Preparation, properties and application of Aspergillus oryzae S1 nuclease covalently bound to aminobutyl-Bio-Gel P-2 through its carbohydrate moiety.

Abstract

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.