Description:

Mutanolysin (EC 3.2.1.17) (N-acetylmuramidase) is a muralytic enzyme that cleaves the β-N-acetylmuramyl-(1->4)-N-acetylglucosamine linkage of the bacterial cell wall polymer peptidoglycanpolysaccharide. Its carboxy terminal moieties are involved in the recognition and binding of unique cell wall structures aboundant in many gram-positive bacteria.

Recombinant mutanolysin effectively lyses particularly problematic bacteria, like:

Streptococcus, Lactobacillus, Lactococcus, Listeria.

Recombinant Mutanolysin and lysozyme activity is synergistic. Using Mutanolysin and lysozyme mixture leads to increased yield of bacteria lysis.

Unit definition:
One unit will produce a ∆A600 nm of 0.01 per minute at 50 mM MES

pH 6.0, 1 mM MgCl2 at 37 °C in a 1 ml volume using suspension of

Streptococcus faecalis cell wall as substrate.

Activity: > 10.000 U/mg

Concentration: Powder (to obtain concentration 10 U/µl dissolve the whole content of vial of mutanolysin lyophilisate in 1000 µl of Mutanolysin storage buffer.

Storage buffer (not included)
20 mM MES pH 6.2, 50 mM NaCl, 50% glycerol (v/v). 1 ml / 10000 U

Shipping: shipped on blue ice

Storage Conditions: store at -20°C

Notes: avoid multiple freeze / thaw cycles

Shelf Life: at least 18 months

• Enzyme is safe, being a product originating from organism recognized by U.S Food and Drug Administration as GRAS (Generally Recognized as Safe).

• Great purity in contrast to native product

- produced in the non-E.coli expression system, an organism with GRAS status. We guarantee no DNA/RNA content.

- an extra purification step and Real-time PCR based tests. DNase and RNase assays are performed with specially designed fluorescent probes.

- traditional methods concerning incubation of Mutanolysin with linear DNA fragments followed by electrophoresis and incubation with RNA followed by fluorometric RNA concentration measurement.

Application:

• Mild conditions formation of spheroplasts of gram-positive bacteria

• Enzymatic cell lysis in DNA and RNA isolation process

Effective lysis of gram-positive bacteria in environmental studies and DNA-based microbial detection.

Protocol:

Recommended protocol (for cell wall digestion):

1. Transfer 0.2-1.0 ml of overnight bacterial culture to 1.5 ml Eppendorf
     tube and centrifuge (i.e. 2500 x g, 5 min).

2. Discard supernatant and suspend the bacterial pellet

     in 100 µl of the digestion buffer (suggested buffer: 50 mMMES pH 6.0,
     1 mM MgCl2).

!! Different digestion buffers may also be tested !!

Note: The mutanolysin acivity may strongly differ among various strains of Gram-positive bacteria tested.

3. Add 50 U of mutanolysin. Mix gently and incubate for 20 min at 50 °C.