Enzymatic Assay of Lysozyme (EC 3.2.1.17)

Description

This procedure may be used for the enzymatic assay of Lysozyme products. The spectrophotometric rate determination (A450, Light path = 1 cm) is based on the following reaction:

                                                               Lysozyme

Micrococcus lysodeikticus Cells (Intact)  ––––––––>  Micrococcus lysodeikticus Cells (Lysed)

Unit Definition – One unit of Lysozyme will produce a ΔA450 of 0.001 per minute at pH 6.24 at 25 °C using a suspension of Micrococcus lysodeikticus as substrate in a 2.6 ml reaction mixture.

Reagents and Equipment Required

1.0 M Potassium phosphate monobasic solution (Catalog Number P8709)

1.0 M Potassium phosphate dibasic solution (Catalog Number P8584)

1 M Potassium hydroxide (KOH) solution

1 M HCl solution

Micrococcus lysodeikticus, ATCC No. 4698, lyophilized cells (Catalog Number M3770)

Precautions

Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Preparation Instructions

Use ultrapure water (≥18 MΩxcm resistivity at 25 °C) for the preparation of reagents.

Buffer (50 mM Potassium Phosphate Buffer, pH 6.24, at 25 °C) – To prepared 550 ml:

Substrate Suspension (0.015% [w/v] Micrococcus lysodeikticus Cell Suspension) – Prepare a 0.15 mg/ml suspension in Buffer using Micrococcus lysodeikticus, ATCC No. 4698, lyophilized cells (Catalog Number M3770).

Substrate Suitability: The A450 of this suspension must be between 0.6–0.7 versus a Buffer blank. If necessary, adjust the absorbance using appropriate amount of Buffer or Micrococcus lysodeikticus cells.

Enzyme Solution (Lysozyme) – Immediately before use, prepare a solution containing 200‑400 units/ml of Lysozyme in cold (2–8 °C) Buffer.

Procedure

1. Pipette the following reagent into suitable cuvettes and equilibrate to 25 °C using a suitably thermostatted spectrophotometer:

Reagent Blank (ml) Test 1 (ml) Test 2 (ml) Test 3 (ml)
Substrate Suspension 2.50 2.50 2.50 2.50

 

2. Then add:

Reagent Blank (ml) Test 1 (ml) Test 2 (ml) Test 3 (ml)
Buffer 0.10
Enzyme Solution 0.10 0.10 0.10

 

3. Immediately mix by inversion and record the decrease in A450 for 5 minutes. Obtain the maximum linear rate (ΔA450/minute) for all the Tests and the Blank using at least a one minute interval and a minimum of 4 data points.

Results

Calculation

1.      

where:

df = dilution factor

0.001 = Change in absorbance (ΔA450) as per the Unit Definition

0.1 = Volume (in milliliters) of Enzyme Solution

 

2.      

 

Materials

     

 Reference

  • Shugar, D., Biochimica et Biophysica Acta, 8, 302-309 (1952).

 

11/17-1

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