Benzonase

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Benzonase is an enzyme from the group of nucleases . It is a genetically modified form of the extracellular nuclease from Serratia marcescens , which in E. coli strain W3110 expressed will. In E. coli , Benzonase is encoded by the plasmid pNUC1 . Benzonase is used in the course of protein purification to break down nucleic acids in cell lysates and thus to reduce their viscosity , to facilitate the purification of proteins and to meet official requirements for nucleic acid-free protein pharmaceuticals. Sometimes it is also used in an immobilized manner .

structure

Benzonase is a homodimer , the subunits have 245 amino acids and a relative molecular mass of 30 kDa. The enzyme requires Mg 2+ (1–2 mM) as a cofactor and has two essential disulfide bonds . The enzyme can work in the presence of detergents , urea , phenol , PMSF and thiols . It is inhibited by salt concentrations> 20 mM. The optimal pH value is 8 (working range 6–10), the optimal temperature is 35 ° C (working range 0–42 ° C).

Specificity

Benzonase has broad substrate specificity against DNA and RNA (linear, circular, single or double stranded) with a slight preference for G / C-rich segments, these are cleaved into oligonucleotides that are 2-5 nucleotides in length and a 5 'monophosphate . Benzonase has no measurable protease activity. Theoretically, it is also possible to use Benzonase to make bacteria and viruses non-infectious in vaccine production .

Individual evidence

  1. ↑ CAS 9025-65-4, PDB 4e3y, EC 3.1.30.2 at BRENDA .
  2. ↑ P. Friedhoff, O. Gimadutdinow, T. Rüter, W. Wende, C. Urbanke, H. Thole, A. Pingoud: A procedure for renaturation and purification of the extracellular Serratia marcescens nuclease from genetically engineered Escherichia coli. In: Protein expression and purification. Volume 5, Number 1, February 1994, pp. 37-43, doi : 10.1006 / prep.1994.1005 , PMID 8167472 .
  3. ↑ Molin, S., Givskov, M. & Riise, E .: Production in Escherichia coli of extracellular Serratia spp. hydrolases. US Patent 5,173,418 of December 22nd, 1992, Benzon Pharma A / S, Hvidovre, Denmark, https://patents.google.com/patent/US5173418A/en
  4. ↑ JM Moreno, JM Sanchez-Montero, A. Ballesteros, JV Sinisterra: Hydrolysis of nucleic acids in single-cell protein concentrates using immobilized benzonase. In: Applied biochemistry and biotechnology. Volume 31, Number 1, October 1991, pp. 43-51, PMID 1665680 .
  5. ↑ JM Moreno, JM Sanchez-Montero, JV Sinisterra, LB Nielsen: Contribution to the study of the enzymatic activity of benzonase. In: Journal of Molecular Catalysis. 69, 1991, p. 419, doi : 10.1016 / 0304-5102 (91) 80120-R .
  6. ^ M. Nestle, WK Roberts: An extracellular nuclease from Serratia marcescens. II. Specificity of the enzyme. In: Journal of Biological Chemistry . Volume 244, Number 19, October 1969, pp. 5219-5225, PMID 4310088 .