The unicellular cyanobacterium Microcystis aeruginosa K-81 has two types of restriction barrier, an extracellular nuclease and sequence-specific endonucleases. The nuclease was detected in the culture supernatant and it was easily released from the cells by washing with water or buffer containing Triton X-100. This nuclease was identified as a polypeptide of about 28 kDa that digested covalently closed circular and linear double-stranded DNAs, including chromosomal DNA from M. aeruginosa K-81. Among another 13 Microcystis strains examined, 3 produced an extracellular nuclease. Furthermore, M. aeruginosa K-81 contained two sequence-specific endonucleases, MaeK81I and MaeK81II, which were isoschizomers of SplI and Sau96I, respectively.

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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