Lactase is an enzyme that is commonly used for the breakdown of lactose (a sugar within dairy products) into glucose and galactose. Lactase can be extracted from yeasts and fungi such as Aspergillus niger, Aspergillus oryzaei, Kluyveromyces fragilis and Kluyveromyces lactisii. Lactose-intolerance affects nearly 75% of all adults, who cannot digest dairy products since they can not metabolize lactose. Thus, it is useful to know how different amounts of the enzyme lactase affects lactose levels because lactase is used in creating “lactose-free” products. The purpose of the investigation was to determine how the amount of lactase affects rate of the breakdown of lactose.

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Hypothesis: By increasing the amount of lactase, the rate of lactose breakdown will increase at a uniform rate until the amount of lactase is greater than that of the amount of lactose, after which the rate of reaction will remain constant.

Table 1: The Effect and Use of Variables

Independent Variables Dependant Variables Controlled Variables

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Variable Information Variable Information Variable Information

Amount of

Lactase

The enzyme used in the

break down of lactose.

Different amounts such as 0.1 g, 0.2 g, 0.4

[Glucose] Glucose is a product of

lactose breakdown.

Hence, glucose

levels can be

Amount of Lactose

Time

Temperature

Lactose powder was weighed (0.1g) on the electronic balance (±0.01g)

A stop watch timer on the iPod touch 2G(±0.02sec) was used to record the 10 sec interval

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g, 0.5 g, 0.6 g, 0.8 g were used. These amounts were measured

out using the electronic

balance (±0.01). By changing the

used as an accurate

measure of the rate of

breakdown. This was

measured using glucose

Water Volume

Two thermometers were used to keep the temperature of the water and air consistent for all trials.

Water was poured into a graduated cylinder (±0.02ml) to be measured

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amount of lactase, the rate of breakdown of

lactose can be observed

test strips to compare

their colour to the colour

chart provided

pH Level

and then poured into a medium sized test tube.

Although the levels were not measured, since no additional substances were added to the solution, the pH level should have been the same for all trials

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Figure 1: Work Area

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Graduated Cylinder

Test Tube Rack with Test Tubes

Glucose Test StripsPaperTweezersPestleStopwatch (Ipod)

Colour Chart

Lactase Tablets

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iii

Materials:

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Water

39- Tablets of ‘Dairy Digestive’ containing 9,000 FCC lactase units of the enzyme lactase per tablet. Weight: 0.2g/tablet. Made by: Perrigo International. Brand name: Life. (±450 FCC lactase units)

18 grams of the 50g lactose powder –Brand: Lice 1- 10ml Graduated cylinder –Brand: Pyrex (±0.02ml) 1- Electronic balance- Brand: Sargent Welch SW-410. Max weight: 410g (±0.01g) 2 - Eversafe Thermometer (-20°C to 110°C; +/- 0.1C) 1- Stopwatch. IPod touch 2G. Made by Apple Incorporated (±0.02seconds) 1- Test tube rack 1- Medium sized test tube –Brand: Pyrex

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39- Glucose reagent strips. Made by: Bayer HealthCare LLC. Brand name: Diastix (±5mmol/L) 1- Tweezers 1- Pestle 3- Sheets of paper- 8 by 11 inches 1-400ml Beaker – Brand: Pyrex (±5%) 1- Stirring rod

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Procedure:1. A thermometer was left on the table to monitor air temperature2. One beaker was filled with approximately 270ml of water3. A second thermometer was left in the beaker to monitor water temperature

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4. 15ml of water was measure out using the graduated cylinder and then poured into a medium sized test tube

5. 1g of lactose powder was measured on a piece of paper using the electronic balance6. One 0.2g lactase tablet was placed on the other piece of paper and crushed lightly into a powder

using the pestle7. Two 0.1g halves of the tablet were measured out using the electronic balance and placed on

separate pieces of paper.8. One glucose test strip was placed in the tweezers and set beside the medium sized test tube9. Lactose was poured into the test tube and stirred using the stirring rod

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Observations Time in Procedure Description

Initial Observations

Once lactose was added to the water, but before lactase

was added

Lactose- a white powder, crystallized, opaque and dry. Placed into the water, did not appear to dissolve, it just settled to the bottom. Water was diluted and turned translucent.

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Experimental Observations

After the addition of lactase

Lactase- an opaque tablet crushed into a white, opaque, dry powder. Placed into the water and was stirred, but did not dissolve, and no significant observable event occurred. Eventually settled on the bottom as well.

The observations from table 2 showed that there was no effect when adding lactase to lactose, while by using equipment mentioned in the procedure, the effects can be recorded from the molecular level.

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Table 3: Effect on Rate of Breakdown as the Amount of Lactase is Increased

Amount of Lactase

Trial

[Glucose]

(mmol/L)

(± 5mmol/L)*

Time

(seconds)

(±0.02sec)

Amount of Lactose

(Grams)

(±0.01g)

Trial’s Breakdown

Rate**

(mmol/L/sec)

Average Breakdown

Rate**

(mmol/L/sec)

FCC lactase units

(±450 FCC lactase units)

Grams

(±0.01g)

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4,500 0.1

1 6 10 1.0 0.60 (±0.50)

0.667 (±0.866)***

2 7 10 1.0 0.70 (±0.50)

3 7 10 1.0 0.70 (±0.50)

9,000 0.21 15 10 1.0 1.50 (±0.50)

1.53 (± 0.866)2 17 10 1.0 1.70 (±0.50)

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3 14 10 1.0 1.40 (±0.50)

18,000 0.4

1 30 10 1.0 3.00 (±0.50)

2.97 (± 0.866)2 29 10 1.0 2.90 (±0.50)

3 30 10 1.0 3.00 (±0.50)

22,500 0.5 1 39 10 1.0 3.90 (±0.50) 3.87 (± 0.866)

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2 37 10 1.0 3.70 (±0.50)

3 40 10 1.0 4.00 (±0.50)

27,000 0.6

1 50 10 1.0 5.00 (±0.50)

4.80 (± 0.866)2 46 10 1.0 4.60 (±0.50)

3 48 10 1.0 4.80 (±0.50)

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36,000 0.8

1 56 10 1.0 5.60 (±0.50)

5.60 (± 0.866)2 56 10 1.0 5.60 (±0.50)

3 56 10 1.0 5.60 (±0.50)

*See Limitations and Errors **Uncertainties appear to be identical, but result variation can be seen by the fifth or higher decimal place

***Due to the limitations of glucose concentration (which was used in calculations), the uncertainty is unreliable

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In table 3, all the trials had similar results in terms of breaking down the lactose. All of the trials had been deemed successful since glucose was present after testing for glucose. The values for glucose concentration appear to be increasing at a uniform rate, but a graph is required to clarify these results.

Sample Calculations:

Average Glucose Concentration: 9,000 FCC lactase units Time Uncertainty:

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Trial 1 = 15 Average = Trial1+Trial2+Trial3

3 Smallest unit of

measurement: 0.1

Trial 2 = 17 Average = 15+17+14

3 0.1sec * 0.2 (20% rule)

Trial 3 = 14 Average = 463

≈ 15.3 = ± 0.02sec

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Trial’s Rate of Breakdown: Average Rate of Breakdown:

Trial 1 – 9,000 FCC lactase units 9,000 FCC lactase units

Glucose concentration: 15mmol/L Average Glucose concentration: 15.3

Time: 10 seconds Time: 10 seconds

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15mmol /L10 sec

=¿ 1.5mmol/L/second15.3mmol /L10 sec

=¿

1.53mmol/L/second

Weight Uncertainty: grams (g)

Given - Uncertainty of electronic balance: ± 0.01g

Amount of Lactase Uncertainty: FCC lactase units

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Given – Uncertainty: ± 450 FCC lactase units

Glucose Concentration Uncertainty: mmol/L

Estimated - ± 5mmol/L Please refer to Limitations section

Rate of Breakdown Uncertainty: Trials and Average- 4,500 FCC lactase units

Trial 1 – 6mmol/L Glucose uncertainty – ±5mmol/L

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Time –10 seconds Time uncertainty – ±0.02 seconds

Breakdown rate in trial 1 – 0.6mmol/L

Relative uncertainty = r = AbsoluteUncertainty

Magniude → r1 =

56

≈ 0.833(glucose), r2 = 0.0210

= 0.002

(time)

√r12+r22=√0.8332+0.0022≈0.833 (Percent error)

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Percent error * Breakdown rate = Breakdown rate uncertainty (trial 1)

0.833*0.6≈0.5

∴ the uncertainty for the breakdown rate of lactose in trial 1 is approximately ±0.5mmol/L/sec

Average: 4,500 FCC lactase units

Trial 1 ≈ 0.5(e1) Trial 2 ≈ 0.5(e2) Trial 3 ≈ 0.5(e3)

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Uncertainty for average breakdown rate = e4= √e12+e22+e32≈√3∗0.52≈0.866

∴ the uncertainty for the average breakdown rate of lactose is approximately ±0.866mmol/L/sec

Graph 1: Rate of Breakdown of Lactose (mmol/L/sec) vs. Amount of Lactase (FCC lactase units)

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0 5 10 15 20 25 30 35 400

1

2

3

4

5

6

Trial 1

Trendline (Trial 1)

Trial 2

Trendline (Trial 2)

Trial 3

Trendline (Trial 3)

Amount of Lactase (FCC lactase units- in 1000's)

Rate

of B

reak

dow

n of

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(mm

ol/L

/sec

)

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In graph 1, it appears as if there is a direct relation between the rate of breakdown and the amount of lactase. All of the trials appear to be relatively similar, and appear to increase at a constant linear rate as seen above. All three trends appear to be nearly identical to each other, so a graph of the average of the trials should look very similar.

Graph 2: Average Rate of Breakdown of Lactose (mmol/L/sec) vs. Amount of Lactase (FCC lactase units)

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0 5 10 15 20 25 30 35 400

1

2

3

4

5

6

Average of TrialsTrendline (Average Trial)

Amount of Lactase (LCC lactase units- in 1000's)

Aver

age

Rate

of B

reak

dow

n of

Lact

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(mm

ol/L

/sec

)

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Similarly to graph 1, graph 2 appears to have a direct relation between the average rate of breakdown and the amount of lactase forming a linear trend. Both graphs showed that by increasing the amount of lactase, the rate of breakdown will increase as well. By observing the expected results, it can be seen that both graphs 1 and 2 only show the beginning of the accepted trend.

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Figure 2: Rate of Reaction vs. Enzyme concentration

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iv

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In the AB second in Figure 2, the rate of breakdown can be seen as a direct relation to the amount of lactase as shown by its trend, which the dotted line emphasizes. In the BC section, the rate of reaction begins to plateau, and this section is where the saturation point lies. As the enzyme molecules begin to out number the substrate molecules, the rate of reaction beings to plateau and the addition of any more enzyme molecules will not increase the rate of breakdown.v

In graphs 1 and 2, the trend appears to be a linear relation. Both graphs depict the AB section of figure 1. In graphs 1 and 2, the point of saturation would appear somewhere after final point on the graph

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(36,000 FCC lactase units), since the trend doesn’t appear to be curving yet. If the amount of lactase was increased, the graphs would have shown the plateau of the trend.

Table 3’s observations show the results of mixing various concentrations of lactase and a set concentration of lactose in a 15ml test tube of water. When the lactase is mixed with the lactose, two products are produced: glucose and galactose.

Lactose+Water Lactase→

Glucose+Galactose+Heat

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C12H 22O11+H 2OLactase→

C6H 12O6+C6H 12O6+Heat

Observations from graphs 1 and 2 show that as the amount of lactase increases, the rate of breakdown increases as well, up until the saturation point where the lactase molecules begin to outnumber the lactose molecules, stopping the increase of the breakdown rate. By comparing it to the accepted trend (Figure 2), it can be noted that graph’s 1 and 2 only give a partial view of the overall trend. If higher amounts of lactase were tested, the accepted trend (Figure 2) may have been more evident in both graphs 1 and 2.

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Conclusion

An enzyme is a protein that speeds up reactions that would otherwise take a much longer period of time to occur. It does this by binding the substrate to its active site, which lowers the amount of energy required to reach the transition state. In this case the substrate is broken down into two different products, which are released and the enzyme moves on to the next substrate to repeat the process. As the amount of enzyme molecules increase and surpass the substrate molecules, there are enough active sites for every substrate molecule. This is show in figure 2 when the enzyme concentration reaches a certain concentration and the rate of reaction begins to slow down.

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Overall this experiment was partially successful. It proved part of the hypothesis to be true and partially fulfilled the purpose of this experiment which was to understand how the concentration of the enzyme lactase, affected the rate of reaction when mixed with the sugar, lactose. Since it did not give the desired amount of detail like for example, the plateau of the increase in the rate of breakdown, it can only be considered a semi-successful experiment.

A limiting factor within this experiment was the accuracy of the glucose concentration. Glucose concentration was measured by dipping the glucose test strip into a solution, then to remove the strip, wait 30 seconds and then compare the colour of the test strip to the colour chart and its corresponding

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value on the glucose test strips bottle. Normally the uncertainty would be easy to calculate if the values were in uniform increments, but unfortunately the values to compare your results to were not in uniform increments. The bottle had 6 colours on the colour chart; 0, 6, 14, 28, 56, and 111mmol/L. It can be seen that the differences between the values are not uniform and if it could be calculated, the uncertainty would change as the glucose concentration increased. A colorimeter could have been used to compare the colour given by the test strips to the colour chart much more accurately.

Another limiting factor was the procedure to measure the colour of the glucose test strip. After waiting 30 seconds before comparing the colour to the colour chart, there was only one instant where the

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colour could be compared. The longer the wait was after 30 seconds, the more the colour changed, so there was only one opportunity to compare. This was evident because long after the experiment had finished, the glucose test strips had turned dark brown, which indicated that the glucose concentration was above 111mmol/L, which was untrue. A way to improve this limitation would be to keep a closer watch on the stopwatch to ensure that the colour was compared at 29 seconds to give the eyes and brain time to process the information.

Some of the equipment used limited the experiment. For measuring the volumes of water, a graduated cylinder was used. The graduated cylinder is only accurate to a certain extent (±0.02ml) which may have

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reduced the accuracy of the experiment and could have been avoided by using a pipette which is much accurate. Although, a pipette would have improved the experiment, it is not necessary, since water volume was not a major factor in the experiment.

Crushing the lactase pill into a powder form created another limitation. It is impossible to crush the pill identically, so when it was crushed, the smaller pieces that made up the powder varied in size. This variance in size meant that there were different surface areas for different trials. Since surface area has an effect on reaction rate (the greater the surface area, the faster reaction rate), this variance may have influenced the accuracy of the results. Since the human arm always varies in strength, the surface area

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of each tablet for each trial would be slightly different. To prevent this, the tablet could have been placed in a container (preferably plastic to avoid damage of the container and to prevent the pieces of the tablet from flying everywhere) and had a heavy object (such as a text book, or a rock) dropped from a small predetermined height on the tablet, until it is in a powdered form.

In some of the trials, some human error occurred. After pouring the lactase into the solution, the tweezers holding the glucose test strip was not picked up in before 10 seconds resulting in the solution not to be stirred before testing for glucose. There is no purposeful way to improve this since it takes two hands to pour in the lactase (which eliminates the possibility of holding the tweezers in the free hand),

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and it was accidental, which leaves the only possibility to fix this error is to strengthen hand-eye co-ordination.

Since the solution was not stirred in some of the trials as stated previously, the lactase stayed on the surface of the solution for a short time period. Usually the stirring rod would have pushed the lactase down, and stirred it to spread it throughout the solution, but the stirring rod was not present, which resulted in the lactase not being equally present throughout the solution. To improve this would be the same as the previous error: to strengthen hand-eye co-ordination to ensure the tweezers and the glucose test strip were picked up in time to allow time for stirring.

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Three trials were done for each amount of lactase, which is not very accurate. This is a limitation of the experiment’s design. If more trials had been done, the results would have been more accurate. Three days were given for this experiment, where the first day was wasted, which limited the amount of trials that could be completed. To improve, there should be more preparation in advance such as have a more detailed plan of the experimental procedure, and to purchase more equipment like lactase tablets, since there were not enough.

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i Callahan, Larry N. "Lactase, chemical structure, molecular formula, Reference Standards."Newdruginfo.com. Web. 17 Jan. 2010. <http://www.newdruginfo.com/pharmacopeia/usp28/v28230/usp28nf23s0_m44050.htm>.ii Fenton, Dennis M. "Lactase preparation - Patent 4329429." Patent Searching and Inventing Resources. Web. 17 Jan. 2010. <http://www.freepatentsonline.com/4329429.html>.iii "Chemical Change, Chemical Reactions and Equations, Science, Science Tutoring, Science Help, Science, Online Science TutoringT | TutorVista." Tutorvista.com - Online Tutoring, Homework Help for Math, Science, English from Best Online Tutor. Web. 19 Jan. 2010. <http://www.tutorvista.com/content/science/science-ii/chemical-reactions-equations/chemical-change.php>.iv "Enzyme Concentration (Introduction to Enzymes)." Enzymes, Biochemicals: Worthington Biochemical Corporation. Worthington Biochemical Corporation. Web. 14 Jan. 2010. <http://www.worthington-biochem.com/introbiochem/enzymeconc.html>.v Gregory, Michael J. "Enzymes." Clinton Community College. Web. 14 Jan. 2010. <http://faculty.clintoncc.suny.edu/faculty/michael.gregory/files/Bio%20101/Bio%20101%20Laboratory/Enzymes/Enzymes.htm>.