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?
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.
Product Number L1414 (powder) From Streptomyces fulvissimus
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)
Figure 2.Lysozyme and Mutanolysin
Product Number L6876 (dialyzed, lyophilized, approx 50,000 un/mg)
Product Number L7651 (molecular biology grade, lyophilized, approx 50,000 un/mg)
Product Number L/CA/en7001 (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)
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)
Product Number H9395
Figure 3.Staphylococcus aureus
The study of microbial communities has recently been revolutionized by the widespread adoption of techniques such as 16S rRNA gene sequencing and metagenomics. Since DNA contamination during microbiome sample preparation is a major challenge of these sequence-based approaches, DNA extraction reagents free of additional DNA contaminants are essential.
Another major challenge in processing microbiome samples is that microbes are difficult to disrupt - the cell walls can form capsules or resistant spores when processed. DNA can be extracted from the microbes by using lysing enzymes to induce partial spheroplast formation. These spheroplasts are subsequently lysed to release DNA.
We offer a growing list of microbial DNA-free lytic enzymes and enzyme mixes that will help improve lysis of microbiome samples without random DNA contamination.
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.
Lysing Enzymes from Rhizoctonia solani (Kitalase)
Product Number L8757 (powder)
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)
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.
Product Number H9395 (lyophilized powder, ≥10,000 units/mg protein)
Figure 5.Streptococcus Pyogenes
Product Number T5319 (lyophilized powder)
Figure 6.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.
Figure 7.Cellulose, Hemicellulose and Pectin structures
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 niger
Product Number C1184 (powder, min. 0.3 un/mg)
from Basidiomycetes sp
Product Number D9515 (Driselase, powder)
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.
from Aspergillus aculeatus
Product Number P2611 (Novozymes Pectinex Ultra SPL)