New enzymes

We identified a bacterial strain, Brevundimonas mongoliensis 53, which produces a new Type II restriction endonuclease, designated BmoI. BmoI recognizes the palindromic heptanucleotide sequence 5’-GG^GWCCC-3’ (W = A or T) and cleaves at the indicated position to generate three-nucleotide 5′ overhangs. BmoI is an isoschizomer of the previously described restriction enzyme SanDI. The producing strain was identified based on morphological and biochemical characteristics and by sequencing a fragment of the 16S rRNA gene. A preparation of BmoI with an activity of 2000 U/ml was obtained using a three-step chromatographic purification workflow. Optimal reaction conditions for BmoI were determined to be SE buffer W (10 mM Tris-HCl, pH 8.5; 10 mM MgCl₂; 100 mM NaCl; 1 mM DTT) and an incubation temperature of 37°C.

We have discovered a bacterial strain, Peribacillus species 31, which produces a novel restriction endonuclease named Pse31I. The enzyme recognizes a six-nucleotide non-palindromic DNA sequence, 5’-GGTCTC-3’, and cleaves it away from the recognition site at positions 1/5: 5’-GGTCTC(N)1^/3’-CCAGAG(N)5^-5’. Thus, Pse31I is a true isoschizomer of the restriction enzyme Eco31I . The producer strain was identified based on morphological and biochemical characteristics, as well as by analyzing the primary structure of a fragment of the 16S rRNA gene. Pse31I exhibits maximal activity at 37°C and is inactivated by incubation at 55°C and 65°C. The enzyme does not cleave sites containing a methylated cytosine in the top strand (5’-GGTCT(5mC)-3’) and/or in the bottom strand (3’-(5mC)CAGAG). The optimal reaction conditions for the enzyme include SE-buffer 5 (Y) (33 mM Tris-acetate (pH 7.9), 10 mM Mg-acetate, 66 mM K-acetate, 1 mM DTT) and a temperature of 37°C

We identified a bacterial strain, Paracoccus species 12, which produces a novel restriction endonuclease, named PecI. This enzyme recognizes the hexanucleotide palindromic DNA sequence 5’-TTA^TAA-3’ and cleaves it, as indicated by the arrow, generating blunt ends. Thus, PecI is a true isoschizomer of the restriction enzyme PsiI. A preparation of PecI restriction endonuclease at a concentration of 10,000 U/mL was obtained through a four-step chromatographic purification process. The optimal reaction conditions for the enzyme are SE-buffer 5 (Y) (33 mM Tris-acetate (pH 7.9), 10 mM Mg-acetate, 66 mM K-acetate, 1 mM DTT) and a temperature of 37oC.

We have discovered a bacterial strain Lysinibacillus manganicus An22 that produces the new prototype of restriction endonuclease named LmnI. This enzyme recognizes a nonpalindromic DNA sequence 5’-GCTCC-3’/3’-CGAGG-5’. The LmnI restriction endonuclease preparation with the concentration of 1000 units/ml was isolated using four chromatographic steps. It was shown that new enzyme cuts its recognition sequence forming 3’-protruding ends as indicated by the arrows: 5’-GCTCCN↓-3’/3’-CGAG↑GN-5’, and can be applied to a IIS type of restriction-modification systems.