Differential behaviour of recombinant wild-type and altered amidases on immobilized metal-ion affinity chromatography

The amidase gene from a wild-type strain 8602 of Pseudomonas aeruginosa was altered by site-directed mutagenesis at positions C91A, T103I, W138G, W138S, H275K, H281K and W138G-T103I and expressed in E. coli. The recombinant, wild-type, and altered enzymes bound to Cu(II)-IDA agarose column; the mutant H281K amidase bound partially. The wild-type enzyme did not bind to Ni(II)-IDA agarose column at pH 7.0 whereas the altered T103I amidase did. The binding of amidases onto immobilized metal chelates was pH dependent since an increase in the binding of amidase was observed as the pH varied from 6.0 to 8.0 for the wild-type and mutant enzymes. Recombinant altered (T103I and W138G) amidases were purified by immobilized metal-ion affinity chromatography with a final recovery of enzyme activity of about 70%. The purified T103I and W138G enzyme preparations were apparently homogeneous on SDS-PAGE with a Mr of 39.000 Dalton whereas the W138G enzyme exhibited two main protein bands on native PAGE with Mr of 110.000 and 150.000 Dalton.