PREPARATION, PROPERTIES AND APPLICATION OF ASPERGILLUS-ORYZAE S1 NUCLEASE COVALENTLY BOUND TO AMINOBUTYL-BIO-GEL P-2 THROUGH ITS CARBOHYDRATE MOIETY

Purified Aspergillus oryzae S1 nuclease, when covalently coupled to am inobutyl-(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 wi th 1 ml (packed volume) of AB-Bio-Gel P-2 at 4-degrees-C, in the prese nce of 20% (v/v) ethylene glycol, for 15 h. Immobilization did not cha nge the pH and temperature optima of the enzyme, but it increased the temperature-stability. Immobilization brought about an approx. 2-fold increase in the K(m) and a slight decrease in the V(max). On repeated use, the bound enzyme retained 60-65% of its initial activity after si x cycles. Immobilized S1 nuclease could be stored, in a wet state, for more than 45 days without any significant loss in its initial activit y. The application of AB-Bio-Gel- and concanavalin A-Sepharose-bound S 1 nuclease in removing restriction-endonuclease-generated single-stran ded tails in plasmid DNA is demonstrated.