Immobilization of the Improved DCase

Escherichia coli JM109 (pAD108) and JM109 (pAD404) cells were harvested by centrifugation, suspended in 50 mL of 0.1 M KP buffer, pH 7.0, containing 5 mM DTT, and then disrupted by sonication. The cell debris was removed and the supernatant was obtained, as was the enzyme solution. Duolite A-568 (Rohm and Haas) was washed with 1 M NaCl, water, and then 0.1 M KP buffer, pH 7.0, and then equilibrated with the same buffer for 18 h at room temperature. The wet resin was subjected to filtration, and then DTT (final concentration of 5 mM) was added to the enzyme solution, followed by stirring at 4 °C for 20 h under nitrogen sealing. For adsorption, the weight of the wet resin and that of protein in the enzyme solution were in the ratio of 1 to 0.04. After adsorption, the resin was washed three times with a fivefold volume of 0.1 M KP buffer, pH 7.0, containing 10 mM DTT, cross-linked with a fivefold volume of 0.2% (w/w) of glutaraldehyde at 4 °C for 10 min, washed three times with a fivefold volume of the same buffer at 4 °C, and then filtered (17,18).

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