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 a 1 mL volume using a suspension of Streptococcus faecalis cell walls as the substrate.

Precautions

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

Reagents and Equipment Required

MES hydrate (Catalog No. M8250)
1.0 M Magnesium chloride solution (Catalog No. M1028)
TES (Catalog No. T1375)
Mutanolysin Assay Substrate (Catalog 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 (Catalog No. M8250) in 190 mL of ultrapure water and then add 0.2 mL of 1.0 M Magnesium chloride solution (Catalog 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 (Catalog No. T1375) in 190 mL of ultrapure water and then add 0.2 mL of 1.0 M Magnesium chloride solution (Catalog 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 (Catalog 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 time period and a minimum of 4 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.