Enzymatic Activity of Lysozyme (EC 3.2.1.17)
1. OBJECTIVE
The objective of this procedure is to standardize a method for the enzymatic assay of Lysozyme.
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.
4. DISCUSSION
Micrococcus lysodeikticus Cells (Intact) Lysozyme> Micrococcus lysodeikticus Cells (Lysed)
5. RESPONSIBILITIES
All Analytical Services employees are responsible for running this procedure as written.
6. SAFETY
Please refer to the Material Safety Data Sheet (MSDS) for hazards and appropriate handling precautions.
7.1. Conditions
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. Method
7.2.1. Enzymatic Rate Determination
7.3. Reagents
7.3.1. 66 mM Potassium Phosphate Buffer, pH 6.24 at 25°C (Buffer)
7.3.1.1 Prepare 100 ml in purified water using Potassium Phosphate, Monobasic, Anhydrous, Sigma-Aldrich Product Number P5379.
7.3.1.2 Adjust the pH to 6.2 at 25°C using 1M Potassium Hydroxide (KOH)
7.3.2 Micrococcus lysodeikticus Cell Suspension (Substrate)
7.3.2.1. Prepare 25 ml in Reagent 7.3.1 using Micrococcus lysodeikticus, ATCC 4698 lyophilized cells, Sigma-Aldrich Product Number M3770.
7.3.2.2. 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. Procedure
7.4.1. Pipette (in milliliters) the following reagents into suitable cuvettes:
| Blank | Test | |
| 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. Calculations
| 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) |
| (% protein/100) |
8. REFERENCES & ATTACHMENTS
Shugar, D. (1952) Biochimica et Biophysica Acta 8, 302-309
9. APPROVAL
Review, approvals and signatures for this document will be generated electronically using DocCompliance (QUMAS). Print a “For Use” copy if hardcopy with signature verification is required
