Gite S, Shankar V
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Biotechnol Appl Biochem. 1993 Jun;17(3):373-82.
Purified Aspergillus oryzae S1 nuclease, when covalently coupled to aminobutyl-(AB)-Bio-Gel P-2, via its carbohydrate moiety, retained 40-50% activity of the soluble enzyme. Optimization of coupling conditions showed that the most active immobilized preparations are obtained when 50-60 units of 1 mM periodate-oxidized enzyme are allowed to react with 1 ml (packed volume) of AB-Bio-Gel P-2 at 4 degrees C, in the presence of 20% (v/v) ethylene glycol, for 15 h. Immobilization did not change the pH and temperature optima of the enzyme, but it increased the temperature-stability. Immobilization brought about an approx. 2-fold increase in the Km and a slight decrease in the Vmax. On repeated use, the bound enzyme retained 60-65% of its initial activity after six cycles. Immobilized S1 nuclease could be stored, in a wet state, for more than 45 days without any significant loss in its initial activity. The application of AB-Bio-Gel- and concanavalin A-Sepharose-bound S1 nuclease in removing restriction-endonuclease-generated single-stranded tails in plasmid DNA is demonstrated.
纯化的米曲霉S1核酸酶通过其碳水化合物部分与氨丁基（AB）-生物凝胶P-2共价偶联后，保留了可溶性酶40%-50%的活性。偶联条件的优化表明，当50-60单位的1 mM高碘酸盐氧化酶在4℃、20%（v/v）乙二醇存在下与1 ml（填充体积）AB-生物凝胶P-2反应15小时时，可获得活性最高的固定化制剂。固定化没有改变酶的最适pH值和温度，但提高了温度稳定性。固定化使Km增加了约2倍，Vmax略有下降。重复使用时，结合的酶在六个循环后保留了其初始活性的60%-65%。固定化的S1核酸酶可以在湿态下储存超过45天，其初始活性没有任何显著损失。证明了AB-生物凝胶和伴刀豆球蛋白A-琼脂糖结合的S1核酸酶在去除质粒DNA中限制性内切酶产生的单链尾巴方面的应用。
