effect of temperature on the breakdown of lipid by lipase activity using ph sensor

Candidate Name: Tony Hong (Seung Mo Hong) IB Biology HL

Candidate Number: 00213-021

Date: December 16, 2010

How will changing the temperature affect the rate of lipase activity of

digesting milk fat into fatty acid and glycerol measured using a pH probe?

Aim

To investigate the relationship between temperature and the rate of lipase activity of the digestion of

milk fat into fatty acid and glycerol using a pH probe.

Introduction

Enzymes are proteins that act as a catalyst to speed up chemical reactions. Hence the enzyme activity

is the rate in which the particular enzyme catalyzes a particular chemical reaction.

The rate of enzyme activity is affected by the following:

a) pH – most enzyme have different optimum pH. But most of enzymes would denature in

extreme acidic or basic pH.

b) Temperature – as temperature rises, molecules will have a greater kinetic energy and hence,

more molecules will have a greater energy than the activation energy, leading to more a

greater enzyme activity. However when temperature becomes too high, the enzyme would

start to denature, leading to slower enzyme activity. Most enzymes have an optimum

temperature of 37 ℃.

c) Enzyme concentration – As the concentration increases the enzyme activity increases. As

the concentration decreases, the enzyme activity decreases.

d) Substrate concentration – As the substrate concentration increases, the enzyme activity

would increase as well. However, when it reaches its maximum rate, it would remain

constant despite increasing the substrate concentration.

e) Presence of inhibitors – Presence of inhibitors would consequently decrease the enzyme

activity or may even stop the enzyme activity. It is categorized into three different groups:

competitive, non-competitive and substrate inhibition.

Hence, if any of these variables are changed, the rate of enzyme activity would alter as well.

Lipase is a water-soluble enzyme that catalyzes the hydrolysis of lipid substrates. It plays an important

role in digestion, processing and transporting of dietary lipids.1

1 "Lipase." Wikipedia. 20 Dec 2010. Wikipedia Foundation. 16 Dec 2010 <http://en.wikipedia.org/wiki/Lipase>.




Variables

Variable Measured Method of measuring /controlling the variable

Controlled

Variables

Volume of Milk 5ml of milk was added to each test tube.

Room temperature All trials are experimented in room temperature which is

approximately 28℃

Lipase Concentration 2g of lipase was measured using a electronic weighing

machine and was place inside 100ml of distilled water to

make 2% lipase solution

Amount of lipase and bile

mixture

20ml of bile solution was added to 50ml of 2% lipase.

Then 5ml of lipase and bile mixture was added to each

test tube

Amount of sodium

carbonate

1ml of sodium carbonate was added to each test tube in

order to increase the overall pH of the milk

Independent

Variables

Temperature of lipase The temperature of the lipase solutions were changed

accordingly by 5℃, 25℃, 35℃, 45℃, 55℃

Dependent

Variables

Rate of enzyme activity

of lipase

ΔpH/t (change of pH over

period of time)

As the temperature of the lipase solution changed, the

rate of lipase activity also changes. Change of pH (over

time) was measured through Logger Pro by using a pH

probe to calculate the rate of lipase activity.

Table 1: List of Variables

Hypothesis

The rate of lipase activity is the measure of how fast the lipase enzyme can catalyze the procedure of

breaking down the lipid into triglyceride and fatty acids. The lipase solution would convert the milk

fat into triglyceride and fatty acids, causing the pH to decrease. Five different variables that could

change the rate of the lipase activity are shown in the introduction. However, as it can be seen in table

1, the independent variable is temperature. Hence by changing the temperature of the lipase solution

the change of rate of lipase activity would be measurable.

The following graph would display the rate of enzyme activity against temperature:

Figure 1: Graph representing the ideal relationship between rate of enzyme activity and temperature2

2 "Enzyme activity." Bio Quick Notes. 2010. Rankin County School Distric. 16 Dec 2010

<http://www.rcsd.ms/~rawest/Bio%20Quick%20Notes.htm>.




As it can be seen in figure 1, there is an increase in rate of enzyme activity as the temperature

increases. However, as it reaches its optimum temperature value (approximately 37℃ for majority of

enzymes), the enzyme activity begins to rapidly decrease as the enzyme starts to denature. Hence it

extreme cold or hot temperatures, the enzyme completely denatures and the enzyme activity stops.

As such, the hypothesis for this experiment is that as the temperature of the lipase solution increases

up to its optimum temperature (approximately 37℃), the rate of lipase activity would exponentially

increase. And as the temperature increases over the optimum temperature value, the rate of lipid

activity would rapidly decrease exponentially. Hence, showing a parabolic shape; similar to figure 1.

Apparatus

- Logger Pro

- pH probe

- Milk (not skim milk)

- Lipase

- Bile solution

- Sodium carbonate solution (Na2Co3)

- Electronic thermometer (±0.1)℃

- 10.0cm3 pipette (±0.05)cm

3

- 25.0cm3 pipette (±0.1)cm

3

- Test tubes

- Test tube rack

- Micropipette (±0.006cm³)

- 100.00 cm3 conical flask (±0.10)cm

3

- Distilled Water

- Incubator

- Refrigerator

- Beaker

Procedure

Preparation of making 2% Lipase solution

1. Measure 2.00g of lipase using an electric weighing machine.

2. Measure 100 cm3 of distilled water using a conical flask and put the 2.00g of lipase inside it

3. Stir well until all the lipase is completely dissolved.

Preparation of making the lipase and bile mixture

1. Measure 50 cm3 of 2% lipase solution using a 25.0 cm

3 pipette.

2. Measure 20 cm3 of bile solution using 10.0 cm

3 pipette.

3. Mix two solutions in a beaker to make the lipase and bile mixture

Process of measuring the pH of the different temperature of 2% of lipase solution

1. Measure 5 cm3 of milk into a test tube using a micropipette.

2. Measure 1 cm3 of sodium carbonate (Na2Co3) using a micropipette and add it to the test tube

containing the 5 cm3 milk (in order to increase the pH of the milk)

3. Measure 5 cm3 of the 2% lipase and bile mixture and incubate it to 25℃.

4. Add the 5 cm3 25℃ of lipase and bile mixture to the test tube containing the milk and sodium

carbonate

5. Measure the change of the pH of the milk by using Logger Pro and a pH probe

6. Repeat steps 1-5 three times in order to collect triplicate data.

7. Repeat steps 1-6 with 5℃, 35℃, 45℃, and 55℃ of 2% lipase solutions.




Figure 2: Diagram for the process of measuring the change of pH using pH probes

pH probe

Milk with

sodium

carbonate and

lipase and

bile mixture

Test tube

Test tube

rack




Data collection

Table 2: pH measured for every 50 seconds of the triplicate trial of each different temperature of lipase solution

Time,

t/s pH of milk mixed with 5℃

of 2% lipase solution

p/ (±0.1)

pH of milk mixed with

25℃ of 2% lipase solution

p/ (±0.1)



p/ (±0.1)



p/ (±0.1)



p/ (±0.1)

Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3

0 7.63 7.58 7.60 7.80 8.01 7.80 7.68 7.64 7.62 7.75 7.68 7.68 7.70 7.66 7.66

50 7.70 7.59 7.52 7.81 7.98 7.81 7.68 7.63 7.63 7.67 7.65 7.67 7.64 7.41 7.67

100 7.65 7.58 7.58 7.81 7.97 7.81 7.67 7.61 7.76 7.67 7.61 7.62 7.66 7.57 7.61

150 7.65 7.57 7.57 7.80 7.96 7.80 7.65 7.58 7.58 7.66 7.59 7.61 7.66 7.58 7.62

200 7.63 7.56 7.58 7.79 7.94 7.78 7.62 7.57 7.58 7.65 7.58 7.59 7.64 7.59 7.63

250 7.62 7.55 7.55 7.77 7.93 7.76 7.60 7.55 7.56 7.64 7.56 7.58 7.66 7.57 7.62

300 7.61 7.53 7.53 7.76 7.91 7.75 7.58 7.53 7.52 7.62 7.56 7.56 7.65 7.57 7.63

350 7.60 7.52 7.51 7.74 7.89 7.73 7.56 7.51 7.51 7.60 7.55 7.57 7.65 7.57 7.62

400 7.58 7.51 7.52 7.73 7.88 7.72 7.54 7.49 7.50 7.59 7.54 7.56 7.65 7.57 7.62

450 7.57 7.50 7.50 7.72 7.86 7.71 7.54 7.48 7.48 7.59 7.53 7.55 7.65 7.59 7.61

500 7.56 7.49 7.50 7.70 7.85 7.69 7.52 7.46 7.46 7.58 7.51 7.53 7.66 7.58 7.61

550 7.55 7.48 7.47 7.69 7.83 7.68 7.50 7.45 7.45 7.57 7.52 7.52 7.66 7.57 7.61

600 7.55 7.46 7.47 7.68 7.82 7.67 7.49 7.43 7.44 7.56 7.51 7.50 7.65 7.58 7.63

650 7.53 7.46 7.45 - - - 7.48 7.43 7.40 7.56 7.50 7.49 7.65 7.58 7.62

700 7.52 7.45 7.44 - - - 7.47 7.41 7.41 7.55 7.49 7.48 7.65 7.58 7.63

750 7.51 7.44 7.43 - - - 7.44 7.40 7.41 7.54 7.48 7.48 7.66 7.59 7.62

800 7.51 7.43 7.42 - - - 7.44 7.39 7.39 7.54 7.46 7.47 7.66 7.60 7.62




Figure 3: Graph showing the change of pH against time for every trial of five different temperature of lipase solution




Quantitative Data

Table 3: Rate of lipase activity of triplicate trial of each different temperature of lipase solution

Qualitative Data

Observations

Temperature of lipase

solution, T/℃

(±0.1)℃

Before reaction of milk and lipase

After reaction of milk and lipase

5.0 White, opaque liquid White, opaque liquid (no change)





Table 4: Observation of milk solution before and after the reaction with lipase

Data processing

The average rates of lipase activity of the three trials were calculated by the following equation:

Temperature of

lipase solution,

T/℃

(±0.1)℃

Calculation

Average rate of lipase

Activity, r/(s-1

)

(s-1

± s.d4)

5.0

≈ 0.000249

2.49x10-4

± 0.000023

25.0

≈ 0.000312

3.12x10-4

± 0.000027

35.0

≈ 0.000377

3.77x10-4

± 0.000031

45.0

≈ 0.000219

2.19x10-4

± 0.000029

55.0

≈ 0.000012

1.20x10-5

± 0.000002

Table 5: Calculation of average rate of lipase activity

3 Absolute value was taken for each of the rate

4 s.d - Abbreviated form of Standard Deviation

Temperature of

lipase solution,

T/℃

(±0.1)℃

Rate of lipase activity, r/( s-1

)3

Trial 1 Trial 2 Trial 3

5.0 0.000264 ± 0.000006 0.000260 ± 0.000007 0.000222 ± 0.000017

25.0 0.000281 ± 0.000003 0.000324 ± 0.000003 0.000332 ± 0.000004

35.0 0.000398 ± 0.000009 0.000391 ± 0.000005 0.000342 ± 0.000002

45.0 0.000252 ± 0.000006 0.000199 ± 0.000010 0.000206 ± 0.000011

55.0 0.000014 ± 0.000008 0.000010 ± 0.000014 0.000011 ± 0.000009




Data Presentation

Figure 5:Graph showing the average rate of diffusion against the surface area of NaCl solution

5 Error bar representing the standard deviation of the average rate of diffusion

y = -9E-09x3 + 5E-07x2 - 3E-06x + 0.0003

R² = 0.96380

0.00005

0.0001

0.00015

0.0002

0.00025

0.0003

0.00035

0.0004

0.00045

0 10 20 30 40 50 60

Rate

of

lipase

act

ivity (

s-1)

Temperature (℃)

Rate of lipase activity, r/(s-1) against temperature, T/℃ of

lipase solution

5




Uncertainty/ Error Analysis

Table 6: Uncertainty of the temperature of lipase solution using the electronic thermometer

Temperature of

lipase solution,

T/℃

% uncertainty in temperature, T/℃

ΔT (%)

Absolute % uncertainty in temperature, T/℃

ΔT (℃)

Surface Area, SA/cm2 and uncertainty

ΔSA (cm2)

5.0 2.00% (2.00/100) x 5.0 = 0.1 5.0 ± 0.1

25.0 0.40% (0.40/100) x 25.0 = 0.1 25.0 ± 0.1

35.0 0.29% (0.29/100) x 35.0 = 0.1 35.0 ± 0.1

45.0 0.22% (0.22/100) x 45.0 = 0.1 45.0 ± 0.1

55.0 0.18% (0.18/100) x 55.0 = 0.1 55.0 ± 0.1

Table 7: The absolute uncertainty for each temperature of lipase solution

Temperature of

lipase solution,

T/℃

% uncertainty of temperature

Total % of Uncertainty

Temperature with uncertainty Temperature of lipase

solution using a electronic

thermometer

(ΔT = ± 0.1) cm

% uncertainty of

temperature ( % )

5.0 5.0 ± 0.1 (0.1/5.0) x 100

= 2.00%

2.00% 5.0 ± 2.00%

25.0 25.0 ± 0.1 (0.1/25.0) x 100

= 0.40%

0.40% 25.0 ± 0.40%

35.0 35.0 ± 0.1 (0.1/35.0) x 100

= 0.29%

0.29% 35.0 ± 0.29%

45.0 45.0 ± 0.1 (0.1/45.0) x 100

= 0.22%

0.22% 45.0 ± 0.22%

55.0 55.0 ± 0.1 (0.1/55.0) x 100

= 0.18%

0.18% 55.0 ± 0.18%




Enzyme

Temperature, T/℃

Average rate of lipase activity, r/(s-1

)

(s-1

± s.d)

Lipase

5.0 ± 0.1 2.49x10-4

± 0.000023

25.0 ± 0.1 3.12x10-4

± 0.000027

35.0 ± 0.1 3.77x10-4

± 0.000031

45.0 ± 0.1 2.19x10-4

± 0.000029

55.0 ± 0.1 1.20x10-5

± 0.000002

Table 8: Relationship between the temperature with absolute uncertainty and the average rate of lipase activity




Conclusion

The relationship between the average rate of lipase activity and the different temperature values can

be seen in Figure 5. As the temperature increases, the average rate of lipase activity also increases up

to a certain point (which is known as the optimum value). Then as it reaches its optimum temperature

value it starts to rapidly decrease as the enzyme starts to denature at high temperatures. As it can be

seen in the figure 5, the equation of the line is a cubic equation. Although logically it may not make

much sense as a cubic equation would imply that the graph would decrease, increase, and decrease

again. However, when putting a limit on the domain (as the temperature would be greater than 0, {x| x

> 0}), I believe the cubic equation is a better fit than a quadratic equation. In addition, the R2 value for

the cubic is higher than the quadratic equation. However, when looking at the theoretical, ideal graph

the graph should show a quadratic equation. The problem of my data collection is the value of the rate

of lipase activity when the temperature is 5℃. Theoretically, it should show a much lower rate of

lipase activity. If it did, the data points would almost perfectly fit the ideal graph showing the rate of

lipase activity against temperature. Also, the systematic error and random error must be taken in

consideration. As it can be seen on figure 5, the cubic equation shows that the optimum temperature is

32℃, where as the theoretical optimum temperature of lipase should be approximately 37℃. Hence,

this shows that there are some systematic errors included in the experiment. In addition when

observing figure 5, the error bars are relatively big and when looking at table 8 the standard deviation

of the average rate of lipase activity is also relatively big. This concludes that the experiment consists

of high random error. I believe that this is because the data deals with very small values with five

decimal places. Although the graph on figure 5 somewhat demonstrates the description of the

hypothesis and has a fairly similar trend to the predicted trend (figure 1), due to the fact that it does

not completely follow the trend and the graph mentioned in the hypothesis (optimum temperature is

not close to 37℃ etc) and that it contains high uncertainties/errors, the hypothesis is rejected.

Evaluation

When looking at table 5, 6 the uncertainty through random error is relatively big as the data deals with

values up to five decimal places. In addition when looking at the value of the average rate of lipase

activity when the temperature is 5℃, it can be seen that the presence of systemic error is quite high.

Hence, I believe that the data provided through this example is not very reliable. Here are some of the

weaknesses of the experiment and the ways to improve them:

Weaknesses Improvements

Uncertainty due to systemic error is high because

of the fact that all experiments was run at room

temperature. Hence when taking the lipase

solution from the incubator, the lipase solution

would have either cooled down or heated and the

temperature of it would have changed.

Could have run each experiment in different

atmosphere so that the temperature of the lipase

solution would not have altered by much

(especially for the 5℃ experiment). It is quite

obvious that the average rate of lipase activity is

relatively high for such a low temperature

because it has warmed from 5℃ to a higher

temperature due to the higher room temperature.

The change of pH was relatively very slow.

Hence the difference between the average rates of

lipase activity was relatively small.

Could have used a greater concentration of lipase

solution to speed up the overall reaction of the

digestion of fat and show a greater difference in

the average rate of lipase activity.

The fluctuating initial reactions made it

impossible to take the average initial rate of

lipase activity. This was caused by the method

where the lipase and bile mixture was added and

immediately the lid closed. Hence, the human

error caused the fluctuation.

A more mechanical method is preferred over the

method where humans have to try to close the lid

as soon as possible. Hence with the help of better

apparatus the method could be adjusted allowing

the collection of the initial average rate of lipase

activity.

effect of temperature on the breakdown of lipid by lipase activity using ph sensor

Education

lipase enzyme

enzyme activity increases

greater enzyme activity

enzyme activity decreases

enzyme activity stops

slower enzyme activity

milk lipase

lipase solutions