Serratia marcescens nuclease
| Serratia marcescens nuclease | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Identifiers | |||||||||
| EC no. | 3.1.30.2 | ||||||||
| CAS no. | 9025-65-4 | ||||||||
| Databases | |||||||||
| BRENDA | enzyme data | ||||||||
| ExPASy | NiceZyme view | ||||||||
| KEGG | enzyme entry | ||||||||
| MetaCyc | metabolic pathway | ||||||||
| Rhea | reactions | ||||||||
| PDB structures | RCSB PDB PDBe PDBsum | ||||||||
| |||||||||
| Serratia marcescens nuclease | |||||||
|---|---|---|---|---|---|---|---|
| Identifiers | |||||||
| Organism | |||||||
| Symbol | nucA | ||||||
| Orthologs | OMA: entry | ||||||
| UniProt | P13717 | ||||||
| |||||||
Serratia marcescens nuclease (EC 3.1.30.2, endonuclease (Serratia marcescens), barley nuclease, plant nuclease I, nucleate endonuclease) is an enzyme.[1][2][3][4] This enzyme catalyses the following chemical reaction
- Endonucleolytic cleavage to 5′-phosphomononucleotide and 5′-phosphooligonucleotide end-products
Hydrolyses double- or single-stranded substrate DNA or RNA. It is a representative of the DNA/RNA non-specific endonuclease family.
It is commercially available.
Characteristics
[edit]Serratia nuclease was first purified from its native source in 1969.[5] It was cloned in 1987 and shown to consist of a 266 protein precursor,[6] which is further cleaved and secreted as a 245 amino acid active nuclease.[7] Its active form in solution is a homodimer.[8] It has two disulfide bonds, the first between cysteine 30 and 34 and the second between cysteine 222 and 264.[7] Reduction of these disulfides or site directed mutagenesis of their residues to serine, specifically the first one, leads to a large loss in nuclease activity,[8] and a loss of the ability to reversibly regain activity after inactivating 40–60 °C heat treatments.[7] It has a much higher catalytic efficiency than other nucleases, about 4 times greater than staphylococcal nuclease, and about 34 times greater than bovine pancreatic DNase I.[8] The enzyme cleaves single or double stranded DNA and RNA with similar rates, so long as the substrate DNA or RNA contains no fewer than 5 nucleotides (or basepairs).[8] Magnesium(II) (Mg2+) is an essential cofactor for its nuclease activity.[8] Serratia nuclease is activated by up to 4 M urea.[9] At 5 M urea the initial activity is decreased from its peak although still above its baseline, and the enzyme is significantly inhibited after 60 minutes. At 6 M urea, the nuclease activity is below baseline and almost completely inactivated within 60 minutes. At 7 M the nuclease becomes essentially completely inactivated within 15 minutes, but significant and workable degradation of nucleic acids can occur before the nuclease is inactivated.[9] 8 M urea causes a complete inactivation of the enzyme within 5 minutes.[7]
Optimal conditions
[edit]| Condition[9] | Optimal1 | Effective2 |
|---|---|---|
| Mg2+ concentration | 1–2 mM | 1–10 mM |
| pH | 8.2–9.2 | 6.0–10.0 |
| Temperature | 37 °C | 0–42 °C |
| Dithiothreitol (DTT) | < 100 mM | > 100 mM |
| β-Mercaptoethanol (BME) | < 100 mM | > 100 mM |
| Monovalent cation concentration (Na+, K+, etc.) | 0–20 mM | 0–150 mM |
| PO3−4 | 0–10 mM | 0–100 mM |
| Urea | < 4 M | > 4 M |
- 1. "Optimal" is the condition in which Serratia nuclease retains over 90% of its activity.
- 2. "Effective" is the condition in which Serratia nuclease retains over 15% of its activity.
Inhibitory conditions
[edit]Some inhibitory conditions are known:[9]
- > 300 mM monovalent cations (Na+, K+, etc.)
- > 100 mM phosphate
- > 100 mM ammonium sulfate
- > 100 mM guanidine HCl
- > 2 mM EDTA
- > 4 mM EGTA
- > 0.4% w/v Triton X-100 (no effect below 0.4%, slight activation above 0.4%)
- > 0.4% w/v Sodium deoxycholate (70% activity at 0.4%, steady inactivation below and above 0.4%)
- > 0.1% w/v SDS (inactivation kinetics allow for Serratia nuclease to still degrade some nucleic acids before inactivation)
Use in biotechnology
[edit]Given its high activity, high stability & reversible inactivation to heat treatments, rate enhancement or otherwise compatibility with some denaturing reagents like urea, Serratia nuclease was recognized early on to have industrial & commercialization potential. A patent covering the recombinant expression of Serratia nuclease in E. coli was submitted by Benzon Pharma in 1986, granted in 1992, & expired in 2006.[10] This recombinant Serratia nuclease was commercialized as Benzonase, and is still available from and a registered trademark of Merck KGaA.[11] Notably, the patented sequence[10][12] for Benzonase is slightly different (1 amino acid substitution) from the Serratia marcescens nuclease which was cloned publicly.[13]
As the benzonase patent is now expired, and in fact was never submitted nor granted in the United States, several commercial alternatives for recombinantly produced Serratia marcescens nuclease are now available:
- Basemuncher, from Westburg Life Sciences[14]
- Benzo Nuclease, from Tinzyme[15]
- Benz-Neburase, from GenScript[16]
- Decontaminase, from AG Scientific[17]
- Denarase, from c-LEcta[18]
- Dr. Nuclease, from Syd Labs[19]
- GENIUS Nuclease, from ACROBiosystems[20]
- Pierce Universal Nuclease, from Thermo Fisher Scientific[21]
- TurboNuclease, from Accelagen[22]
- MaxNuclease™, from KACTUS[23]
(A current notable non-producer is New England Biolabs)[24]
See also
[edit]References
[edit]- ↑ Mikulski AJ, Laskowski M (October 1970). "Mung bean nuclease I. 3. Purification procedure and (3') omega monophosphatase activity". The Journal of Biological Chemistry. 245 (19): 5026–5031. doi:10.1016/S0021-9258(18)62813-3. PMID 4319109.
- ↑ Stevens A, Hilmoe RJ (1960). "Studies on a nuclease from Azotobacter agilis. I. Isolation and mode of action". Journal of Biological Chemistry. 235 (10): 3016–3022. doi:10.1016/S0021-9258(18)64581-8.
- ↑ Stevens A, Hilmoe RJ (1960). "Studies on a nuclease from Azotobacter agilis. II. Hydrolysis of ribonucleic and deoxyribonucleic acids". Journal of Biological Chemistry. 235 (10): 3023–3027. doi:10.1016/S0021-9258(18)64582-X.
- ↑ Wechter WJ, Mikulski AJ, Laskowski M (February 1968). "Gradation of specificity with regard to sugar among nucleases". Biochemical and Biophysical Research Communications. 30 (3): 318–322. Bibcode:1968BBRC...30..318W. doi:10.1016/0006-291x(68)90453-1. PMID 4296679.
- ↑ Nestle M, Roberts WK (October 1969). "An extracellular nuclease from Serratia marcescens. I. Purification and some properties of the enzyme". The Journal of Biological Chemistry. 244 (19). Elsevier BV: 5213–5218. doi:10.1016/s0021-9258(18)63648-8. PMID 4899013.
- ↑ Ball TK, Saurugger PN, Benedik MJ (1987). "The extracellular nuclease gene of Serratia marcescens and its secretion from Escherichia coli". Gene. 57 (2–3). Elsevier BV: 183–192. doi:10.1016/0378-1119(87)90121-1. PMID 3319779.
- 1 2 3 4 Biedermann K, Jepsen PK, Riise E, Svendsen I (1989). "Purification and characterization of a Serratia marcescens nuclease produced by Escherichia coli". Carlsberg Research Communications. 54 (1). Springer Science and Business Media LLC: 17–27. doi:10.1007/bf02910469. PMID 2665765. S2CID 12831178.
- 1 2 3 4 5 Benedik MJ, Strych U (August 1998). "Serratia marcescens and its extracellular nuclease". FEMS Microbiology Letters. 165 (1). Oxford University Press (OUP): 1–13. doi:10.1111/j.1574-6968.1998.tb13120.x. PMID 9711834.
- 1 2 3 4 "Benzonase® Nuclease - Effective removal of nucleic acids and viscosity reduction from protein solutions" (PDF). EMD Biosciences. SigmaAldrich. Retrieved 29 April 2023.
- 1 2 EP 0229866A1, Molin S, Givskov M, Riise E, "Bacterial enzymes and method for their production", issued 9 December 1992, assigned to Benzon Pharma AS and Takeda Pharma AS
- ↑ "Benzonase® Nuclease HC, Purity > 99% - 71206". MilliporeSigma. Retrieved 2023-04-29.
- ↑ "UniProt". UniProt. Retrieved 2023-04-29.
- ↑ "UniProt". UniProt. Retrieved 2023-04-29.
- ↑ "Basemuncher Benzonase". Westburg. January 2001. Retrieved 2023-04-29.
- ↑ "Benzo Nuclease". Tinzyme Ltd – Enzymes, dNTP and rNTP. 2021-12-24. Retrieved 2023-04-29.
- ↑ "Benz-Neburase™, His". GenScript. 2021-08-12. Retrieved 2023-04-29.
- ↑ "B-1400-5KU - Decontaminase™, 5 KU". AG Scientific. 2022-12-13. Retrieved 2023-04-29.
- ↑ "Denarase". c-LEcta. 2022-12-13. Retrieved 2023-04-29.
- ↑ "Benzonase Nuclease Alternative, DENARASE Nuclease Alternative". Syd Labs. 2020-05-01. Retrieved 2023-04-29.
- ↑ "GENIUS™Nuclease DMF Filed". ACROBiosystems. Retrieved 2023-04-29.
- ↑ "Pierce™ Universal Nuclease for Cell Lysis". Thermo Fisher Scientific. 2023-04-29. Retrieved 2023-04-29.
- ↑ "TurboNuclease". Accelagen. 2023-04-29. Retrieved 2023-04-29.
- ↑ "MaxNuclease™ Benzonase Nuclease Alterantive". KACTUS. 2025-02-19. Retrieved 2025-02-19.
- ↑ Biolabs, New England. "DNA Modifying Enzymes & Cloning Technologies - Exonucleases and Non-specific Endonucleases". New England Biolabs. Retrieved 30 April 2023.
External links
[edit]- Serratia+marcescens+nuclease at the U.S. National Library of Medicine Medical Subject Headings (MeSH)
- Serratia marcescens endonuclease at Protean Ltd., highly active
