December 2025

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.

A bacterial strain of Microbacterium oxydans was identified as a producer of a 5-methylcytosine-dependent, site-specific DNA endonuclease, MoxI. The enzyme recognizes and cleaves the DNA sequence 5′-R(5mC)↓GY-3′ / 3′-YG↓(5mC)R-5′. The moxI gene was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified and biochemically characterized. The recognition sequence of MoxI was shown to be identical to that of the previously described 5mC-directed site-specific DNA endonuclease GlaI. In contrast to GlaI, however, MoxI exhibits maximal catalytic activity at 37 °C and demonstrates higher cleavage efficiency toward substrates containing two 5-methylcytosine residues within the recognition site