Immobilization of Nuclease P_1 on Inorganic Supports by Titanium Complex Method : Studies on Immobilized Enzyme (III)

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Nuclease P_1 was immobilized on such inorganic supports as macroporous silica gelbeads, porous glass beads and pumice by the titanium complex method. Scanning electron microscopy confirmed that titanium was uniformly combined with the surface of the support. A pore diameter of least 100-120 Å seemed to required for efficient immobilization of nuclease P_1.Titanium (IV) chloride was the most efficient activator of support among the metal salts tested. The optimum pH of immobilizing reaction was 4.5-5.0,and the reaction was 80% complete within 10 min at room temperature. From the efficiency of expression of the activity, the proper amount of the immobilized enzyme was considered to be 8-10 mg/g support. The immobilized enzyme on pumice had similar enzymatic properties to the free enzyme but higher thermal stability. The immobilized enzyme protein was quantitatively liberated from the supports by treatment with 1N sodium hydroxide at 70℃ for 30 min. Titanium was not liberated by 1N sodium hydroxide but was by 18 N sulfuric acid. On the basis of these findings, repeated use of the support was attempted. The rate of co-ordination binding was so fast that continuous reactivation of the support and immobilization of the enzyme in a column were feasible. The immobilized nuclease P_1 column could quantitatively hydrolyze 1% RNA for 25-28 days and the support could be repeatedly used by regeneration.
社団法人日本生物工学会の論文
1980-12-25