Title of practice

Effect of temperature in lactase capacity to catalyze lactose hydrolysis.

Introduction

Enzymes are biological catalysts, that is, they speed up reactions that are normally too slow or that have very high activation energies. Enzymes are proteins and they work by lowering the activation energy needed for a certain reaction to occur, by giving the substrate what is called an active site, a site where the reactants can be brought to react.

Enzymes have a very important role in the human body and industry, let’s take for example Lactase. Lactase is the enzyme that speeds up the hydrolysis of lactose, a disaccharide that can be found in dairy products. This hydrolysis reaction leads to the production of two monosaccharides, Galactose and Glucose.

Figure 1 Hydrolysis of lactose[1]

Lactase is naturally produced in the small intestine but several people lack a good production of this enzyme, which leads to a high level of lactose, this is called lactose intolerance. This disease can lead to nausea, diarrhea, gas, abdominal pain, among other symptoms.

Lactase is also commercially produced and it is used for the creation of Lactose-free milk, ice cream, lactase tablets for lactose intolerant people, among other uses. Enzymes need certain optimum conditions to work at their best; they can be affected by temperature, pH, enzyme concentration and substrate concentration.

The objective of the following practice is to study the effect of temperature on the rate of lactose hydrolysis in presence of the lactase enzyme.

Research Question

How will the different temperatures (9 ˚C, 26 ˚C and 38 ˚C) affect the hydrolysis rate of lactose in presence of lactase?

Hypothesis

At higher temperatures the concentration of glucose will be greater, which demonstrates a higher hydrolysis rate of lactose. This will happen because at higher temperatures the kinetic energy of the system will be greater; this will lead to stronger and faster energetic collisions, which will cause a faster breaking of the reactants bonds[2]. There’s a point in which kinetic energy is so much that the active site of the enzyme is “deformed” causing the inability of the enzyme to function as a catalyst, this is called denaturation. The optimum temperature for lactase is 45 ˚C before it starts to denature[3]. With all that explained the hypothesis of this experiment is that the reaction at 38 ˚C will present a higher level of glucose in the samples and therefore a higher rate of lactose hydrolysis and the samples at 9 ˚C will have the lowest rate.

Variables

There are 4 main factors that affect enzyme activity and they are: pH, temperature, and enzyme and substrate concentration[4]; this four factors will be controlled during all the experiment as can be seen in the following table:

Table 1 Variables involved

Type of variable / Variable / Units / Control Method
Independent Variable / Temperature / Celsius / Temperature will be monitored during all the experiment with a thermometer (± 0.5 ˚C)
Dependent Variable / Glucose / Mg/dL / Glucose will be monitored every 10 minutes during the experiment with a Contour® TS Bayer® Glucometer (±5mg/dL).
Controlled Variable / Enzyme concentration
Substrate concentration / UFL/mL
Milliliters of milk / The amount of UFL/mL will be controlled when making the enzyme solution. Lactofin® lactase will be used with a concentration of 9000 UFL per pill.
The quantity of milk will be controlled, to control the estimated substrate concentration.
Acidity/Basicity / pH / pH Strips

Materials

·  3 Test tube racks

·  9 Test tubes

·  9 Thermometers (± 0.5 ˚C)

·  1 Contour® TS Bayer® Glucometer (±5mg/dL)

·  63 Contour® TS Test Strips

·  1 Mortar

·  1 Beaker (±5%)

·  10 mL Pipettes (±5%)

·  Incubator

Reagents

·  Lactofin® Lactase pills

·  A liter of LALA® Whole Milk

Procedure

Based on the reaction of lactose hydrolysis, the method was designed to measure glucose, a product of the reaction, to measure the rate of lactose hydrolysis. They interact in a proportional way, the more glucose present; the more lactase was used to hydrolyze the lactose. The following is the procedure that was used for the experiment:

1.  Grind 1 Lactase pill on the mortar, put it on a 100mL beaker and add distilled water to complete 100mL; this will give a 90 UFL/mL solution.

2.  Label 3 test tubes as Sample1, Sample2 and Sample3, in front of this write the temperature that will be tested.

3.  Add 1mL of milk to each of the test tubes; this will be the substrate solution.

4.  Place a thermometer in each of the test tubes.

5.  Wait for the milk and the enzyme solution to get to the desired temperature:

5.1  For the 9 ˚C experiment put both solutions on the refrigerator at 6 ˚C, this will make the solutions to get a 9 ˚C (± 0.5 ˚C) temperature. Monitor temperature of the solutions with the thermometers.

5.2  For the 26 ˚C use a water bath to get to the desired temperature. Monitor temperature of the solutions with the thermometers and add water to the bath to control it.

5.3  For the 38 ˚C use an incubator and monitor temperature with the thermometers.

6.  Add 2mL of the enzyme solution to each of the test tubes.

7.  Measure temperature and glucose level each 10 minutes for 1 hour and one initial measure, a total of 7 test strips will be used test tube. The glucose is measured by placing a test strip on the glucometer[5], taking a drop from the thermometer and placing the drop in the test strip.

8.  Collect Data.[6]

Figure 2 Test tubes with enzyme and substrate Solutions

Figure 3 Test tubes of enzyme and substrate solutions with thermometers on the refrigerator for the 9 ˚Csamples

Safety

None of the reactants needed for the experiment are dangerous, so the use of gloves is not necessary. Heat gloves are not necessary, the temperatures used are tolerable.

Data collection

In the following tables the raw data collected from the experiments will be represented. The tables contain glucose concentration that was taken every ten minutes with the glucometer, with an uncertainty of ±5mg/dL. In the table it will be also the temperature every 10 minutes taken from the thermometers in the test tubes. The temperature was recorded thermometers with an uncertainty of ± 0.5 ˚C.

Table 2 Samples at 9 ˚C

Sample1 / Sample2 / Sample3
Time
(minutes) / Glucose Concentration (mg/dL) / Temperature
(Celsius) / Glucose Concentration (mg/dL) / Temperature
(Celsius) / Glucose Concentration (mg/dL) / Temperature
(Celsius)
0 / 12 / 9 / 10 / 9 / 10 / 9
10 / 12 / 9 / 10 / 7 / 11 / 8
20 / 20 / 8 / 18 / 9 / 16 / 9
30 / 22 / 9 / 23 / 9 / 22 / 9
40 / 27 / 8 / 26 / 9 / 26 / 9
50 / 32 / 9 / 30 / 8 / 32 / 8
60 / 36 / 9 / 35 / 9 / 36 / 9

Table 3 Samples at 26 ˚C

Sample4 / Sample5 / Sample6
Time
(minutes) / Glucose Concentration (mg/dL) / Temperature
(Celsius) / Glucose Concentration (mg/dL) / Temperature
(Celsius) / Glucose Concentration (mg/dL) / Temperature
(Celsius)
0 / 12 / 26 / 10 / 26 / 11 / 26
10 / 32 / 25 / 36 / 26 / 38 / 25
20 / 57 / 25 / 58 / 26 / 62 / 26
30 / 87 / 26 / 92 / 25 / 88 / 26
40 / 116 / 26 / 107 / 26 / 114 / 27
50 / 135 / 26 / 139 / 27 / 133 / 26
60 / 154 / 25 / 153 / 26 / 154 / 26

Table 4 Samples at 38 ˚C

Sample7 / Sample8 / Sample9
Time
(minutes) / Glucose Concentration (mg/dL) / Temperature
(Celsius) / Glucose Concentration (mg/dL) / Temperature
(Celsius) / Glucose Concentration (mg/dL) / Temperature
(Celsius)
0 / 10 / 36 / 11 / 37 / 10 / 37
10 / 67 / 38 / 59 / 38 / 64 / 37
20 / 119 / 38 / 103 / 37 / 114 / 38
30 / 156 / 38 / 148 / 38 / 146 / 38
40 / 163 / 37 / 164 / 38 / 171 / 37
50 / 175 / 38 / 177 / 38 / 169 / 39
60 / 184 / 38 / 182 / 37 / 185 / 38

The pH of milk was checked with ph strips. It recorded a ph of 6.3 which is good comparing it to the optimum ph for Lactase which is 6.7[7].

Data processing

To graph the raw data and analyze the results more easily, the mean of the samples of each temperature is needed. The mean can be obtained by a simple formula:

Example:

To get the mean of the results at 20 minutes of the samples at 9 C˚ the next step should be done:

As mean is greatly influenced by outliers, a standard deviation test is needed, to see how far away from mean the data items are. Sample Standard deviation can be obtained by the next formula:

Where represents the data item.

Example:

The standard deviation of the results at 20 minutes of the samples at 9 C˚ is obtained by using the formula:

The mean and standard deviation for the other samples are represented in the following tables; the glucose concentration column contains the means of the samples at each temperature (e.g. Table 5 contains the mean of Sample 1, 2 and 3):

Table 5 Glucose Concentration of sample at 9 ˚C

Time
(minutes) / Glucose Concentration (mg/dL) / Standard Deviation
0 / 10.667 / 1.15470054
10 / 11 / 1
20 / 18 / 2
30 / 22.333 / 0.57735027
40 / 26.333 / 0.57735027
50 / 31.333 / 1.15470054
60 / 35.667 / 0.57735027

Graph 1 - Graph of Glucose Concentration of sample at 9 ˚C

Graph1 represents the data from Table 5, the equation of best fit for the information was obtained by logistic regression which was calculated with the graphic calculator. The logistic model was used to draw the line of best fit of these points because at some time during the hydrolysis the level of glucose will slowly stop growing until it totally stops, this happens because of the level of lactase available to catalyze the hydrolysis combined with the temperature. At high temperature the glucose will stop growing at a higher value of glucose concentration because less lactase is needed for the reaction to occur, so the glucose level will go higher in the same time. The next graph represents the whole logistic model of Graph1:

Graph 2 Complete logistic model of Graph 1

As it can be seen from the graph the hydrolysis reaction will probably stop at about 150 minutes at a glucose concentration of 48.6 mg/dL, the reaction cannot continue because the lactase capacity to catalyze is rapidly removed because of the low temperature

.

The following are the table and graphs of the other temperatures samples:

Table 6 Glucose Concentration of sample at 26 ˚C

Time / Glucose Concentration (mg/dL) / Standard Deviation
(minutes)
0 / 11 / 1
10 / 35.333 / 3.05505046
20 / 59 / 2.64575131
30 / 89 / 2.64575131
40 / 112.333 / 4.72581563
50 / 135.667 / 3.05505046
60 / 153.667 / 0.57735027

Graph 3 Graph of Glucose Concentration of sample at 26 ˚C

Table 7 Glucose Concentration of sample at 38 ˚C

Time
(minutes) / Glucose Concentration (mg/dL) / Standard Deviation
0 / 10.333 / 0.57735027
10 / 63.333 / 4.04145188
20 / 112 / 8.18535277
30 / 150 / 5.29150262
40 / 166 / 4.35889894
50 / 173.667 / 4.163332
60 / 183.667 / 1.52752523

Graph 4 Graph of Glucose Concentration of sample at 38 ˚C

The next graph represents the comparison between the 3 temperatures:

Graph 5 Comparison of glucose concentration between the reactions at different temperatures

As it can be seen from the graph the best temperature for the enzyme is 38 ˚C, which agrees to the literature previously cited that states that the optimum temperature for lactase is 45 ˚C. The lactase at 38 ˚C catalyses and generate a very fast lactose hydrolysis, it’s important to mention that there’s only a difference of 30 mg/dL between the samples at 26 ˚C and the samples at 38 ˚C but the hydrolysis at 26 ˚C was considerably slower; both reactions went to about 152 mg/dL, but the samples at 38 went to that concentration in 30 minutes and the samples at 26 ˚C in 60 minutes. That is the rate of reaction was as twice as fast at 38 ˚C until the lactase concentration and temperature didn’t make possible more hydrolysis. The reaction at 9 ˚C was very slow; it went to a maximum of 35.667 in 60 minutes.

Conclusion and evaluation

According to the evaluation of the Graph 5, the hypothesis proposed at the beginning of the practice is correctly supported.

A higher difference between the 26 ˚C samples and the 38 ˚C was expected but as seen from the graphs the quantity of lactose hydrolyzed is not very different, the important difference resides in the speed of the reaction; the 38 ˚C samples went to 152mg/dL twice as fast as the 26 ˚C samples.

The experiment is really expensive because of the necessity of the glucometer strips; because of this, the practice was done using just 7 strips per sample, to record the initial glucose concentration and every ten minutes for one hour. If possible, the experiment should be done by taking the glucose concentration every 10 minutes but for 2 hours to be able to see the complete model.