MMEI, A CLASS-IIS RESTRICTION-ENDONUCLEASE - PURIFICATION AND CHARACTERIZATION

Two restriction endonucleases, MmeI and MmeII, from Methylophilus meth ylotrophus were purified to homogeneity. Both enzymes belong to the cl ass-II restriction endonucleases (ENases) but exhibit very different e nzymatic and physical properties. MmeII is a typical member of class-I I ENases. It is a polymeric protein composed of 50-kDa subunits. In co ntrast to MmeII, MmeI is a monomeric protein of 101 kDa, cleaving a DN A molecule 20/18 nucleotides away from the asymmetric recognition sequ ence (5'-TCCRAC-3'); therefore, it is classified as a member of subcla ss-IIS. MmeI has an pi of 7.85 and is active in the pH range 6.5 to 10 with the optimum at 7 to 8. Increasing salt concentration creates an inhibitory effect on MmeI: 40 mM KCl decreases activity by 50%, 100 mM completely inhibits DNA cleavage. Tris HCl (pH 7.5) at a concentratio n exceeding 20 mM inhibits MmeI activity. Mg2+ stimulates MmeI in the range of 0.2 to 35 mM, with the optimum between 0.5 and 10 mM.