This procedure may be used for mutanolysin products. The continuous spectrophotometric rate determination (A600, Light path = 1 cm) is based on the following reaction:
Streptococcus faecalis Cells Walls (Insoluble) -------------------> Soluble Products
Unit Definition: One unit of mutanolysin will produce a ΔA600 of 0.01 per minute at pH 6.0 at 37 °C in 1 mL using a suspension of Streptococcus faecalis cell walls as the substrate.
Use ultrapure water (≥18 MΩxcm resistivity at 25 °C) for the preparation of reagents.
Buffer (50 mM MES Buffer with 1 mM magnesium chloride, pH 6.0, at 37 °C) – Dissolve 1.95 grams of MES hydrate (Product No. M8250) in 190 mL of ultrapure water and then add 0.2 mL of 1.0 M Magnesium chloride solution (Product No. M1028). Adjust the pH to 6.0 at 37 °C using 1 M NaOH and then bring the volume of the entire solution to 200 mL using ultrapure water.
Enzyme Diluent (50 mM TES with 1 mM magnesium chloride, pH 7.0 at 37 °C) – Dissolve 2.29 grams of TES (Product No. T1375) in 190 mL of ultrapure water and then add 0.2 mL of 1.0 M Magnesium chloride solution (Product No. M1028). Adjust the pH to 7.0 at 37 °C using 1 M NaOH and then bring the volume of the entire solution to 200 mL using ultrapure water.
Substrate (Streptococcus faecalis cell wall suspension) – Prepare a cell wall suspension of log-phase Streptococcus Faecalis STF-3 (ATCC® 12784) cells in ~10 mL of Buffer to a concentration where the A600 is in the range of 0.48–0.52. Alternatively, mutanolysin assay substrate (Product No. M3440) may be prepared according to the instructions in the product datasheet.
Enzyme Solution (Mutanolysin) – Immediately before use, prepare a solution containing 500–700 units/mL of mutanolysin in cold (2–8 °C) Enzyme Diluent.
In a 3.04 mL reaction mix, the final concentrations are 49 mM MES, 1 mM MgCl2, 0.66 mM TES, and 6.5–9.2 units of mutanolysin.
1. Pipette the Substrate into suitable cuvettes.
2. Equilibrate at 37 °C and monitor the A600 until constant using a suitably thermostatted spectrophotometer. Then add:
3. Immediately mix by inversion and record the decrease in A600 for 10 minutes. Using a 1 minute period and a minimum of four data points, obtain the ΔA600/minute using the maximum linear rate for both the Test and the Blank.
3.04 = Volume (mL) of reaction mix
df = Dilution Factor
0.01 = Decrease in absorbance per minute as defined by the unit definition
0.04 = Volume (mL) of Enzyme Solution used