 10oC, the mean change in pH is -6.0955 whereas at 28oC, the mean change in pH is - 11.036. This is because as temperature increases, particle gain more kinetic energy, causing more vibrations and collisions. More Enzyme Substrate Complex (ESC) formed. Therefore more products are formed.

More lipids are hydrolyzed to fatty acids and glycerol. Fatty acids decreases pH of mixture.

Above 40oC, the enzyme is denatured, so less decrease in pH is noted.

Different errors have been committed during the experiment. Those possible errors and their suggestion for improvement are shown in the Table 6.

Possible Source of Error

Suggestions for Improvements

Only the substrate was acclimatized at the set temperature.

Both the substrate and the enzyme should have been acclimatized with the set temperature so as to obtain accurate results.

http://repositorio.ucp.pt/bitstream/10400.14/6604/3/Lipase%20catalyzed%20modification.pdf [accessed 30.4.13]