Experimental Procedure for Determination of Km and Vmax (Enzyme Kinetics)

A. Preparation of Substrate Solutions of Different Concentrations

1. Select the rack of microcentrifuge tubes.
2. Select the micropipette.
3. Set the micropipette volume to 5 µL using the adjustment wheel.
4. Pipette 5 µL of substrate solution into each microcentrifuge tube.
5. Adjust the micropipette volume to 995 µL to measure the buffer solution.
6. Pipette 995 µL of buffer solution into the same tubes to obtain a final volume of 1 mL with a concentration of 0.5 mM substrate (as per the table).
7. Click the Run option to prepare the remaining substrate solutions of different concentrations according to the given table.
8. Proceed to the next step.

B. Enzyme–Substrate Reaction Setup

1. Select the prepared substrate rack.
2. Choose the micropipette.
3. Set the micropipette volume to 5 µL and transfer 5 µL of substrate into a clean cuvette.
4. Adjust the micropipette volume to 895 µL to measure the buffer solution.
5. Transfer 895 µL of buffer solution into the same cuvette.
6. Set the micropipette volume to 100 µL to measure the enzyme solution.
7. Add 100 µL of lysozyme enzyme solution into the cuvette.
8. Immediately proceed for absorbance measurement after enzyme addition.

C. Measurement of Absorbance at 450 nm

1. Open the spectrometer lid.
2. Insert the micro-cuvette containing the reaction mixture into the spectrometer.
3. Set the instrument to Test mode by pressing the Test button.
4. Press the A450 button to record the absorbance at 450 nm.
5. Note the absorbance reading every 30 seconds.
6. Click Next Step to proceed with the enzyme kinetics analysis for determination of Km and Vmax.