Enzymes and Inhibitors

<h2>What Are Nucleases?</h2>
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Nucleases are enzymes that cleave phosphodiester bonds between nucleotide subunits in nucleic acids. Deoxyribonucleases (DNases) act on DNA; ribonucleases (RNases) act on RNA; and some nucleases (S1 Nuclease, Mung Bean Nuclease) act on both single-stranded DNA and RNA. Nucleases are further divided into endonucleases, which cleave internal phosphodiester bonds, and exonucleases, which progressively remove nucleotides from the 5' or 3' terminus of a strand. Major research nucleases include DNase I (non-specific endonuclease for both ss- and ds-DNA), RNase A (cleaves single-stranded RNA at pyrimidine residues), RNase H (degrades the RNA strand of RNA: DNA hybrids), Exonuclease III (removes nucleotides from 3'-recessed or blunt ends of duplex DNA), S1 Nuclease (degrades single-stranded DNA or RNA), and RNase R (degrades linear RNA but not circular RNA). Match the nuclease to the substrate type (single-stranded vs. double-stranded, DNA vs. RNA), the directionality required (endo vs. exo), and cofactor compatibility with your reaction buffer.
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<h2 dir="ltr">What you will find:</h2>
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PureRec RNase A (Recombinant): A ribonuclease free of animal-derived components, designed for the swift elimination of RNA from DNA samples, ensuring no threat of viral contamination.
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DNase I Set (RNase-free): A highly effective endonuclease formulated for complete DNA digestion in delicate RNA applications while preserving the integrity of the transcript.
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PureRec Duplex-Specific Nuclease (DSN): A unique enzyme designed to equalize high-abundance transcripts, greatly enhancing the identification of rare variants in cDNA libraries.
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RNase R: A distinctive 3'-to-5' exoribonuclease that breaks down linear RNA but preserves circular RNA forms, crucial for the identification of circRNA.
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RNaseOFF Inhibitor: A strong protein-based barrier that counteracts a wide range of RNases, maintaining the delicate transcriptional environment of your samples.
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<h2>How to Choose Nucleases</h2>
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DNase selection
DNase I (bovine pancreatic, recombinant) non-specifically cleaves both single- and double-stranded DNA in the presence of Mg2+ (random cleavage) or Mn2+ (double-strand cuts at approximately the same position on both strands). Use RNase-free DNase I for RNA preparations to remove genomic DNA contamination. Use Plasmid-Safe ATP-Dependent DNase to selectively degrade linear double-stranded DNA while preserving supercoiled and relaxed circular plasmid DNA. Baseline-ZERO DNase is optimized for the complete removal of trace DNA without damaging RNA.
RNase selection
RNase A cleaves single-stranded RNA at pyrimidine (C and U) residues to produce 3'-pyrimidine nucleotides; it is used to remove RNA from DNA preps, to perform RNase protection assays, and to analyze RNA secondary structure. RNase H degrades the RNA strand of RNA: DNA duplexes and is used after first-strand cDNA synthesis to remove the mRNA template. RNase R (3'-&gt;5' exoribonuclease) degrades all linear RNA without affecting circular RNA, and is used in circular RNA enrichment workflows and to characterize circular RNA splice junctions. Terminator 5'-Phosphate-Dependent Exonuclease (Terminator) digests linear RNA with a 5'-monophosphate, enriching for capped mRNA or circular RNA.
Exonuclease selection
Exonuclease III (Exo III) removes nucleotides from the 3' terminus of duplex DNA and generates single-stranded regions used in unidirectional nested deletions, mapping regulatory elements, and generating single-stranded templates for sequencing. Exonuclease I (Exo I) digests single-stranded DNA in the 3'-&gt;5' direction and is used after PCR to remove unincorporated primers before Sanger sequencing. Lambda Exonuclease removes nucleotides from 5'-phosphorylated duplex DNA in the 5'-&gt;3' direction and is used in isothermal amplification and strand displacement workflows.
Cofactor and inhibitor sensitivity
DNase I requires Mg2+ or Ca2+ for activity and is completely inhibited by EDTA at a 5 mM concentration. RNase A is active across a wide pH range (optimum 7.0-7.5) and is exceptionally stable; inactivation requires proteinase K digestion, phenol-chloroform extraction, or RNase inhibitor protein. RNase H requires Mg2+; RNase R requires Mg2+ and is most active between 37-50 degrees C.
Purity and application grade
For RNA-focused applications, all nucleases used on DNA substrates must be certified RNase-free. For DNA-focused work, DNase-free grades of RNases are required. Confirm lot-specific QC contamination data from the manufacturer before use in sensitive workflows.
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<h2>Specifications Context</h2>
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DNase I activity is expressed in Kunitz units: 1 unit increases A260 by 0.001 per minute per mL during DNA hydrolysis at pH 5.0. RNase A activity is expressed as the amount of enzyme hydrolyzing 50% of an RNA substrate under defined conditions; it remains active at a wide range of temperatures and is notoriously difficult to inactivate (requires boiling in SDS, proteinase K digestion, or phenol-chloroform extraction). RNase R from E. coli degrades linear RNA processively in the 3'-&gt;5' direction and is used at 2-10 U per microgram of total RNA to enrich for circular RNA species. RNase R treatment before library construction is the standard method for circular RNA sequencing (circRNA-seq) workflows on both short-read and long-read platforms. For protecting RNA from RNase contamination during nuclease-adjacent workflows, see&nbsp;<a href="https://mbpinc.net/reagents/molecular-biology/enzymes-and-inhibitors">RNase inhibitors</a>&nbsp;in the parent Enzymes and Inhibitors category.
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Safeguard your molecular images&mdash;<a href="https://mbpinc.net/contact-us">reach out to the MBP team</a> now to obtain a quote for our sophisticated nucleases and inhibitors.

Nucleases

MSDS

2X DSN Stop Solution (50 ml)

E1020-4-50 by Zymo Research — 2X DSN Stop Solution; 50 mL; designed to inactivate PureRec Duplex-Specific Nuclease (DSN) in DSN normalization and cDNA library subtraction workflows; made in USA.

Manufacturer

Shipping Condition

Storage Condition

DataSheet

Protocol

DNase I Set (RNase-free)

E1010 by Zymo Research is a recombinant DNase I Set (RNase-free), supplied lyophilized with DNA Digestion Buffer. One unit = 12 Kunitz units. Guaranteed RNase-free and protease-free; stable through ≥10 freeze-thaw cycles. Heat-inactivated at 75°C/10 min. Available in 250 U, 1500 U, and 5×1500 U.

Unit Definition

Storage

Reconstitution

Inactivation

DNase I Set (250 U) w/ DNA Digestion Buffer (4 ml)

DNase I Set (1500 U) w/ DNA Digestion Buffer (4 ml)

DNase I Set (5 x 1500 U) w/ DNA Digestion Buffer (16 ml)

PureRec Duplex-Specific Nuclease(Liquid)

E1020-200 by Zymo Research is PureRec Duplex-Specific Nuclease (DSN), 200&nbsp;U at 1&nbsp;U/&micro;l (200&nbsp;&micro;l, liquid). Selectively digests dsDNA and DNA-RNA hybrids; spares ssDNA and RNA. Purity &gt;95%, RNase-free. Optimal at 60&deg;C. Also available as E1020-1000.

Concentration

Optimal Temperature

Reaction Buffer

Stop Solution

PureRec Duplex-Specific Nuclease (1 U/µl, 200 µl) (Liquid)

PureRec Duplex-Specific Nuclease (1 U/ul, 1000 ul) (Liquid)

PureRec RNase A (Recombinant)

PureRec RNase A Recombinant (E1019-5) by Zymo Research — 5 mg at 1 mg/ml, tag-free recombinant RNase A. DNase-free, protease-free, and animal-free. Active across a wide range of buffer conditions. For RNA removal from DNA and protein samples, NGS, and cloning workflows. Made in the USA.

PureRec RNase A (0.1 mg, 100 µl) (Liquid)

PureRec RNase A (5 mg, 1 mg/ml (Liquid)

PureRec RNase A (20 mg, 1 mg/ml) (Liquid)

RNase A (30 mg)

RNase A (30 mg) (E1008-30) by Zymo Research — lyophilized, DNase-free, protease-free bovine pancreas RNase A, reconstitutable to 10 mg/ml. Thermostable; cleaves single-stranded RNA at 3' pyrimidine residues for RNA removal in DNA/plasmid purification workflows.

<h2>What Are Lytic Enzymes?</h2>
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Lytic enzymes degrade the structural polysaccharide scaffolding of microbial cell walls, enabling cell disruption under mild conditions without mechanical force. Lysozyme (N-acetylmuramide glycanohydrolase) cleaves the beta-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine residues in bacterial peptidoglycan, lysing gram-positive bacteria efficiently and gram-negative bacteria when paired with EDTA to destabilize the outer membrane. Lyticase from Arthrobacter luteus hydrolyzes beta-1,3-glucan in yeast cell walls and is the standard reagent for generating spheroplasts from Saccharomyces cerevisiae and other yeast species. Lysostaphin is a glycyl-glycine endopeptidase that specifically cleaves the polyglycine cross-links in Staphylococcus aureus peptidoglycan. Mutanolysin cleaves beta-1,4 linkages in the alternating N-acetylmuramyl-N-acetylglucosamine backbone of gram-positive bacteria, complementing lysozyme for complete lysis of resistant species. Choose the lytic enzyme based on organism type (bacterial vs. yeast/fungal), Gram stain classification, and intended downstream workflow (DNA extraction, protein isolation, or transformation).
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<h2 dir="ltr">What you will find:</h2>
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Zymolyase&trade; Ultra: An elite, high-activity enzyme optimized for rapid fungal cell wall digestion and maximum nucleic acid recovery.
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Flash-Extract Lysis Solution: A streamlined reagent for immediate genomic DNA release from diverse samples, ideal for NGS integration.
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Zymolyase&trade; w/ Storage Buffer: A stabilized formulation designed to maintain long-term enzymatic potency and consistent performance across all assays.
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<h2>How to Choose Lytic Enzymes</h2>
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Gram-positive vs. gram-negative bacteria
Lysozyme alone lyses most gram-positive bacteria efficiently at concentrations of 0.1-1 mg/mL in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA at 37 degrees C for 30-60 minutes. Gram-negative bacteria require EDTA (typically 10-50 mM) to chelate divalent cations and disrupt the outer membrane before lysozyme can access the peptidoglycan. Species with thick, atypical cell walls, such as Micrococcus, Staphylococcus, and Streptococcus, may respond better to lysostaphin, mutanolysin, or mechanical disruption combined with enzymatic lysis.
Yeast and fungal lysis
Lyticase (minimum 2,000 U/mg protein) is the reagent of choice for yeast spheroplast generation and DNA/RNA extraction from Saccharomyces, Candida, Pichia, Kluyveromyces, and related species. A typical protocol uses 10-100 U of lyticase per OD600 unit of yeast cells in 1 M sorbitol buffer at 30 degrees C for 30-60 minutes. Kitalase (from Rhizoctonia solani) combines beta-1,3-glucanase with protease, pectinase, and amylase activity, providing broader lytic activity for resistant fungal species.
DNA purity requirements
For microbiome research and 16S/18S rDNA amplification, lytic enzymes must be certified free of contaminating DNA. Lyticase lots used in microbiome studies are QC-tested by 35-cycle PCR amplification of 16S and 18S rDNA using universal primer sets; confirm this certification with each lot before use in amplicon sequencing workflows.
Downstream compatibility
Most lytic enzymes are inactivated by subsequent SDS treatment or heating to 95 degrees C for 5 minutes, making them compatible with standard nucleic acid extraction kits. Lyticase requires removal before silica column binding; heat inactivation at 70 degrees C for 10 minutes or proteinase K digestion at 55 degrees C for 30 minutes accomplishes this. Confirm inactivation conditions in the manufacturer's protocol before proceeding.
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<h2>Specifications Context</h2>
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Lysozyme activity is measured in units per milligram of protein, where 1 unit is defined as the decrease in absorbance of 0.001 per minute at 450 nm when Micrococcus lysodeikticus cells are used as substrate at pH 6.24 and 25 degrees C. Lyticase activity is expressed as units per milligram protein; 1 unit releases 1 nmol of glucose from yeast cell walls per minute at 30 degrees C. Lysozyme is stable for 12 months at 4 degrees C as a lyophilized powder and should be dissolved in 10 mM sodium acetate, pH 4.5-5.0, for storage in solution. Lytic enzyme combinations with mechanical disruption (bead beating) are standard for complete lysis of difficult samples, including Mycobacterium, spore-forming bacteria, and thick-walled fungi in clinical and microbiome research workflows. For downstream removal of protein from lysates, see <a href="https://mbpinc.net/reagents/molecular-biology/enzymes-and-inhibitors/proteases">proteases,</a> including Proteinase K, for nucleic acid cleanup.
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Streamline your path to discovery&mdash;<a href="https://mbpinc.net/contact-us">contact the MBP team</a> today to secure a quote for our advanced lytic reagents.

Lytic Enzymes

Zymolyase Ultra

Zymolyase Ultra (E1007T) by Zymo Research is a lyophilized multi-enzyme cell wall lytic preparation (100 U) combining β-1,3-glucanase, β-1,6-glucanase, chitinase, and trace protease. 50× more efficient than lyticase; up to 70× less contaminating DNA. Reconstitutes in water at ≤5 U/µl; stable through freeze-thaw; optimal at pH 7.3–8.0, 37°C.

Activity

Format

Optimum pH

Optimum Temperature

Solubility

Zymolyase Ultra (100 U)

Zymolyase Ultra (2000 U)

Zymolyase Ultra (10,000 U)

Protocol - E1004/E1005

MSDS - (E1004/E1005)

MSDS - E1006

Protocol - E1006

Zymolyase™ w/ Storage Buffer

Zymolyase w/ Storage Buffer (E1004) by Zymo Research contains 1,000 units of lyophilized Zymolyase (β-1,3-glucanase) for yeast cell wall digestion across 17 fungal genera, with no detectable DNase or RNase activity and optimal activity at 35°C, pH 7.5.