Enzymatic Activity of Lysozyme (EC 188.8.131.52)
2.1 The scope of this procedure includes products that have a specification for Lysozyme activity.
2.2 This assay procedure is not to be used to assay Lysozyme, Bovine Recombinant Expressed in Pichia pastoris, Sigma-Aldrich Product Number L9772, Lysozyme, Human Recombinant Expressed in Pichia pastoris, Sigma-Aldrich Product Number L2026, and Lysozyme Insoluble Enzyme on Agarose, Sigma Product Number L1129.
3.1. Purified Water: Water from a deionizing system, resistivity > or = 18MΩ.cm @ 25°C.
3.2. Unit Definition: One unit will produce a ΔA450nm of 0.001 per minute at pH 6.24 at 25°C using a suspension of Micrococcus lysodeikticus as substrate, in a 2.6ml reaction mixture.
7.1.1. Temperature = 25°C
7.1.2. pH = 6.24
7.1.3. Absorbance = 450 nm
7.1.4. Light Path = 1 cm
7.2.1. Enzymatic Rate Determination
7.3.1. 66 mM Potassium Phosphate Buffer, pH 6.24 at 25°C (Buffer)
184.108.40.206 Prepare 100 ml in purified water using Potassium Phosphate, Monobasic, Anhydrous, Sigma-Aldrich Product Number P5379.
220.127.116.11 Adjust the pH to 6.2 at 25°C using 1M Potassium Hydroxide (KOH)
7.3.2 Micrococcus lysodeikticus Cell Suspension (Substrate)
18.104.22.168. Prepare 25 ml in Reagent 7.3.1 using Micrococcus lysodeikticus, ATCC 4698 lyophilized cells, Sigma-Aldrich Product Number M3770.
22.214.171.124. The A450nm of this suspension should be between 0.6-0.7 versus a buffer blank. This is typically a 0.01% (w/v) solution, but could deviate based on the purity of the cell suspension used.
7.3.3. Lysozyme Enzyme Solution (Enzyme)
Immediately before use, prepare a solution containing 200-400 units/ml of Lysozyme in cold Reagent 7.3.1.
7.4.1. Pipette (in milliliters) the following reagents into suitable cuvettes:
|Reagent 7.3.2 (Substrate)||2.50 ml||2.50 ml|
7.4.2. Equilibrate cuvettes to 25°C. Monitor the A450nm until constant, using a suitably thermostatted spectrophotometer. Then add:
|Reagent 7.3.1 (Buffer)||0.10 ml||------|
|Reagent 7.3.3 (Enzyme)||-------||0.10 ml|
7.4.3. Immediately mix by inversion and record the decrease in A450nm for approximately 5 minutes. Obtain the maximum linear rate (ΔA450nm/minute) for both the test and blank using at least a one minute interval and a minimum of 4 data points.
|7.5.1.||Units/mg solid =||(A450nm/min Test - A450nm/min Blank)(df)|
|(0.001)(0.1)(mg sample used)|
where: df = dilution factor
0.001 = Change in absorbance at A450nm as per the Unit Definition
0.1 = Volume (in milliliters) of enzyme used
|7.5.2.||units/mg protein =||(units/mg solid)|