A new sequence specific endonuclease, Zan I has been purified from Zymomonas anaerobia (NCI B8227). The purified protein was homogeneous as judged by 10% polyacrylamide gel electrophoresis in the presence of 0.1% SDS, and the subunit molecular weight was 30,000 ± 1,000 daltons.
For the purification of the enzyme, 300 g of cells (wet weight) were used and broken by French Press at 20,000P.S.I. After ammonium sulfate fractionation, the enzyme was further purified by phospho cellulose column chromatography, DEAE cellulose column chromatography, Hydroxyl-apatite column chromatography and phospho cellulose column chromatography.
From the dideoxy sequencing it was demonstrated that the new enzyme Zan I endonuclease (an isoschizomer of Eco RII, Bst NI, etc.), recognize 5'- CC$^{\downarrow{A}}_T$ GG-3' and cleaves at the site indicated by the arrows. Also Zan I endonuclease was able to cleave dcm-methylated DNA which is resistant to the digestion with Eco RII endonuclease. In order to study the catalytic properties of Zan I endonuclease, ($^3H$)- labeled lambda DNA was used as a substrate. The enzyme showed maximum activity at pH values between 7.0 and 9.0 in the presence of 10 mM $MgCl_2$, 10 mM NaCl. The optimum temperature for Zan I endonuclease activity is 42℃, which is different from Bst NI endonuclease. Also the enzyme activity was not changed in the presence of 150 mM NaCl.
Zymomonas anaerobia 로 부터 새로운 type Ⅱ 제한효소인 Zan I endonuclease 를 순수 정제하였다. 300g (wet weight) 의 cells로 부터 얻은 crude extract를 ammonium sulfate fractionation을 거친후 phospho-cellulose, DEAE-cellulose, hydroxylapatite, phosphocellulose 등 chromatography 를 거쳐 최종적으로 0.2mg 의 효소를 얻었다. 얻어진 Zan I endonuclease 는 SDS-polyacrylamide gel 에서 한개의 band 를 보였고, 그 subunit의 분자량은 30,000±1,000 daltons 이었으며, specific activity 는 $6\times10^5$ units/mg 이었다. Zan I endonuclease 는 Bst NI endonuclease 및 Eco RII endonuclease 와 같은 인식 부위를 가지고 있으며, 아래 화살표와 같은 염기 사이를 절단했다. $(5'-CC^A_{T_{\uparrow}}GG-3')$ 그 효소 활성의 적정 pH 는 8 이었고 적정 온도는 45℃ 였으며, $MgCl_2$ 및 NaCl 를 효소 활성을 위해 꼭 필요로 하였다.
