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 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.
Gite
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Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Shankar
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D001236:Aspergillus oryzae / Q000201:enzymology*; D001665:Binding Sites; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010957:Plasmids / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000737:chemistry* / Q000378:metabolism
D004800:Enzymes, Immobilized; D015719:Single-Strand Specific DNA and RNA Endonucleases