Procedure

Simulator-Guided Step-by-Step Procedure of the Experiment

Preparation of Reagents and Solutions

Phosphate Buffer (50 mM, pH 6.2):

• Reagents: Sodium phosphate monobasic (NaH₂PO₄); Sodium phosphate dibasic (Na₂HPO₄);Distilled water.
• Preparation: Dissolve 6.9 mM NaH₂PO₄ and 43.1 mM Na₂HPO₄ in distilled water. Adjust the pH to 6.2 using NaOH or HCl if needed. Make up the final volume to 100 mL and store at 4°C until use.

Substrate Solution (Micrococcus luteus Suspension, 0.2 mg/mL):

• Reagents: Micrococcus luteus (lyophilized powder or fresh culture) Phosphate buffer (50 mM, pH 6.2)
• Preparation: Suspend 0.2 mg of M. luteus powder in 1 mL of phosphate buffer. Vortex the solution to achieve a uniform suspension. Store on ice and mix well before use to prevent settling.

Lysozyme Solution (0.1 mg/mL):

• Reagents: Lysozyme (from egg white or recombinant source) Phosphate buffer (50 mM, pH 6.2)
• Preparation: Dissolve 1 mg of lysozyme in 10 mL of phosphate buffer. Vortex gently to ensure complete dissolution. Store on ice or at 4°C if not used immediately.

Experimental Setup

Reaction Setup

• Label test tubes for different substrate concentrations (e.g., 0.05, 0.1, 0.2, 0.3 mg/mL M. luteus).
• Pipette 2.5 mL of substrate solution into each tube.
• Pre-warm the tubes in a 37°C water bath for 5 minutes.

Enzyme Reaction

• Add 0.1 mL of lysozyme solution to each tube.
• Mix well and record time (t = 0 min).
• Measure absorbance at 450 nm using a spectrophotometer every 30 seconds for 5 minutes.
• Record the decrease in absorbance over time, as lysozyme hydrolyzes bacterial cell walls.

Data Analysis and Determination of Km and Vmax

1. Calculate Initial Reaction Velocity (V_o):

• Plot absorbance vs. time to determine the initial linear phase.
• The slope of this phase represents reaction velocity (V_o).

2. Plot Michaelis-Menten Curve:

• Plot V_o against substrate concentration [S] to generate a hyperbolic curve.
• Identify Vmax (the plateau of the curve).
• Estimate Km as the substrate concentration at V_o=Vmax/2.

3. Use Lineweaver-Burk Plot:

• Lineweaver-Burk plot (double reciprocal plot) can also used to determine Km and Vmax more accurately.