Separation Of Arginine De-imino Enzyme Immobilization

Arginine deiminase (ADI) plays an important role in medicine. ADI, which can inhibit proliferation of blood vessel inner wall cell, has been proved to be an inhibitor of cell proliferation. It can convert arginine, a semi-necessary amino acid, into citrulline and ammonia. And the absence of arginine can restrain the growth of cell. In the present research the optimum condition of fermentation of the recombinant strain BL21(DE3)(pET-30a-cit) which was constructed in the lab was studied. ADI was then isolated and purified by breaking the cellswall, precipitation with (NH4)2SO4 and DEAE-cellulose ion-exchange chromatography from the culture broth.. The properties and immobilization of ADI were also studied. The results showed that the optimum induced conditions were: 10% corn flour, 10% soybean extract, 1%NaCl, 160rpm at 37℃ for 3h and then induced by 0. 6% 200mg/mL IPTG for 10h. The purified ADI showed a single band in SDS-PAGE. The molecular weight was 46kD. The ratio of enzyme activity of ADI was enhanced from 8.9U/mg to 33.8 U /mg. The ratio of purification was 3.8 folds. The recovery of the enzyme activity was 82.2%. The optimum condition of ADI was as follows: pH 5.8, 25℃. ADI was immobilized by compound silica. The remainder of the enzyme activity was over 80% after 10 times.