Enzyme Lab Report

Biofuel Enzyme Kit
Katie Adamson
Biochemistry Laboratory, BIO124L
1/29/15

Abstract
The objective in this lab was to determine the effects different conditions had on the enzyme cellobiase. We examined reaction rates in the presence or absence of an enzyme, the effects temperature and pH changes on the enzyme and the effects enzyme concentration and substrate concentration had on the enzyme. As expected results showed us that cellobiase works optimally when conditions are favorable. We see this when we have high substrate concentration, high enzyme concentration, with a 37oC temperature and at 5.0pH. Activity 1 showed us that the presence of an enzyme could dramatically increase the rate of reaction. In activity 2 we tested different temperature
Pipet 1.5ml of 1.5 mM substrate into the “High concentration” conical tube. Into the 15ml “Low concentration substrate” tube pipet 1.25ml of buffer and 250 μl of 1.5 mM substrate and mix. Get six cuvettes, label the first three H1-H3 and the second three L1-L3 for low and high concentration substrate time points. After labeling pipet 500 μl of stop solution into each cuvette. Taking the “High concentration substrate” conical pipet 750 μl of enzyme into it and then pipet the same amount of enzyme into the “Low concentration substrate” conical. Start timer. At 1 minute, 2 minutes and 8 minutes remove 500μl from the “High Concentration substrate” and “Low concentration substrate” conical tubes and add them to the appropriate cuvettes containing stop solution. Analyze samples and record amplitudes with
Time(minutes)
Cuvette
Amount of p-Nitrophenol (nmol) from the Standard Curve
Absorbance at 410nm
0
Start
0
0
8
End
0
0
1
E1
28
0.416
2
E2
45
0.694
4
E3
82
1.264
6
E4
112
1.766
8
E5
135
2.104
The amount of p-nitrophenol in the cuvettes containing cellobiase was significantly higher as time went on from 1 minute (28 nmol) to 8 minutes (135 nmol) in the reaction (Table 4).
Activity 2: Determine the effect of temperature on the reaction rate
Table 5: Determination of p-nitrophenol produces at three different temperatures based on p-nitrophenol standards.
Temperature
Standard that is Most Similar
Amount of p-Nitrophenol Produced (nmol)
0oC
S1
0
~22oC(room temp)
S4
50
37oC
S5
100

Table 6: Determination of p-nitrophenol produced at three different temperatures based on standard curve.
Temperature
Absorbance at 410 nm
Amount of p-Nitrophenol Produced (nmol)
0oC
0.089
9
~22oC(room temp)
0.753
51
37oC
1.562
97

Activity 3: Effect of pH on reaction rate

Table 7: Determination of p-nitrophenol produced at three different pH values based on p-nitrophenol standards. pH Standard That is Most Similar
Amount of p-Nitrophenol Produces (nmol) pH 5.0
S4
50
pH