Enzyme Explorer Lysing Enzymes

Sigma^® offers the most comprehensive line of enzymes for enzymatic cell lysis and protoplast preparation

We also offer a complete range of kits for the purification and amplification of DNA and RNA and extraction and purification of proteins.

Enzyme Resources

• Enzymes for Bacterial Cells
• Enzymes for Yeast Cells
• Protein Toxins for Eukaryotic Cell Permeabilization
• Enzymes for Plant Cells

Additional Resources
Enzymatic Carbohydrate Analysis

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Enzymes for Bacterial Cells

The cell wall of Gram-positive bacteria is composed of multiple layers of peptidoglycan which comprises approx 90% of the cell wall structure. Peptidoglycan is a Polymer of β-(1-4)-N-Acetyl-D-glucosamine units. Alternating residues are modified to form N-acetylmuramic acid with the addition of lactate to form branching links to a tetrapeptide. The tetrapeptides of adjacent polymers are linked by penta-glycine bridges. The cross-linked peptidoglycan polymers form a mesh-like network over a phospholipid bylayer plasma membrane.

The Gram-negative cell wall is composed of an outer lipid bylayer, which, in addition to phospholipids, is also covered with lipopolysaccharide moieties. Lipoproteins link the outer lipid membrane to the thin peptidoglycan layer in the periplasmic space. The inner plasma membrane is a phospholipid bilayer.

Labiase
Labiase from Streptomyces fulvissimus is an enzyme preparation useful for the lysis of many Gram-positive bacteria such as Lactobacillus, Aerococcus and Streptococcus.

Labiase contains β-N-acetyl-D-glucosaminidase and lysozyme activity.

pH Optimum for activity: pH ~ 4
pH Optimum for stability: pH ~ 4-8

From Streptomyces fulvissimus
Product Number L1414 (powder)

Reference:

1. Niwa, t., et al., J. Microbiol. Methods, 61, 251-60 (2005)

Lysostaphin
Lysostaphin is a zinc endopeptidase with a molecular weight of approximately 25 kDa. Because lysostaphin cleaves the polyglycine cross-links in the peptidoglycan layer of the cell wall of Staphylococcus species it has been found useful for cell lysis and also as a potential anti-microbial therapeutic.

pH Optimum for activity: pH ~ 7.5

Product Number L7386 (lyophilized powder, min. 500 units/mg protein)
Product Number L4402 (lyophilized powder, min. 2,000 units/mg protein, >97% by SDS-PAGE)
Product Number L2898 (aseptically filled, lyophilized powder, min. 500 units/mg protein)

References:

1. Trayer, H. R., and Buckley, C. E., J. Biol. Chem., 245, 4842-4846 (1970).
   
2. Browder, H.P., et al., Biochem. Biophys. Res. Commun., 19, 383 (1965).

Lysozyme
Lysozyme is a single chain protein with a MW of 14.3 kD. It hydrolyzes β(1-4)-linkages between N-acetylmuraminic acid and N-acetyl-D-glucosamine residues in peptidoglycans and between N-acetyl-D-glucosamine residues in chitodextrin. Gram-positive cells are quite susceptible to this hydrolysis as their cell walls have a high proportion of peptidoglycan. Gram-negative bacteria are less susceptible due to the presence of an outer membrane and a lower proportion of peptidoglycan. However, these cells may be hydrolyzed more easily in the presence of EDTA that chelates metal ions in the outer bacterial membrane.

Optimal pH: The activity of lysozyme is a function of both pH and ionic strength. The enzyme is active over a broad pH range (6.0 to 9.0). At pH 6.2, maximal activity is observed over a wider range of ionic strengths (0.02 to 0.100 M) than at pH 9.2 (0.01 to 0.06 M).

For the lysis procedure, use a freshly prepared solution of 500,000 units/ml in 10 mM Tris-HCl, pH 8.0. The product is also soluble in water (10 mg/ml) yielding a clear to slightly hazy colorless solution. Aqueous solutions should be stable for at least one month when stored between 2–8 °C refrigerated. 25 µl Of this stock solution will typically lyse E. coli from >1ml of culture media cell pellet resuspended in 350 µl 10 mM Tris-HCl, pH 8.0, with 0.1 M NaCl, 1 mM EDTA, and 5% [w/v] TRITON. X-100. Typical incubation conditions for lysis are 30 min at 37 °C.

From chicken egg white
Product Number L6876 (dialyzed, lyophilized, approx 50,000 un/mg)
Product Number L7651 (molecular biology grade, lyophilized, approx 50,000 un/mg)
Product Number L7001 (lyophilized, approx 50,000 un/mg)
Product Number L7773 (aseptically filled, lyophilized, approx 50,000 un/mg)
Product Number L2879 (chloride, lyophilized, approx 60,000 un/mg)

Human Lysozyme
Product Number L1667 (recombinant expressed in rice)
Product Number L6394 (from human milk)
Product Number L8402 (from human neutrophils) 

References:

1. Jolles, P., Angewandte Chemie, International Edition, 8, 227-239 (1969).
2. Rupley, J.A., Biochim. Biophys. Acta, 83, 245-255 (1964).
3. Holler, H., et al., Biochem., 14, 2377-2385 (1975).
4. Canfield, R.E., J. Biol. Chem., 238, 2698-2707 (1963).
5. Davies, R.C., et al., Biochim., Biophys. Acta, 178, 294-305 (1969).

Achromopeptidase
Achromopeptidase is a lysyl endopeptidase with a MW of ~27kD. It is useful for lysis of Gram-positive bacteria that are resistant to lysozyme.

pH Optimum for activity: pH ~ 8.5 - 9

Approximately 500-1,500 un/ml achromopeptidase can be used to lyse cells at a density of A₆₀₀=0.6 over 2 hours at 37 °C

Product Number A3422 (partially purified powder, 20,000-40,000 units/mg solid)
Product Number A3547 ((crude powder, 800-3,200 units/mg solid)
Product Number A7550 ((crude powder, 300-600 units/mg solid)

Reference:

1. Ezaki, T and Suzuki, S., J. Clin. Microbiol., 16, 844–846 (1982)

Mutanolysin from Streptomyces globisporus
Mutanolysin provides gentle cell lysis for the isolation of easily degradable biomolecules and RNA from bacteria. It has been used in the formation of spheroplasts for isolation of DNA.

Mutanolysin is a 23kD N-Acetyl Muramidase, like lysozyme, is a muralytic enzyme that cleaves the N-acetylmuramyl-β(1-4)-N-acetylglucosamine linkage of the bacterial cell wall polymer peptidoglycan-polysaccharide. Its carboxy terminal moieties are involved in the recognition and binding of unique cell wall polymers. Mutanolysin lyses Listeria and other gram positive bacteria such as Lactobacillus and Lactococcus.

Product Number M9901( (lyophilized, min 4000un/mg protein)
Product Number M4782( (Aseptically filled, lyophilized, min 4000un/mg protein)

References:

1. Yokogawa, K., et al., Agric. Biol. Chem., 39, 1533-1545 (1975).
2. Calandra, G. B., and Cole, R. M., Lysis and Protoplast Formation of Group B Streptococci by Mutanolysin. Infect. Immun., 28, 1033-1037 (1980).
3. Fliss, I., et al., A Rapid and Efficient Method of Lysis of Listeria and Other Gram-positive Bacteria Using Mutanolysin. Biotechniques, 11, 453-457 (1991).
4. Assaf, N. A., and Dick, W. A., Spheroplast Formation and Plasmid Isolation from Rhodococcus sp. Biotechniques, 15, 1010-1015 (1993).

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Enzymes for Yeast Cells

In Yeast, the cell wall comprises ~30 % of the dry weight of the cell. The yeast cell wall is made of ~25% helical β(1-3) and β(1-6)-D-glucans and ~25% oligo-mannans, ~20 % protein, ~10% lipids, and some chitin. The protein component exists predominantly as a mannoprotein complex. Covalent linkages are reported to exist as β(1-4)-linkages between the reducing ends of chitin and the nonreducing end of b(1-3)-glucans¹ as well as among glycoproteins, β(1-6)-glucans, and β(1-3)-glucans.²

References:

1. Kollàr, R., et al., E. J. Biol. Chem., 270, 1170-1178 (1995)
2. Kapteyn, J. C., et al., Glycobiology., 6, 337-345 (1996)

Lysing Enzymes from Rhizoctonia solani
(Kitalase)
Main enzymatic acitivity is β-1,3-glucanase, also reported to contain protease, pectinase and amylase activities.

Product Number L8757 (powder)

Reference:

1. Tsuchiya, D., and Taga, M., Phytopathology, 91, 354-360 (2001)

Lysing Enzymes from Trichoderma harzianum
(Glucanex^® a product of Novozymes)
Contains β-glucanase with some cellulase, protease and chitinase activities.

Product Number L1412 (lyophilized powder)

Reference:

1. Petit, J., et al., Trends Genet., 10, 4-5 (1994)

Lyticase from Arthrobacter luteus
Contains β-1,3-glucan laminaripentaohydrolase with additional β-1,3-glucanase, protease and mannanase activities. Trace activities may include xylanase, amylase and phosphatase.

Reported to be useful for lysis of Ashbya, Candida, Debaryomyces, Eremothecium, Endomyces, Hansenula, Hanseniaspora, Kloeckera, Kluyveromyces, Lipomyces, Metschikowia, Pichia, Pullularia, Torulopsis, Saccharomyces, Saccharomycopsis, Saccharomycodes, and Schwanniomyces,

Product Number L2524 (powder, min 2,000 un/mg protein)
Product Number L4025 (powder, min 200 un/mg solid)
Product Number L5263 (Zymolyase^® 100Tpowder, min 3,000 un/mg protein)

Reference:

1. Phalip, V., et al., Biotechnol Lett., 26, 409-13 (2004)

Lyticase from Oerskovia xanthinoelytica expressed in E. coli
Purified recombinant β-1,3-glucanase preparation that is protease-free.

Product Number L4276 (powder, min 2,000 un/mg protein)

Reference:

1. DeSampiao, G., Mol Microbiol., 34, 247-56 (1999)

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Protein Toxins for Eukaryotic Cell Permeabilization

α-Hemolysin from Staphylococcus aureus
α-Hemolysin is a 33 kD extracellular protein secreted by most strains of pathogenic Staphylococcus aureus. It is selectively hemolytic and has a marked preference for rabbit red blood cells. It induces dermonecrosis, muscle paralysis, and it is lethal for laboratory animals. The toxin must be in the monomeric form to initially bind to a membrane. Specific receptors are not required for binding. Upon binding to biological membranes and/or artificial membranes, self-oligomerization occurs, resulting in ring structures (hexameric aggregates) believed to be transmembrane pores, which are permeable to ions and small metabolites. It is thought that α-hemolysin stimulates cellular phospholipases and induces a Ca²⁺ influx that can result in membrane disruption, leakage of cytoplasmic components, impaired membrane permeability, and osmotic lysis of the cells.

Product Number H9395 (lyophilized powder, ≥10,000 units/mg protein)

References:

1. Fink, D., et al., Staphylococcus aureus alpha-toxin activates phospholipases and induces a Ca²⁺ influx in PC12 cells., Cell. Signal., 1(4), 387-393 (1989).
2. Harshman, S., et al., Preparation and purification of staphlococcal alpha toxin. Meth. Enzymol., 165, 3-7 (1988).
3. Thelestam, M., and Blomqvist, L., Staphlococcal alpha toxin-recent advances. Toxicon., 26(1), 51-65 (1988).

Streptolysin O from Streptococcus pyogenes
Streptolysin O is a 69 kD immunogenic, oxygen-labile toxin, which is reversibly activated by dithiothreitol. It may be used for cell permeabilization or hemolysis. Erythrocytes from different animal species have significantly different susceptibility to hemolysis by Streptolysin O.

SLO has been used to introduce oligonucleotides into cultured eukaryotic cells by permeabilization. Mouse kidney cells were first trypsinized, then pelleted, and resuspended in a buffered SLO solution. After 5 minutes, permeabilization was stopped. Streptolysin O has also been used to permeabilize human T lymphocytes.

Product Number S5265 (lyophilized powder, 25,000-50,000 U/vial)
Product Number S0149 (γ-irradiated)

References:

1. Bhakdi, S. et al., Infection and Immunity, 46, 394 (1984).
2. Alouf, J.E., and Geoffroy, C., Methods in Enzymology, 165, 52 (1988).
3. Alouf, J.E., Pharmacol. Ther., 11, 661-717 (1980).
4. Barry, E. et al., Biotechniques, 15, 1016 (1993).
5. Graves, J.D. et al., Biochem. J., 265, 407-413 (1990).
6. Kanbayashi, Y. et al., J. Biochem., 71, 227-237 (1972).

Tetanolysin from Clostridium tetani
Cholesterol-binding toxin used to permeabilize cellular membranes to enhance the entry of macromolecules into the interior of the cell. Pores induced reported to be in the range of 20-50 nm.

Product Number T5319 (lyophilized powder)

Reference:

1. Haque, A., et al. Infect. Immun., 60, 71, (1992)

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Enzymes for Plant Cell Lysis

Plant cells are surrounded by a rigid, semi-permeable cell wall. The cell wall is comprised mainly polysaccharides with some proteins and lipids. The three main polysaccharide components of the cell wall are cellulose, an unbranched polymers of β-(1-4)-D-glycopyranosyl units associated in microfibril bundles. The microfibrils are cross-linked by hemicellulose (a branched polymer of β-(1-4)-D-xylopyranosyl units). This cross-linked structure is embedded in a matrix of pectin (primarily containing an α-(1-4) polygalacturonic acid backbone which can be randomly acetylated and methylated.

Reference:

1. Carpita, N., and McCann, M., The cell wall. In Biochemistry and Molecular Biology of Plants., Buchanan, B., et al., (Eds.) pp 52-108 (American Society of Plant Biologists, Rockville, MD, 2000).

Cellulase
Cellulase preparations are typically mixtures of enzymes containing high cellulase activity with some hemicellulase activity. These enzyme mixtures are capable of degrading cellulose, mannans, xylans, galactomannans, pectins and other polysaccharides.

from Aspergillus species
Product Number C2605 (Novozymes Carezyme 1000 L)
Product Number V2010 (Novozymes Viscozyme L)

from Aspergillus niger
Product Number C1184 (powder, min. 0.3 un/mg)

from Basidiomycetes sp
Product Number D9515 (Driselase, powder)

from Trichoderma reesei
Product Number C8546 (powder, min. 1.0 un/mg)
Product Number C2730 (Novozymes Celluclast 1.5 L)

from Trichoderma viride
Product Number C0615 (Yakult Onozuka RS)
Product Number C1794 (cell culture tested, powder, min. 3.0-10 un/mg)
Product Number C9422 (powder, min. 3.0-10 un/mg)

Pectinase and Pectolyase
Pectinase catalyzes the random hydrolysis of 1-4-α-D-galactosiduronic linkages in pectin and other galacturonans. Pectolyase catalyzes the eliminative cleavage of (1-4)-α-D-galacturonan methyl ester to give oligosaccharides with 4-deoxy-6-O-methyl- α-D-galact-4-enuronosyl groups at their non-reducing ends.

Pectinase
from Aspergillus aculeatus
Product Number P2611 (Novozymes Pectinex Ultra SPL)

from Aspergillus niger
Product Number P2736 (Novozymes Pectinex 3XL)
Product Number P4716 (aqueous glycerol solution, min. 5 un/mg protein)
Product Number P0690 (plant cell culture tested)

from Rhizopus species
Product Number P2401 (Macerozyme R10 Crude Powder 400-800 un/g)
Product Number P4300 (plant cell culture tested)

Pectolyase
from Aspergillus japonicus
Product Number P3026 (lyophilized, min. 0.3 un/mg)
Product Number P5431 (lyophilized, min. 2 un/mg)
Product Number P5936 (plant cell culture tested)

References:

1. Okada, G., Methods Enzymol., 160, 259-63 (1988)
2. Potrykus, J. and Shillito, R.D., Methods Enzymol., 118, 549-78 (1986) 
3. Nishimura, M., et al., Preparation of protoplasts from plant tissues. Meth. Enzymol. 148, 27-34, (1987)
4. Barraclough, R. and Ellis, R.J., Eur. J. Biochem., 94, 165-77, (1979)

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