Urea Dehydrogenase of Green Algae (II):Preparation of Enzyme Solution from Lyophilized Powder

概要

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On the basis of some conditions for preparing urea dehydrogenase solution from green algae, the procedure was settled as follows. Algae were freezed and thawed with dry ice several times, lyophilized and ground sufficiently in a ball mill. The powder was suspended in phosphate buffer of pH 8, sonicated at 10KC for 15-20 minutes and extracted at 35°C for 30-60 minutes. The extract was fractionated with acetone at -10°C between 33% and 75%. The pellet was dissolved in cold H2O, after washing it, if deep green color was expected, with cold 75% ethanol to fade. The solution was dialysed against cold running H2O for 3 hours and sonicated at 10KC for 5 minutes immediately or after addition of Tris-HCl buffer of pH 7.5. The supernatant obtained by the centrifugation at 10, 000r. p. m. for 15 minutes was employed as the enzyme solution.
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