EmiI

A bacterial strain, Exiguobacterium mexicanum 6, was identified as a producer of a novel restriction endonuclease designated EmiI. The enzyme recognizes the non-palindromic hexanucleotide DNA sequence 5′-CTCTTC-3′ and cleaves DNA outside the recognition site at positions 1/4, generating three-nucleotide 5′-protruding ends: 5′-CTCTTC(N)₁↓-3′ / 3′-GAGAAG(N)₄↓-5′. Thus, EmiI is an isoschizomer of the restriction endonucleases Ksp632I and Bst6I . The producer strain was identified based on morphological and biochemical characteristics as well as analysis of the primary structure of a fragment of the 16S rRNA gene. A preparation of restriction endonuclease EmiI with an activity of 5000 U/ml was obtained through purification using three chromatographic steps. Optimal reaction conditions for EmiI include SE buffer Y (33 mM Tris–acetate, pH 7.9; 10 mM Mg–acetate; 66 mM potassium acetate; 1 mM DTT) at a temperature of 37 °C.