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NAME: Michael Timson

DATE: 11/09/2014
FORM: L6-4
LAB: #
SUBJECT: Biology
AIM: To investigate the effect of temperature on the hydrolysis of lipids by lipase.

Lipids, such as the fats in milk, are digested by the body. They are broken down into
fatty acids and glycerol by an enzyme called lipase. Triglyceride + 3H 2O --> glycerol + 3
fatty acid chains. It is necessary to break down fat in the digestive system to allow its
absorption through the membranes of the gut and enabling it soluble enough to be transported
in the blood. Gastric lipase, secreted by the stomach lining. Most lipid digestion in the adult
occurs in the upper loop of the small intestine and is accomplished by a lipase secreted by the
pancreas. In recent years, knowledge of lipases has increased dramatically in the areas of
molecular structure and mechanism of action. Lipases are enzymes which catalyse the
hydrolysis of triglyceride to give di- and mono- glycerides, glycerol and free fatty acids.
Lipases perform essential roles in the digestion, transport and processing of dietary lipids
such as triglycerides, fats and oils in most, if not all, living organisms.
Enzymes are proteins that act as catalysts for biological reactions. Enzymes, like all
catalysts, speed up reactions without being used up themselves. They do this by lowering the
activation energy of a reaction. All biochemical reactions are catalysed by enzymes. Since
enzymes are proteins, they can be denatured in a variety of ways, so they are most active
under mild conditions. Most enzymes have optimum activity at a neutral pH and at body
temperature. Enzymes are also very specific. They only act on one substrate or one class of
related substrate molecules. The reason for this is that the active site of the enzyme is

complementary to the shape and polarity of the substrate. Typically, only one kind of
substrate will fit into the active site.
Lipases are involved in diverse biological processes ranging from routine metabolism
of dietary triglycerides to cell signalling and inflammation. Thus, some lipase activities are
confined to specific compartments within cells while others work in extracellular spaces. In
the example of lysosomal lipase, the enzyme is confined within an organelle called
the lysosome.





as pancreatic




into extracellular spaces where they serve to process dietary lipids into more simple forms
that can be more easily absorbed and transported throughout the body. Fungi and bacteria
may secrete lipases to facilitate nutrient absorption from the external medium (or in examples
of pathogenic microbes, to promote invasion of a new host.
For two molecules to react they must collide with one another in the right direction
(orientation) and with sufficient energy. This sufficient energy means that between them they
must have enough energy to overcome the energy barrier to reaction. This is known as
the activation energy. Enzymes have an active site. This is part of the molecule that has just
the right shape and functional groups to bind to one of the reacting molecules. The reacting
molecule that binds to the enzyme is called the substrate. An enzyme-catalysed reaction takes
a different 'route'. The enzyme and substrate form a reaction intermediate. Its formation has a
lower activation energy than the reaction between reactants without the presence of a catalyst.
That is:
Rout A:

Reactant 1 + Reactant 2 Products.

Rout B:

Reactant 1 + Enzyme Intermediate

Intermediate + Reactant 2 Product

Test tubes, Milk, Phenolphthalein, 10cm3 syringe, 1cm3 syringe, Water bath,
Thermometer, Glass rod, Lipase

The test tube with the temperature being investigated was labelled accordingly. 5
drops of phenolphthalein was added to the test tube. 5 cm 3 of milk was measured using a
syringe and was added to the test tube. 7 cm 3 of sodium carbonate solution was measured
using another or syringe and was added to the test tube. A thermometer was placed into the
test tube. The test tube was placed into a water bath and left until the contents reach the same
temperature as the water bath. The thermometer was removed from test tube and replaced
with a glass rod. A 2 cm3 syringe was used to measure 1 cm3 of lipase from the beaker in the
water bath for the temperature investigated. The lipase was added to the test tube. The
stopwatch was then started. The contents was stirred until the solution loses its pink colour.
The stopwatch was stopped and the time taken was noted in a suitable table of results. The
experiment was then repeated for all the other temperatures.

Temperature (C)

Time taken (s)

Initial rate of reaction






























From the table of results shown above, the time taken for the solution to lose its pink
colour decreased from 15 C to 45 C as the temperature was increased and thus, the rate of
the reaction was calculated to have increased. Beyond 45 C, there was an increase in time for
the reaction to occur and a decrease in the initial rate of reaction where 55 C was determined
to be 111 seconds with an initial rate of 0.005 and 70 C to 100 C had no time reading an
thus an initial rate of 0.
From the results, a graph of initial rate of reaction vs temperature was plotted. As
observed from the graph, there was gradual increase in the rate of reaction between 0 C to 25
C. From 15 C to 45 C there was then a rapid incline in the rate of reaction where 45 C was
captured as the peak of the graph. This is thus represents the optimum temperature for
enzyme activity. Beyond the optimum temperature, there was then a drastic decline in the
reaction where at 70 C to 100 C, no enzyme activity was displayed.
In increasing the temperature up to 45 C, the rate of reaction is increased as a result
increasing collision rate between the enzyme, lipase, and the substrate molecules. The highest
rate of reaction is at the optimum temperature for the enzyme. The rate of reaction then
reduces as temperature increases until, the reaction stops altogether. As temperature surpasses
45 C, the protein structure of the enzyme is denatured by heat. Hence the molecule loses its
shape and the enzyme is de-activated. When an enzyme denatures, the substrate no longer fits
into its active site because secondary, tertiary and quaternary structure of the enzyme have
broken down (i.e. the molecule has lost its shape and is simply a strand of its initial
monomers (amino acids)). As the substrate does not fit in, the enzyme no longer functions.
Within this experiment there were various sources of errors. There were contamination
of reagents to some extent which would have affected the time taken in the results and hence
the rate of hydrolysis of sucrose by invertase. The factor of reaction time was also involved in
stopping the stopwatch and the experiment was conducted in an air conditioning environment
and thus temperature fluctuations occurred. Temperature, being one of the factors affecting
initial rate of reactions of the enzymatic activities, would thus cause a slight depletion in the
accuracy of the results. It is recommended that before conducting the experiment, that all
apparatus are washed thoroughly to ensure that there are no residue of chemicals on the
apparatus when conducting the experiment. Also, the experiment could be conducted using
more temperatures, hence acquiring a more accurate observations pertaining to the effects of
the increase of invertase concentration. The experiment could have also been repeated more

than twice to acquire more Tn values and hence receive a more accurate average for time
taken for the hydrolysis of the lipids.

CONCLUSION: Based on the results obtained increasing temperatures from 0 C to 45 C

reduced the time taken for the lipase to break down the fat in milk. Over this temperature, the
time taken was increased until no reading was obtained as the lipase denatured.