Purification and characterization of a novel type Ⅱ restriction endonuclease, Bsp 1894I, from bacillus sphaericus 1894

Abstract

A novel restriction endonuclease, Bsp 1894I, has been isolated from Bacillus sphaericus 1894, and its catalytic properties have been studied. For the purification of this enzyme, 35g of cells were used and disrupted by French Press at 20, 000 P.S.I. After streptomycin sulfate and ammonium sulfate fractionation, DEAE-cellulose, QAE-Sephadex, hydroxyapatite, and Sephadex G-100 chromatography have been carried out. From the dideoxy sequencing and the comparison of digestion pattern on various DNAs, it was demonstrated that the new enzyme Bsp 1894I recognizes 5``-G↓GNCC-3`` and cleaves at the site indicated by the arrow. This enzyme is an isoschizomer of Asu I, Cfr 13I, Nsp 7524IV, and Sau 96I. In catalytic properties, Bsp 1894I shows maximum activity at range between 6 and 7 in the presence of 10 mM MgCl2. The optimum temperature of Bsp 1894I is 42℃. Unlike above isoschizomer, Bsp 1894I does not require NaCl for activity.