Restriction Endonuclease Pse31I from Bacterial Strain Peribacillus Species 31, an Isoschizomer of Eco31I, Recognizes the Non-palindromic DNA Sequence 5’-GGTCTC-3’ (1/5)

We have discovered a bacterial strain, Peribacillus species 31, which produces a novel restriction endonuclease named Pse31I. The enzyme recognizes the non-palindromic hexanucleotide DNA sequence 5’-GGTCTC-3’ and cleaves outside 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 [1]. The producer strain was identified by morphological and biochemical characteristics as well as by analysis of the 16S rRNA gene fragment.
Pse31I shows maximum activity at 37°C and is inactivated by incubation at 55 and 65°C. The enzyme does not cleave sites with methylated cytosine in the top strand (5’-GGTCT(5mC)-3’) and/or in the bottom strand (3’-(5mC)CAGAG).

The optimal conditions for enzyme activity are SE-buffer 5 (Y) (33 mM Tris-acetate (pH 7.9), 10 mM Mg-acetate, 66 mM K-acetate, 1 mM DTT) at 37°C.

A preparation of the restriction endonuclease PecI with a concentration of 10,000 U/mL was obtained through a four-step chromatographic purification. The optimal reaction conditions 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 37^oC.

Abbreviations: BSA – bovine serum albumin; DTT – dithiothreitol; T7 DNA – bacteriophage T7 DNA; λ DNA – bacteriophage λ DNA; Tris – tris(hydroxymethyl)aminomethane; PAGE – polyacrylamide gel; restriction enzyme, RE – restriction endonuclease; EDTA – ethylenediaminetetraacetic acid; SAM – S-adenosyl-L-methionine; M.SssI – SssI DNA methyltransferase; (5mC) – 5-methylcytosine;