Purification and characterization ofAspMDl, an isoschizomer ofSau3AI, from a marine bacterium,Alcaligenessp MD1

Abstract

A new type II restriction endonuclease, Asp MD1, was isolated from a pigmented marine bacterium Alcaligenes species MD1 which was collected from the open sea around Taiwan Island. Asp MD1 is an isoschizomer of Sau3AI which recognizes four palindromic sequences 5'GATC3' (1). Cells were grown at 25°C up to full growth in a modified sea water broth (PPES II) containing 0.2% polypepton (Daigo Eiyo co. Ltd.), 0.1% proteose peptone No.3, 0.1% bacto yeast extract and 0.1% bacto soytone (Difco Laboratories) dissolved in artificial sea water (Jamarin Laboratory). The cell yield from 1 liter of culture broth was about 5-7 g in wet weight. Since non-specific nuclease activity of the cell lyzate was significantly low, the enzyme was purified easily using CMand DEAE-Sephadex chromatography. Restriction enzymic activity was determined at 30°C, in a reaction mixture containing 50 mM Tris-HCI, 5 mM /3-mercaptoethanol, 10 mM MgCl2, 200 mM NaCl and