Aox I

Description

Recognition site and hydrolysis position:

Source: Arthrobacter oxydans 25K
Substrate specificity:
Unit definition: One unit is defined as the amount of enzyme
required to hydrolyze in 1 μg of linearized plasmid pMHaeIII/DriI in 1 hour at 60°C in a total reaction volume of 50 μl. As a result a linearized plasmid pMHaeIII/DriI disappears (see run 4 in the figure).

AoxI activity assay on DNA pMHaeIII/DriI Lanes: 1 – Control DNA pMHaeIII/DriI, 2 – 0.5 μl Aox I 3 – 1 μl Aox I 4 – 2 μl Aox I 5 – 1 Kb SE DNA-markers Products were separated in 1% agarose gel in Buffer TAE.

Assayed on: DNA pMHaeIII/DriI is a linearized plasmid pMHaeIII. pMHaeIII carries a gene of DNA-methyltransferase M.HaeIII, which methylates sites 5`-GGCC-3` producing 5`-GG(5mC)C-3`/3`-C(5mC)GG-5`.
Optimal SE-buffer: SE-buffer AoxI
Enzyme activity (%):

Storage conditions: 10 mM Tris-HCl (pH 7.4); 100 mM NaCl; 0.1 mM EDTA; 7 mM 2-mercaptoethan. Store at -20°C.
Ligation: –
Non-specific hydrolisis: No detectable degradation of 1μg of Lambda DNA was observed after incubation with 1 units of enzyme for 16 hours at 60°C in a total reaction volume of 50 μl. Reagents Supplied with Enzyme 10 X SE-buffer AoxI
Methylation sensitivity: The enzyme cleaves C5-methylated DNA and doesn’t cut unmodified DNA.
Notes: References:
1. Chernukhin V.A., Belichenko O.A., Tarasova M.V., Gonchar D.A., Akishev A.G., Dedkov V.S., Mikhnenkova N.A., Degtyarev S.Kh. Bacterial strain Arthrobacter oxydans – producer of AoxI site specific endonuclease. // Russian Federation patent RU 2399663 C1 (2009).