Enzymatic Assay of Mutanolysin

Description

This procedure may be used for mutanolysin products. The continuous spectrophotometric rate determination (A₆₀₀, Light path = 1 cm) is based on the following reaction:

Mutanolysin
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.

Reagents and Equipment Required

MES hydrate (Product No. M8250)
1.0 M Magnesium chloride solution (Product No. M1028)
TES (Product No. T1375)
Mutanolysin assay substrate (Product No. M3440)

Preparation Instructions

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.

Procedure

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.

Reagent	Test (mL)	Blank (mL)
Substrate	3.00	3.00

2. Equilibrate at 37 °C and monitor the A₆₀₀ until constant using a suitably thermostatted spectrophotometer. Then add:

Reagent	Test (mL)	Blank (mL)
Enzyme Solution	0.04	–
Enzyme Diluent	–	0.04

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.

Results

Calculations

1.

Units/mL enzyme =	(ΔA600/min Test – ΔA600/min Blank) (3.04) (df)
	(0.01) (0.04)

where:
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

2.

Units/mg solid =	units/mL enzyme
	mg solid/mL enzyme

References

1. Calandra G, Cole R. 1980. Lysis and protoplast formation of group B streptococci by mutanolysin. Infection and Immunity. 28, 1033-1037.