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COMSATS University Islamabad: Mid-Term Exam - Asma Zafar

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226 views3 pages

COMSATS University Islamabad: Mid-Term Exam - Asma Zafar

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umairgul841
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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

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