Cell Lysis Enzymes – How to Tear Down the Wall

Enzymes provide a non-mechanical method for cell lysis and protoplast preparation. It may seem like a simple process to throw in your enzyme, stick your tube in the water-bath and walk away, but what is actually going on in that process? What is being disrupted and how can you choose the best enzyme for the job?

Understanding the Bacterial Cell Wall — the Gram Positives and Negatives

Gram Positive Bacterial Cell Wall and Gram Negative Bacterial Cell Wall

Figure 1.Gram Positive Bacterial Cell Wall and Gram Negative Bacterial Cell Wall

Gram-positive bacteria is composed of multiple layers of peptidoglycan. Peptioglycan 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.

Gram-negative cell wall in bacteria 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.

Which Enzymes Will Get the Job Done - Lysing Bacteria

Labiase

Product Number L1414 (powder) From Streptomyces fulvissimus

  • 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

Lysostaphin

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)
Product number L9043 (lyophilized powder min 3000 unit/mg solid protein)

  • 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
Lysozyme and Mutanolysin

Figure 2.Lysozyme and Mutanolysin

Lysozyme 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)

  • 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.

Achromopeptidase

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)

  • 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

Mutanolysin from Streptomyces globisporus

Product Number SAE0092 (lyophilized, min 4000un/mg protein)
Product Number M9901 (lyophilized, min 4000un/mg protein)
Product Number M4782 (Aseptically filled, lyophilized, min 4000un/mg protein)

  • 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.

α-Hemolysin from Staphylococcus aureus

Product Number H9395

  • α-Hemolysin is an extracellular protein secreted by most strains of pathogenic Staphylococcus aureus.
  • The mature protein contains 293 residues with a molecular weight of 33 kD. It is composed of 65% β-sheets and 10% α-helical structures.
  • α-Hemolysin is secreted as a water-soluble monomer which initially binds and incorporates into the target cell membrane. The monomers assemble to form a heptametric pore The pore allows rapid efflux of K+ and influx of NA+ and Ca+. Osmotic swelling of erythrocytes finally results in a rupture.
Staphylococcus aureus

Figure 3.Staphylococcus aureus

Chitinase

Product number C6137 from Streptomyces griseus
Product number C8241 from Trichoderma viride

Chitinase

Figure 4.Chitinase

  • Chitinase is a 30 kDa (approx.) extracellular enzyme complex that degrades chitin.
  • Chitin is degraded to N-acetyl-D-glucosamine in 2 enzymatic reactions. Firstly, chitobiose units are removed from chitin by chitodextrinase-chitinase, a poly(1,4-β-[2-acetamido-2-deoxy-D-glucoside])-glycanohydrolase. The second reaction involves N-acetyl-glucosaminidase-chitobiase, which cleaves the disaccharide to its monomer subunits of N-acetyl-D-glucosamine.
  • The enzyme may be classified into endo- and exochitinase. The endochitinase activity involves random cleavage at internal points in the chitin chain. The exochitinase activity consists of a progressive action which starts at the non-reducing end of chitin and releases chitobiose or N-acetyl-glucosamine units.

The Yeast Cell Wall — Shape and Rigidity

yeast cell wall

Figure 4.Yeast cell wall

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)-glucans as well as among glycoproteins, β(1-6)-glucans, and β(1-3)-glucans.

The Enzymes That Can Break it Down — the Yeast Cell Wall

Lysing Enzymes from Rhizoctonia solani (Kitalase)
Product Number L8757 (powder)

  • Main enzymatic acitivity is β-1,3-glucanase, also reported to contain protease, pectinase and amylase activities.

Lyticase from Arthrobacter luteus
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)

  • 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.

Lysing Enzymes from Trichoderma harzianum
Product Number L1412 (lyophilized powder)

  • (Glucanex® a product of Novozymes)
  • Contains β-glucanase with some cellulase, protease and chitinase activities.
Yeast Beta Glucan Structure

Figure 5.Yeast Beta Glucan Structure

Toxins for Eukaryotic Cells

A toxin is a poisonous small molecule, peptide, or protein produced within living cells. Some toxins interact with molecules on surface of a cell in a way that enhances the entry into the interior of the cell. These toxins are useful tools for researchers that want to disrupt the cell.

Making Eukaryotic Cells Permeable- with a toxin

α-Hemolysin from Staphylococcus aureus

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

  • 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.

Streptolysin O from Streptococcus pyogenes

Product Number SAE0089
Product Number S5265
Product Number S0149

  • Streptolysin O is a toxin that is a single polypeptide chain with a molecular weight of 62 kDa.
  • Streptolysin O binds to membrane cholesterol and oligomerizes to create a ring structure that consists of 45 to 50 units. The ring structure inserts into the membrane to make a large pore (25 to 30 nm), which DNA, RNA, peptides and proteins may pass.

Streptococcus Pyogenes

Figure 6.Streptococcus Pyogenes

Tetanolysin from Clostridium tetani

Product Number T5319 (lyophilized powder)

  • 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.

Enzymes for Plant Cell Lysis

Plant Cell Wall

Figure 7.Plant Cell Wall

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.

Cellulose, Hemicellulose and Pectin structures

Figure 8.Cellulose, Hemicellulose and Pectin structures

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)

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