Immobilization of RNase Rs via its carbohydrate moiety to aminoethyl-Bio-Gel P-2 and its application for the hydrolysis of RNA to 2′,3′cyclic nucleotides

Purified RNase Rs, from Rhizopus stolonifer, when covalently coupled to aminoethyl (AE) Bio-Gel P-2, via its carbohydrate moiety, retained 35-40% activity of the soluble enzyme. Optimization of coupling conditions showed that the most active immobilized preparations are obtained when 400 units of 100 mu M periodate oxidized enzyme are allowed to react with 1 mi (packed volume) of AE-Bio-Gel P-2 at 6 +/- 1 degrees C for 15 h. Immobilization did not change the pH and temperature optima of the enzyme but it increased the temperature stability. Immobilization did not bring about a change in the K-m but resulted in a 2.5-fold decrease in the V-max. Substrate concentrations as high as 25 mg of RNA could be converted to more than 80% 2',3' cyclic nucleotides in 14 h, at pH 5.5 and 37 degrees C. On repeated use, the bound enzyme retained 70% of its initial activity after six cycles of use. The bound enzyme could be stored in wet state for 60 days without any significant loss in its initial activity. (C) 1998 Published by Elsevier Science Ltd. All rights reserved.