Ready-Lyse-Lysozyme Solution, 4,000,000 U

Lyse Gram positive or Gram negative bacteria for protein or nucleic acid purifications from bacteria

• Highly Active: This enzyme is 200X more active than egg white lysozyme, thus improving bacterial lysis and using less enzyme
• Easy: No sample agitation or heat generation necessary during lysis which helps preserve protein function
• Flexible: Usage volumes are easily adjusted based on the scale of the experiment

Applications

• Lysis of Gram-negative or Gram-positive bacteria (Table 1) for protein purification (Fig. 1).
• Purification of nucleic acids from bacteria.

Ready-Lyse™ Lysozyme Solution is a recombinant lysozyme preparation, derived from a nonmammalian, nonavian source, that is useful for the lysis of Gram-negative (such as E. coli) and Gram-positive (such as Bacillus sp.) bacteria. The specific activity of Ready-Lyse Lysozyme is 200-fold higher than the specific activity of egg-white lysozyme and, therefore, less lysozyme is needed in a reaction. Also, unlike egg-white lysozyme, Ready-Lyse Lysozyme Solution is stable at -20°C, eliminating the need to prepare a fresh solution for each use. The use of Ready-Lyse Lysozyme results in higher yields of protein than can be obtained with standard eggwhite lysozyme.

Unit Definition: One unit of Ready-Lyse Lysozyme produces a decrease in A₃₅₀ of 0.001 per minute at 23°C with a 0.5 mg/ml suspension of lyophilized E. coli K802 cells in 50 mM Tris-HCl (pH 7.5).

Storage Buffer: 50% glycerol containing 50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 0.1 mM EDTA, 1 mM DTT, and 0.1% Triton® X-100.

Table 1. Bacteria lysed with Ready-Lyse™ Lysozyme Solution.

Figure 1. Lysis with Ready-Lyse™ Lysozyme increases yields of nucleic acids. 500 µg per ml of pHC79 cosmid DNA was incubated for 15 minutes at 22°C in TE buffer (25 mM Tris (pH 8.0), 10 mM EDTA) containing either 5 µg (30 KU) per ml of Ready-Lyse Lysozyme or 500 µg (25 KU) per ml of egg white lysozyme. The solutions were centrifuged for 10 minutes and the pellets were resuspended in TE buffer containing 0.1% SDS. DNA in supernatants and pellets was separated by electrophoresis in a 0.8% agarose gel. Approximately 50% of the DNA was lost due to precipitation by egg white lysozyme (EW), while Ready-Lyse Lysozyme (RL) caused minimal precipitation losses of DNA compared to control (C) samples without lysozyme.

Figure 2. Use of Ready-Lyse™ Lysozyme Solution to recover recombinant proteins. One ml of induced cells from a recombinant E. coliclone was pelleted by microcentrifugation before induction and at 1 and 3 hours after induction. Each sample was resuspended in 50 µl of cold TEBG buffer. One µl of Ready-Lyse Solution was added to each suspension and the cells were incubated at room temperature for 30 minutes. The cell debris was pelleted and 10 µl of the supernatant were run on an SDS-PAGE gel. Lane 1, molecular weight markers; Lanes 2-4, time points of induction. The induced protein is designated by an arrow.