COMSATS University Islamabad
Mid-Term Exam – Asma zafar
MS/PhD FA2020 SEMESTER- I/II SUBJECT Enzyme Catalysis COURSE CODE Bio766
TIME ALLOWED 24 Hrs TYPE: Open Book Exam MAX MARKS 50
All questions carry equal marks.
Q1. A therapeutic enzyme was obtained from a bacterium by continuous fermentation. The
enzyme was subjected to serial purification. The most active fraction was collected after each
step of purification. Construct the purification table (showing total units of activity, total protein
content, specific activity, purification factor (fold purification) and %-recovery from the
following data:
Fraction Type Total Vol. Activity Units Protein Content
Crude Cell Lysate 222.4 ml 9.56 /100 µl 0.978 mg/100µl
Supernatant 163 ml 10.45 /100 µl 0.972 mg/100µl
(Centrifugation)
50-70% 11.2 ml 34.2 / 100 µl 1.85 mg/100µl
Ammonium
Sulfate pellet
DEAE-Cellulose 216 µl 134 / 10 µl 1.16 mg/10µl
DEAE-Sephacel 74 µl 259.5 / 10 µl 1.61 mg/10µl
Q2. Determine Vmax and Km values from the following data of a purified sample of Chitinase
using Double Reciprocal Plot:
Activity of Chitinase at various Substrate Concentrations
Substrate Conc. (µM) Units min-1 ml-1
0.4 0.39
0.6 0.60
0.8 0.87
1.0 0.94
1.2 1.21
1.4 1.36
1.6 1.51
1.8 1.62
2.0 1.69
2.2 1.7
Assay Conditions:
Incubation time = 30 min at 40 ᵒC in MES buffer 1%, pH = 6.5
Sample volume = 20 µL of the Enzyme solution was used each time.
Q3. Compare values of MM constants (Vmax and Km) with those obtained from Eadie-Hoftsee
and Hanes-Woolf Plots.
Q4. Calculate Kcat of the enzyme using the value of Vmax obtained from Double Reciprocal Plot.
The sample contained 150 µg/ml protein with estimated molecular weight of 60 KDa. 20 µL of
the Enzyme solution was used each time. Also calculate specificity constant of the enzyme.
Q5. Calculate Topt of the enzyme by plotting activity against temperature and also find Ea of
catalysis of the enzyme from the data given below:
Activity of Chitinase at various Temperatures
Temperature (°C) Units min-1 ml-1
25 0.019
30 0.022
35 0.067
40 0.097
45 0.17
50 0.23
55 0.13
60 0.081
65 0.032
Assay Conditions:
Incubation time = 30 min at 40 ᵒC with 1.5uM substrate in MES buffer 1%, pH = 6.5
Sample volume = 20 µL of the Enzyme solution
