The Resulting Effect When Lactase Enzyme

Is subjected to differences Substrate

concentration, Temperature and PH.

Name: Moses Seba

Class: Molecules and Cells
Introduction

Enzymes are proteins that acts as catalyst within living cells. They increase the rate at which a
reaction occurs without being consumed or permanently altered. Enzymes are composed of
specific shapes of proteins that allow them to reaction with specific substrate. One side of an
enzyme is called the active site. This is where a specific substrate would bind to the enzyme and
be broken down.
Lactase Is an enzyme that catalases the substrate lactose. It is breaks down lactose into
galactose and glucose in the small intestine of mammals. In humans, lactase is abundant during
infancy. But after that, the body stops producing it. But there are people whose bodies
possesses a mutation in their genes that allows to produce the lactase enzyme even after they
have grown up. This could due to the fact that lactose is a source of many nutrients, therefore
resulting a population gene drive to an increase of people who drink milk.

Cite your Sources.

- https://lbc.msu.edu/evo-ed/Pages/Lactase/cellbio.html

Experimental purpose and Hypothesis

The purpose of this lab was to determine the effects of substrate concentration, PH, and
temperature on the enzyme reactivity of Lactase. The variable that were being tested changing
PH, Temperature and substrate concentration. The Hypothesis was that changes temperature.

Methodology

Measuring the changing substrate concentration

The spectrophotometer was calibrated. It was turn on and set at 420 nm. The blank cuvette was
located. Six cuvettes were labelled for different substrate concentration. These were 0 mM,
1.88 mM, 3.75 mM, 7.5 mM, 15 mM and 30mM. 1.0 ml of 0.1M phosphate buffer was added to
each cuvette, at a constant 7.3 PH and room temperature. One of the concentration was
selected and 125 L of that solution to the appropriately labelled cuvette. From there, 125 L
of the lactase solution was added to each labelled cuvette. The reaction was time for 60
seconds, and the absorbance was recorded.

Measuring the changing PH

The Spectrophotometer was recalibrated. Three cuvettes were then labelled with PH 5.7, 7.3,
8.0 respectively. A 1.0 ml amount of 0.1 M phosphate buffer was added to each cuvette. One
the PH condition was selected and 125 L of 30 mM ONPG was added to it. A 125 L amount of
lactase solution was added to the selected cuvette. The reaction was time for 60 seconds, and
the absorbance was recorded.

Measuring the Changing Temperature

The Spectrophotometer was recalibrated. Three cuvettes were labelled as 0 o C, 22 o C, and 50 o

C. a 1.0 mL amount of 0.1M phosphate buffer solution with a PH of 7.3 was added to each
cuvette. Two of the Eppendorf tubes were labelled 0 o C and 50 o C lactase. A 150 L amount of
lactase solution was added to one of the tubes and was incubated for 5 minutes. The 22 o C
didn’t need to be incubated. While the lactase was being incubated, 125 L of 30 mM ONPG
was added to the selected solution. After the 5 minutes were completed, 125 L lactase
solution to three cuvettes. The reaction was time for 60 seconds, and the absorbance was
recorded.

Results

After the variable data collected graphs were created to show the model. The variables were
being measured on their effects on lactase reactivity. Data table 1 and graph 1 shows the
effect of substrate concentration. Data table 2 and graph 2 show the effects of PH. And data
table show the effects of temperature. The absorbance is affected the number of molecules in
the cuvette. An increase in the absorbance because more molecules are absorbing the
wavelength. This is how lactase reactivity will be measured.
Tables should have labeled as the table no# and the

Data Table 1 shows the Effects of Substrate concentrations with changing Concentration and
Absorbance
(ONPG) mM Absorbance
0 0.04
1.88 0.134
3.75 0.152
7.5 0.226
15 0.286
30 0.474
 Effects of Substrate Concentration on Lactase Reactivity
0.6

0.5

0.4
Absorbance (A)

0.3

0.2

0.1

0
0 5 10 15 20 25 30 35
y = 0.0131x + 0.0919
Substrate Concentration (ONPG) mM R² = 0.96

The graph shows a positive linear correlation of 0.96003 between substrate concentration and
absorbance. As Substrate concentration increased, the absorbance also increased. This is meant
that more and more products catalyzed by lactase with each increase substrate concentration
in the cuvette. An increase in substrate concentration, more products will be products. Lactase
has an optimum PH of 6. Any PH higher or lower will start to denature the enzyme, thus there
will be less products formed.

Data Table shows the Effects of PH on Enzyme reactivity with changing in PH and Absorbance
PH Absorbance (A)
Blank () 0.336
5.7 0.298
7.3 0.289
8.0 0.255
 Effects of PH on Lactase Reactivity
0.4

0.35

0.3
Absorbance (A)

0.25

0.2

0.15

0.1

0.05

0
0 1 2 3 4 5 6 7 8 9
y = -0.0085x + 0.3392
PH
R² = 0.86

The graph shows a negative proportionality of 0.85996 between PH and absorbance. As PH

increased, the absorbance decreased. This is meant that there were less products catalyzed by
the enzyme. This can be further explained by the fact Lactase has an optimum PH of 6. Any PH
higher or lower will start to denature the enzyme, thus there will be less products formed.

Data Table shows The Effects of Temperature on Enzyme reactivity with changing in
Temperature and Absorbance

Temperature Absorbance
Blank 0.351
0 0.230
22 0.277
50 0.211
 Effects of Temperature of Lactase Reactivity
0.4

0.35

0.3

0.25
Absorbance

0.2

0.15

0.1

0.05

0
0 10 20 30 40 50 60
Temperature (o C )

Discussion and Conclusion

The main purpose of the lab was to describes the effects of changes in substrate concentration,
PH and Temperature. But due to sources of error, the results

The first error was

References

http://study.com/academy/lesson/what-are-enzymes-definition-lesson-quiz.html